ARTICLE pubs.acs.org/JPCC
Kinetics of Activation of Carboxyls to Succinimidyl Ester Groups in Monolayers Grafted on Silicon: An in Situ Real-Time Infrared Spectroscopy Study L. Touahir, J.-N. Chazalviel, S. Sam, A. Moraillon, C. Henry de Villeneuve, P. Allongue, F. Ozanam, and A. C. Gouget-Laemmel* cole Polytechnique, CNRS, 91128 Palaiseau, France Physique de la Matiere Condensee, E ABSTRACT: The kinetics of activation reaction of acid-terminated monolayers grafted onto crystalline (111) silicon surfaces with N-ethylN0 -(dimethylaminopropyl)-carbodiimide in the presence of N-hydroxysuccinimide is investigated using in situ real-time infrared spectroscopy. In the case of fully acid-terminated surfaces, the results show that the reaction rate exhibits a biexponential law, with two characteristic times, a fast one, τ1, and a slow one, τ2. The τ1 and τ2 values depend on temperature and solution composition. Moreover, the reaction pathways related to τ1 are partially suppressed for mixed monolayers, that is, by lowering the density of acid sites. These observations are discussed within the framework of a reaction mechanism.
’ INTRODUCTION Carbodiimides are efficient coupling reagents widely used in organic or peptide synthesis,1,2 and in nano(bio)technology for the immobilization of biomolecules on surfaces and nanoparticles.3-8 They allow for the formation of strong amide bonds under very mild conditions between a primary amine and a carboxylic acid. Notably, the carbodiimide N-ethyl-N0 (dimethylaminopropyl)-carbodiimide (EDC) is among the most popular ones because it is reasonably cheap, non-toxic, and water-soluble. It is generally used in the presence of N-hydroxysuccinimide (NHS)9 so that reaction with an acid-terminated species yields a stable succinimidyl ester intermediate. This active ester can then react with the amino probe through a classical amidation reaction, allowing for increasing the final yield and minimizing side reactions. Although the mechanism of carbodiimide-mediated activation is quite complex,10,11 experimental conditions are well established in homogeneous phase to maximize the activation yield.12 By contrast, a variety of experimental conditions (e.g., reagent concentrations, temperature, etc.) have been used for the activation of acid-terminated monolayers immobilized on surfaces (a brief literature survey is given in the Introduction of ref 13). This reflects the difficulty of transposing the above experimental conditions at a surface, where the reaction byproduct can strongly adsorb and shield active sites, which not only reduces the reaction yield but also makes the chemical composition of the surface uncertain.14 To circumvent this difficulty, a few groups synthesized the relevant functionalized molecular precursor (e.g., an alkane disulfide chain for adsorption on gold substrates15,16 or an alkene chain for direct grafting on H-terminated silicon surfaces17-19) to obtain succinimidyl ester-terminated monolayers in one step. Though this approach appears very attractive, it presents some potential drawbacks: (i) The bulky terminal group probably hampers obtaining a dense underlying molecular r 2011 American Chemical Society
assembly (i.e., made of the aliphatic chains), a critical point for the long-term stability of the monolayer, especially on silicon surfaces.20 (ii) In addition, there is no indication that the succinimidyl ester groups can be uniformly diluted within a mixed monolayer, also a key point for further uses. For various reasons, organic monolayers directly anchored onto silicon substrates are receiving strong interest for bioelectronic applications.8 In this context, and for the above reasons, we have recently undertaken a systematic study of the activation reaction on ω-carboxyalkyl monolayers grafted onto different silicon surfaces (atomically smooth, porous, and amorphous) and found that using solutions with EDC and NHS in equal concentrations in the range of 5-10 mM (operating temperature = 15 C) appears to be a good compromise to maximize the reaction yield and minimize byproduct adsorption. In fact, FTIR characterizations showed that, in the case of porous and amorphous silicon, the content of the resulting surface in succinimidyl ester-terminated sites is ∼100%21,13 but is only ∼70% in the case of well-defined Si(111) surfaces. The remaining molecular chains are, in the latter case, unreacted acid terminal groups. Very crucially, we showed that all of these surfaces are free of unwanted byproduct, as confirmed by atomic force microscopy (AFM) and by ex situ infrared spectroscopy.22 In this work, we investigate the activation reaction kinetics using in situ real-time IR spectroscopy (ATR geometry) as a function of solution composition, temperature, and surface density of acid terminal groups to obtain new insights into the reaction mechanism and explain the above differences.
