Functionalizing Surfaces with Nickel Ions for the Grafting of Proteins

These functionalized surfaces can be used as templates to anchor recombinant proteins engineered to bear a sequence of six histidines (His-tag). To tu...
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Langmuir 2003, 19, 4138-4143

Functionalizing Surfaces with Nickel Ions for the Grafting of Proteins O. Du Roure,† C. Debiemme-Chouvy,‡,§ J. Maltheˆte,† and P. Silberzan*,† Institut Curie, Section de Recherche, UMR CNRS 168, 11, rue Pierre et Marie Curie, 75231 Paris ce´ dex 05, France, and Institut Lavoisier (IREM, UMR CNRS), Universite´ de Versailles St-Quentin en Yvelines, 45, avenue des Etats-Unis, 78000 Versailles, France Received July 15, 2002. In Final Form: February 6, 2003 We study the grafting of newly synthesized nickel-chelating molecules on gold surfaces via their thiol moiety. These functionalized surfaces can be used as templates to anchor recombinant proteins engineered to bear a sequence of six histidines (His-tag). To tune the nickel concentration on the surface and ultimately the protein density, we have grafted mixed monolayers in which these nickel-chelating molecules are diluted by molecules that have a similar structure but are unable to fix Ni2+ ions. We have used different complementary techniques such as contact angle measurements, ellipsometry, and high-resolution X-ray photoelectron spectroscopy (XPS) to characterize the structures of these monolayers. XPS analyses were also performed to quantify the amount of nickel fixed on the surfaces. We find that the surface composition mirrors well the relative concentrations in the grafting solution. The robustness of the complexation of the Ni2+ ions was probed by exchange and competition experiments. His-tagged protein fragments could be strongly anchored on these surfaces as probed by atomic force microscopy experiments.

Introduction In many experiments designed to study the nature or the function of proteins, the first step is often to immobilize them on plane surfaces. This is the case for instance in the fabrication of protein arrays1 or for experiments probing the interactions between single proteins with tools such as the atomic force microscope (AFM).2 This situation emphasizes the need for a reliable, nondestructive grafting of these fragile biological objects. This fixation has then to fulfill two seemingly antagonistic properties: it has to be strong enough to keep the protein anchored during the experiment, yet it has to be delicate enough not to alter its biological function and to keep it exposed toward the outside. This last point has to be even overemphasized when dealing with nanostructures, which is the natural trend in the making of protein arrays. Most of the numerous protocols described in the literature rely on biochemical interactions.3 We rather focus here on the ones involving a chemical modification of the surfaces. To keep a fraction of the proteins with the correct orientation, a widely used strategy is to graft them via a long flexible spacer (most commonly poly(ethylene glycol) (PEG)) chemically grafted on the surface by one end and whose other extremity bears a chemical group such as N-hydroxysuccinimide acid (NHS) or pyridyldithiopropionate (PDP) able to react with any amino or thiol group of the protein.4 This grafting being random on the * To whom correspondence should be addressed. Tel: +33 (0)1 42 34 67 83. Fax: +33 (0)1 40 51 06 36. E-mail: Pascal.Silberzan@ curie.fr. † Institut Curie, Section de Recherche, UMR CNRS 168. ‡ Institut Lavoisier (IREM - UMR CNRS) - Universite ´ de Versailles St-Quentin en Yvelines. § Present address: Laboratoire Interfaces et Syste ` mes Electrochimiques, Universite´ Paris VI, 4 pl. Jussieu, 75252 Paris Cedex 05, France. (1) MacBeath, G.; Schreiber, S. C. Science 2000, 289, 1760. (2) Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283, 1727. (3) A good practical review of these different strategies can be found in: Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand Techniques; Academic Press: London, 1992.