Received: January 6, 2011 Revised: February 21, 2011 Published: March 14, 2011 6782
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Figure 1. Scheme of the flow cell used for in situ FTIR kinetic characterizations. The cell is initially filled with pure water, and the activation reaction is initiated by injection of the NHS/EDC solution at time t = 0.
’ EXPERIMENTAL SECTION Materials. The chemicals EDC (∼98%), NHS (98%), and ethyl undecylenate (97%) were purchased from Sigma-Aldrich, undecylenic acid (99%) from Acros Organics, and 1-decene (97%) from Fluka. All cleaning (hydrogen peroxide, H2O2, 30%; sulfuric acid, H2SO4, 96%; acetic acid, 100%), and etching (NH4F, 40%; HF, 50%) reagents were of VLSI grade and supplied by Carlo Erba. Ultrapure water (Milli-Q, 18 MΩ cm) was used for the preparation of the solutions and for all rinses. The silicon samples (Siltronix, France) were cut from doubleside polished float zone 800 Ω cm n-type (111) silicon. They were shaped as 45 bevelled platelets (15 15 0.5 mm3) for characterization by ATR-IR spectroscopy. Sample Preparation. The (111) silicon platelets were cleaned in a 1/3 (by volume) H2O2/H2SO4 piranha solution at 100 C and rinsed with water. They were subsequently etched either in an oxygen-free 40% NH4F solution (to obtain atomically flat surfaces, referred to as SiH surfaces)23 or in a 50% HF solution (to obtain atomically rough surfaces, referred to as SiHx surfaces).24 Organic Modifications of the Hydrogenated Silicon Surfaces. The hydrogenated surfaces were grafted with 10-carboxydecyl chains by photochemical hydrosilylation of undecylenic acid (312 nm, 6 mW cm-2, 3 h), followed by a final rinse in hot acetic acid (75 C, 30 min).25 In some cases, the acid-terminated surfaces were also prepared in two steps. The silicon surface was first reacted in oxygen-free neat ethyl undecylenate at 200 C during 20 h. After cooling, the sample was copiously rinsed in THF and CH2Cl2. The ester terminal groups were finally hydrolyzed into acid groups by immersion in deoxygenated 5.6 M HCl aqueous solution at 40 C for 3 h, followed by a final rinse in water.26 To obtain dilute acid monolayers, the grafting was performed in mixtures of ethyl undecylenate and 1-decene in variable proportions. The procedure to activate carboxyl groups in EDC/NHS solution was reported earlier.13 Sample Characterizations. The chemically modified surfaces were characterized by infrared spectroscopy (FTIR) in the ATR geometry, using a Bruker Equinox 55 spectrometer equipped with a liquid-nitrogen-cooled MCT photovoltaic detector. The surface concentrations of grafted carboxydecyl and succinimidyl ester chains were quantitatively determined by calibration of the
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Figure 2. IR spectra (in black) and fits (in blue) of the carbonyl peak of an acid surface (bottom) and of the succinimidyl ester triplet of a surface activated at 15 C (top). The reference is a hydrogenated surface obtained in HF.
characteristic IR band intensities.25,27 The activation yield was deduced from the ratio of the surface concentration of succinimidyl ester groups to that of initial acid groups. In situ measurements were performed by using a homemade vitroceramic IR flow cell with a volume of ∼0.1 mL fitted with inlet and outlet PTFE tubes (0.8 mm inner diameter) to circulate the solution (Figure 1). The silicon prism was pressed on the IR cell via a nitrile O-ring seal (diameter = 10 mm). The temperature of the solution was regulated thanks to a thermometric Ptresistance, incorporated into the body of the cell, and a heat sink placed at the back side of the cell (water- or air-cooled aluminum radiator) through a 20 W Peltier module. The cell temperature was thermostatted in the range of 5-25 C with a stability better than (0. 1 C. For kinetic measurements, the cell was initially filled with pure water. The NHS/EDC solution was then injected at time t = 0, and spectra were continuously recorded at subsequent times (with a resolution of 4 cm-1) for monitoring the evolution of the surface composition.
’ RESULTS AND DISCUSSION Ex Situ Characterizations of Acid- and Succinimidyl EsterTerminated Monolayers. Figure 2 presents narrow infrared
spectra in the carbonyl region of a SiHx surface after formation of the acid-terminated monolayer (bottom) and that of the latter surface after reaction with EDC/NHS (top). The reaction conditions were as follows: [EDC] = [NHS] = 5 mM at 15 C for 1 h 30. The reference spectrum is that of the SiHx surface obtained before grafting. The band at 1714 cm-1 (bottom) is consistent with the carbonyl stretching νCdO mode of the acid group, and the characteristic triplet band at 1745, 1789, and 1825 cm-1 (top) is assigned to the antisymmetric and symmetric νCdO modes of the carbonyl groups of the succinimidyl cycle and to the νCdO mode of the ester unit, respectively.28,29 Notice, however, that the activation is not total, as indicated by a small shoulder on the low-energy side of the 1745 cm-1 peak. This feature is attributed to unreacted acid groups. From a quantitative analysis of the carboxyl and succinimidyl ester 6783
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The Journal of Physical Chemistry C absorption bands,25,27 we obtain a surface concentration of 2.1 1014 cm-2 in carboxydecyl groups on the acid-terminated surface and 1.8 1014 cm-2 in succinimidyl ester groups after activation. These values correspond to an activation yield of 85%. This yield drops to 70-80% at atomically flat SiH surfaces. In Situ Infrared Study. Figure 3A shows successive in situ spectra recorded during the activation treatment of a carboxydecyl-terminated monolayer grafted on SiH at 5 C in a [EDC] = [NHS] = 5 mM solution. The reaction time is indicated next to each spectrum. The reference spectrum is that of the acidterminated surface recorded in water. Therefore, the positive IR bands are related to the creation of new surface species. Conversely, the negative bands are related to the disappearance of existing surface species. We clearly observe the progressive appearance of the characteristic triplet band of the succinimidyl ester (1745, 1789, and 1825 cm-1) together with a lowering of the acid-related IR absorption band at 1722 cm-1(weak feature). Other contributions are also observable. The most prominent one is the broad and negative water absorption (δOH2 scissor mode of water) at 1650 cm-1. This indicates that the density of molecular water is decreasing in the immediate vicinity of the surface, an effect that may be attributed to the increase in monolayer thickness during the reaction and possibly (though not necessarily) to the enhanced hydrophobic character of the surface upon increasing NHS ester coverage.30 The positive absorptions of EDC (1700 cm-1) and NHS (1707 cm-1) molecules present in the solution are also discernible (they were unambiguously identified from the spectra of EDC and NHS solutions; results not shown). We also observe the positive contribution from carboxylate absorption at 1550 cm-1 in the last spectrum. This is probably attributable to a small change in solution pH and the associated partial ionization of acid groups. For the purpose of a quantitative analysis, the obtained spectra were fitted as the superposition of five pseudo-Voigt peaks (accounting for the succinimidyl ester, carboxyl, and carboxylate absorptions), a quadratic baseline, and a contribution proportional to electrolyte absorption. The result of the fit is shown superimposed on the bottom spectrum in Figure 3A. Figure 3B presents the time evolution of the acid and the three
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Figure 4. Time dependence of the activation yield at 5 C (symbols are experimental data) on a SiH surface with [EDC] = [NHS] = 5 mM. The solid line is the best fit of the experimental data using a biexponential law given in the inset (see eq 1) with τ1 = 10 min and τ2 = 80 min. The vertical scaling accounts for the measured reaction yield of 0.63 after 90 min using ex situ measurements (see text).
succinimidyl ester band intensities as a function of reaction time. As expected, the acid loss is correlated with the simultaneous formation of the succinimidyl ester. In the following, we characterize the advancement of the reaction using a faster fitting procedure that also reduces the number of adjustable parameters. Spectra Si(σ) are fitted as a linear combination of the last spectrum [Sf(σ)] and the spectra of the different species contained in the electrolyte and in the ambient (liquid and vapor H2O, EDC, NHS). A constant-slope background (of general form aσ þ b, with a and b adjustable parameters) was also taken into account in the fitting procedure. Within this analysis, the coefficient of Sf(σ) in the combination varies from 0 to 1 and represents the advancement of the reaction relative to the final state. After scaling to the final activation yield determined from independent ex situ measurements on a similar
Figure 3. (A) Set of successive in situ spectra recorded at the various steps of the activation reaction of a SiH surface at 5 C in a solution with [EDC] = [NHS] = 5 mM. The reaction time is indicated for each spectrum on the right part of the graph. The curves are shifted vertically relative to one another for clarity. The fit of the last spectrum is represented in blue. (B) Evolution of the succinimidyl ester and COOH contributions as a function of the reaction time, as obtained from the fit of all recorded spectra. 6784
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Figure 5. Influence of acid-chain dilution on the activation yield: 100% carboxydecyl surface (green circle), 25% carboxydecyl/75% decyl surface (blue square), and 10% carboxydecyl/90% decyl surface (red diamond). Monolayers were grafted on SiHx ([EDC] = [NHS] = 5 mM at 15 C). The solid lines are the best fits of the data to a biexponential law. The A1 values are plotted in Figure 6.