protein, the flexibility of the spacer is used to get a reasonable probability for it to exhibit the right orientation. However, it may be necessary in some circumstances to get rid of this flexible linker and graft the protein via a shorter molecule if only to increase the protein density. The problem of orientation is then of prime importance as one has to control which part of the protein is to be anchored to the surface so that its “active” site remains accessible. In the present study, we have taken advantage of the large amount of work put together in the biology community interested in purifying proteins overexpressed in bacteria or cells. A commonly used technique is to genetically add a sequence of six histidines to their extremity. This (His)6 group (thereafter called His-tag) has the property to strongly bind to divalent metallic ions, the most widely used being Ni2+. Elution of lysates on resins or membranes bearing such ions will then only retain these tagged proteins. They are released at a later stage using either a different pH or a chemical having an even stronger affinity to nickel ions (imidazole most of the time). This technique, called immobilized metal ion affinity chromatography (IMAC),5 is now a routine purification technique, and the molecular biology tools used for the His-tagging are themselves widely accessible. This makes our approach aimed at the grafting of these particular proteins a very versatile one. Indeed, following previous approaches, the strategy we have used in the present work consists in chemically grafting a Ni2+ chelator to the substrates.5-7 For this purpose, we have used a derivative of nitrilotriacetic acid (4) Haselgru¨bler, T.; Amerstorfer, A.; Schindler, H.; Gruber, H. J. Bioconjugate Chem. 1995, 6, 242. Hinterdorfer, P.; Gruber, H. J.; Kienberger, F.; Kada, G.; Riener, C.; Borken, C.; Schindler, H. Colloids Surf., B 2002, 23, 115. Perret, E.; Leung, A.; Morel, A.; Feracci, H.; Nassoy, P. Langmuir 2002, 18, 846. (5) Hochuli, E.; Do¨beli, H.; Schacher, A. J. Chromatogr. 1987, 411, 177. Hochuli, E. Gen. Eng. 1990, 12, 87. Schmitt, L.; Dietrich, C.; Tampe´, R. J. Am. Chem. Soc. 1994, 116, 8485. (6) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490. (7) Kro¨ger, D.; Liley, M.; Schiweck, W.; Skerra, A.; Vogel, H. Biosens. Bioelectron. 1999, 14, 155.

10.1021/la020636z CCC: $25.00 © 2003 American Chemical Society Published on Web 04/18/2003

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Figure 1. Synthesis of HS-NTA and HS-CO2H: (i) (a) BrCH2CO2H, aqueous NaOH; (b) HCl. (ii) H2-Pd/C, aqueous MeOH. (iii) (a) 4-butyrothiolactone, aqueous NaHCO3; (b) AcOH.

(NTA), a chemical group already widely used in affinity chromatography. Here, we have synthesized a molecule whose other extremity bears a thiol moiety that can react with gold and eventually form well-defined self-assembled monolayers (SAMs).8,9 As the surface concentration of proteins may have to be finely tuned, we have also made mixed monolayers of this chelator and a “diluting” molecule similar in structure but unable to chelate Ni2+ ions. A similar approach was used in ref 7 where the diluting molecule exposed an alcohol group at the surface. Practically, we have used (1S)-N-[5-[(4-mercaptobutanoyl)amino]-1-carboxypentyl]iminodiacetic acid (denoted HSNTA) for the Ni2+ chelator and N-[8-(4-mercaptobutanoyl)]aminooctanoic acid (denoted HS-CO2H) as a diluting molecule; their structures and syntheses are outlined in Figure 1. Note that the HS-CO2H molecules not only regulate the quantity of nickel present on the surface but also offer some protection against nonspecific adsorption of proteins in comparison with bare surfaces or with many common chemical groups.10,11 This protection however is by far not as good as what can be obtained with more suitable chemical moieties such as ethylene glycol.10 In this paper, we describe the syntheses of HS-NTA and HS-CO2H molecules and characterize the mixed SAMs formed by the coadsorption of these molecules on gold surfaces. We also investigate the fixation of nickel and its stability on these monolayers. Experimental Section Synthesis of HS-NTA and HS-CO2H. (1S)-N-(5-Carbobenzyloxyamino-1-carboxypentyl)iminodiacetic Acid (2). Compound 2 was prepared according to ref 5. N6-CarbobenzyloxyL-lysine (Aldrich) 1 (8.4 g, 30 mmol) was dissolved in 45 mL of 2 N NaOH, and the solution was added dropwise (10 min) with stirring to a cooled solution (0 °C) of 8.34 g (30 mmol) of bromoacetic acid (Aldrich) in 2 N NaOH (30 mL). The solution was stirred overnight at 25 °C, and after heating for 2 h at 70 °C, 1 N HCl (90 mL) was added to the cooled solution. The precipitate was filtered off and dried to afford a crude white powder. This was purified by further dissolution in 1 N NaOH (100 mL) and precipitation with 1 N HCl (100 mL) to give 12.37 g (89.5%) of pure 2, mp 170 °C. 1H NMR (250 MHz, DMSO-d6, (8) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3. (9) Ulman, A. Introduction to Ultrathin Films; Academic Press: Boston, 1991. (10) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (11) Jordan, C. E.; Corn, R. M. Anal. Chem. 1997, 69, 1449.

297 K): δ 7.34 (m, 5H), 5.00 (s, 2H), 3.44 (m, 5H), 2.96 (m, 2H), 1.59 and 1.38 (2 m, 6H). (1S)-N-(5-Amino-1-carboxypentyl)iminodiacetic Acid (3). A solution of the triacid 2 (6 g, 15 mmol) in MeOH (95 mL)/H2O (5 mL) and 10% Pd/C catalyst (0.6 g) was stirred in H2 at 25 °C and 760 mmHg for 7 h. The catalyst was filtered off and rinsed with H2O (50 mL), and the solvents were removed from the filtrate to give 3.8 g of a colorless paste which crystallized when triturated with pentane. The product was redissolved in H2O (20 mL), and then EtOH (15 mL) was added until the solution became cloudy; after heating to give a limpid solution, the mixture was allowed to stand at -20 °C with seeds. The white crystals were filtered off and dried to afford 2.86 g (72.5%) of 3. 1H NMR (250 MHz, D2O, 297 K): δ 3.77 (m, 5H), 2.85 (t, 2H), 1.76 and 1.54 (2m, 6H). (1S)-N-[5-[(4-Mercaptobutanoyl)amino]-1-carboxypentyl]iminodiacetic Acid (HS-NTA). The amino derivative 3 (1 g, 3.8 mmol) was dissolved in H2O (10 mL), together with NaHCO3 (1 g, 11.9 mmol) and 4-butyrothiolactone (Aldrich, 0.6 g, 5.9 mmol). After heating for 15 h at 72 °C, the solution was cooled, acidified to pH 3 with AcOH (1 mL), and concentrated under reduced pressure to give thiol HS-NTA as a pale orange paste. The crude product was crystallized in absolute EtOH as a light beige hydroscopic solid, which was filtered, washed with absolute EtOH and then with pentane, and dried under vacuum to give 0.97 g (70%) of HS-NTA. 1H NMR (300 MHz, D2O, 300 K): δ 3.62 (m, 5H), 3.06 (t, 2H), 2.40 (t, 2H), 2.21 (t, 2H), 1.74 and 1.42 (2 m, 8H). MALDIHRMS (dihydroxybenzyl alcohol) m/z: 387.1150 (M + Na+, C14H24N2O7S requires m/z 387.1196). [R]25D + 6.7 (c 1.23, H2O). N-[8-(4-Mercaptobutanoyl)]aminooctanoic Acid (HS-CO2H). A solution of 8-aminooctanoic acid 4 (Aldrich, 1.6 g, 10 mmol), 4-butyrothiolactone (Aldrich, 1.02 g, 10 mmol), and NaHCO3 (1.8 g, 21 mmol) in H2O (20 mL) was stirred for 4 h at 95 °C. The cooled solution was washed with AcOEt, and the aqueous phase was acidified with 2 N HCl (30 mL) and extracted with AcOEt (2 × 30 mL). The combined extracts were washed with saturated NaCl and dried over MgSO4. The solvent was removed under reduced pressure to give 2.15 g (82%) of HS-CO2H (white solid). This was purified by recrystallization from aqueous EtOH, mp 64 °C. 1H NMR (300 MHz, DMSO-d6, 300 K): δ 11.96 (broad s, 1H), 7.76 (t, 1H), 2.99 (q, 2H), 2.42 (q, 2H), 2.26 (t, 1H), 2.14 (m, 5H), 1.72 (m, 2H), 1.46 (t, 2H), 1.34 (t, 2H), 1.22 (broad s, 5H). Found: C, 55.1; H, 9.1; N, 5.4; S, 12.1. C12H23NO3S requires: C, 55.14; H, 8.87; N, 5.36; S, 12.27. Preparation of the Samples. Glass slides or silicon wafers (Siltronix, France) were used indifferently as substrates. After careful cleaning (sulfochromic acid followed by O2 plasma cleaning), a 15 nm gold layer was sputtered on top of a 5 nm chromium adhesion layer, following classical procedures. Immediately after coating, the grafting was performed by immersing the substrates at 20 °C for times ranging from 15 min to 12 h in the solution of thiols at a nominal total concentration of 2.75 mM in a H2O/absolute EtOH (10%-90% v/v) mixture. After grafting, the substrates were thoroughly rinsed in the reaction solvent and then in absolute EtOH and finally dried in a stream