Figure 6. Fast-process contribution (pre-exponential factor A1 in eq 1) as a function of the acid stoichiometry as determined from the fits of the curves shown in Figure 5 (b); simulation of the fraction of acid sites able to react through the anhydride pathway as a function of the fraction of acid sites on the initial surface (solid line).
surface, we can plot the time dependence of the reaction yield in real time, as illustrated in Figure 4. The shape of this plot, as discussed below, is closely similar to those displayed in Figure 3B, with a somewhat better signal-to-noise ratio. The curve in Figure 4 was fitted as the linear combination of two exponential functions using the expression below I ¼ R¥ ð1 - A1 expð - t=τ1 Þ - A2 expð - t=τ2 ÞÞ with A1 þ A2 ¼ 1
ð1Þ
where t is the reaction time after injection of the NHS/EDC solution, and A1 and A2 are, respectively, related to the fast- and slow-process contribution. R¥ is the asymptotic value of the reaction yield. Note that R¥ usually comes out close to unity, which represents a good check of the calibration from ex situ measurements. In the case of Figure 4, the short characteristic time is τ1 ∼ 10 min, whereas the long characteristic time is τ2 ∼ 1-2 h. Similar values are found when fitting the curves displayed in Figure 3B. In given conditions, we also found that the values of τ1 and τ2 are independent of the initial structure of the hydrogenated surface (the typical density of acid groups is 2 1014 cm-2 on a SiHx surface and 2.5 1014 cm-2 on a SiH surface). More surprisingly, τ1 and τ2 are weakly depending on EDC and NHS concentrations.31 On the contrary, the temperature has a sizable effect on τ1 and τ2. At 15 C, τ1 = 2-4 min and τ2 = 50-100 min. Note that the accuracy on τ1 is reasonably good at 5 C but becomes limited at 15 C by the time required for recording one spectrum (ca. 2 min). The rather large dispersion of the values of τ2 arises, in part, from the strong dependence of the fit results on the details of the procedure (e.g., setting R¥ to unity or letting it be a free parameter). Some physicochemical phenomena not taken into account in the model (such as the hydrolysis of the succimimidyl ester) could also contribute to this dispersion. Next, we investigated the influence of the density of acid sites by using mixed monolayers obtained by thermal hydrosilylation in a mixture of undecylenate ester and 1-decene in various proportions, followed by hydrolysis of the ester groups.26 The advantage of this two-step route is that the acid chains are thought to be homogenously distributed among the decyl chains
Figure 7. In situ IR spectrum of an acid-terminated surface in the presence of 10 mM EDC at different reaction times and at 15 C. The reference is the acid-terminated surface grafted on SiHx in H2O. The reaction time is indicated next to each spectrum. The curves are shifted vertically relative to one another for clarity. Notice the fast appearance of the anhydride signature in the spectra.
without formation of acid-terminated molecular domains.32 Figure 5 shows the evolution of the reaction yield for mixed layers of various compositions. They were analyzed as explained above to extract the final yield R¥ and the τ’s and A’s. Vertical scaling of the plots was performed as previously explained by measuring the reaction yield ex situ after 90 min of activation. As shown in Figure 6, the contribution of the fast exponential process (as estimated from the A1 parameter deduced from the fit) decreases when the carboxydecyl concentration in the monolayer is lower. As a final test experiment, we investigated the reaction of a carboxydecyl-terminated surface with EDC only. As shown in Figure 7, the reaction leads to fast formation of anhydride 6785
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Figure 8. Reaction scheme of the EDC/NHS activation of carboxylterminated surfaces.
because the intensity of the associated characteristic IR band (νsCO at 1813 cm-1) is constant after only 3 min (the lowfrequency mode νasCO at 1750 cm-1 has an absorbance too small to be reliably detected). This short reaction time is consistent with past measurements on similar self-assembled monolayers on gold.33 Kinetic Behavior and Reaction Pathways. The mechanism of the EDC/NHS reaction on acid groups has been extensively studied for amide-bond formation in homogeneous phase.10-12 At an acid-terminated silicon surface, the same mechanism has been used for interpreting spectroscopic data.13 It is summarized in Figure 8. Briefly, we recall that the reaction involves the competitive reaction of O-acylisourea (formed by reaction of EDC with surface acid groups, reaction 1), either with a neighboring surface acid group to form an anhydride intermediate (reaction 2), subsequently converted into a succinimidyl ester group by reaction with NHS (reaction 3), or directly with NHS to form the succinimidyl ester product (reaction 4). The biexponential kinetic law found in this work may be related to the existence of distinct reaction rates for the direct reaction of O-acylisourea with NHS (step 4) and anhydride formation (and subsequent reaction with NHS, steps 2 and 3), provided that above a threshold value of the reaction yield, the fast process becomes hindered and only the slow one remains efficient (otherwise, only the fast process would be observed). According to Figure 7, which evidences a fast formation of anhydride, it is then tempting to associate τ1 to anhydride formation (step 2). In this case, the need for an O-acylisourea species to find an unreacted acid neighbor to form an anhydride species provides a straightforward mechanism for hindering anhydride formation above a given reaction yield. The influence of acid chain dilution (Figure 5) corroborates this conjecture. Specifically, the contribution of the fast exponential process to the total activation yield decreases when the carboxydecyl concentration in the monolayer is lower (see Figure 6, symbols). The fast reaction mechanism, therefore, appears to be related to the anhydride pathway. A corollary is then that the direct Oacylisourea reaction with NHS would be identified to the slow pathway. To test further the above conclusions, we tried to estimate the threshold value limiting the fast process when this process is identified to anhydride formation. For that purpose, we performed a simple Monte Carlo simulation of the indirect reaction pathway (steps 2 þ 3). For the sake of simplification, the acidterminated surface was modeled as a square lattice of sites and the
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direct pathway was neglected (i.e., assumed infinitely slow). In addition, it was assumed that each acid site can form an anhydride with any of its four nearest neighbors. Once an anhydride group has been formed, its reaction with NHS leads to activation of either one of the two paired sites, the other one being regenerated as an acid group. This two-step process was simulated by choosing a site at random, triggering anhydride formation with one (randomly chosen) among its free neighbors (if one is available), then blocking one (randomly chosen) of the two sites involved in anhydride formation and leaving the other one free. Iteration of the process leads to a final state of the surface where none of the remaining acid sites has a free neighbor to react with; that is, all of the remaining acid sites left are isolated. The simulation was carried out either on a pristine square lattice or on a square lattice where a variable proportion of sites, chosen at random, had been removed prior to starting the simulation, a means to simulate the activation of mixed acid/alkyl surfaces. The result of the simulation gives the number of sites able to be activated through the anhydride pathway (the “blocked” sites) relative to the number of initial acid sites (see the solid line in Figure 6). The excellent agreement with the experimental variations of A1, with no adjustable parameter, gives strong support to our interpretation that the fast process reaction corresponds to the indirect pathway and that the slow process corresponds to the direct pathway.
’ CONCLUSION We have reported on the kinetics of the activation reaction of carboxydecyl-terminated monolayers grafted onto hydrogenated silicon surfaces using in situ infrared spectroscopy in ATR geometry. In the nominal conditions used in this work (5 mM < [EDC] ∼ [NHS] < 10 mM), the reaction rate obeys biexponential kinetics. This behavior arises from the competition between two reaction pathways: a fast one, unambiguously attributable to the fast formation of anhydride, and a slower one, attributed to the direct reaction of O-acylisourea with NHS. A pending question is whether the activation yield can be made complete at a surface. Increasing the reaction time may yield limited improvement if competitive hydrolysis of the succinimidyl ester comes into play. Increasing the reactant concentrations has been found to lead to problems of contamination with reaction byproducts. As mentioned in the Introduction, these difficulties could be overcome by grafting succinimidyl esterterminated monolayers in one step. An alternate attractive possibility would consist in increasing the kinetics of the direct activation pathway to increase the overall reaction yield. ’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Phone: þ33 1 69 33 46 80. Fax: þ33 1 69 33 47 99.
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