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of clean nitrogen. Nickel fixation was performed by exposing the surfaces to a 100 mM NiSO4 (nickel(II) sulfate heptahydrate, Aldrich) aqueous solution for 10 min. Substrates were then rinsed in pure water and dried under a stream of nitrogen. All these operations were performed in a class 10 000 clean room at 20 °C, 60% relative humidity. Experiments of exchange or unfixation of Ni2+ by competitive chelators in solution were performed immediately after Ni2+ fixation for 1 h, at room temperature. As we were interested in the potential effect of biological buffers, experiments were conducted in Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol, Aldrich] 10 mM, NaCl 100 mM. We have checked the stability of Ni2+ against pure buffer and solutions of EDTA (100 mM), imidazole (200 mM), and CaCl2 (100 mM). Surface Analysis. Ellipsometry was performed on a Plasmos SD2300 ellipsometer at 70° incidence using a He-Ne laser as the light source. For each measurement, the “effective” optical constants of the substrates immediately after the sputtering of gold were measured and these values were used as a reference for the determination of the thickness after grafting.12 The index of refraction of the SAMs was constantly fixed at 1.5. Each point is the average of at least 300 independent measurements on at least three different substrates. Contact angles of water were measured with a homemade projection device. Small (typically 1-10 µL) drops of ultrapure water were deposited on the surfaces with a syringe. All angles refer to advancing contact angles. The X-ray photoelectron spectroscopy (XPS) measurements were performed within a few days after sample preparation on a VG ESCALAB 220i-XL spectrometer using monochromatic Al KR radiation as the X-ray source. The survey and the highresolution spectra were recorded with a pass energy of 100 and 20 eV, respectively, in the constant analyzer energy mode. The detection angle was 90° with respect to the plane of the sample. The XPS peak areas were measured after background subtraction according to Shirley’s method.13 For each value of β, at least three samples were measured giving consistent results. We give here the average of these measurements. For some samples, several spots on the surface were analyzed and were found to give identical results within our experimental uncertainty, showing the homogeneity of the surface composition on the millimeter scale. Protein Fixation and Force Measurement Experiments. In the AFM experiments reported here, we have used E-cadherin fragments14 synthesized with a (His)6 sequence at their C-terminal. The silicon nitride tip of the instrument was functionalized using the procedure described above. Proteins were then adsorbed for 10 min on this functionalized tip from PBS 1 X (phosphate-buffered saline, pH ∼ 7.4) at a typical concentration of 1 µM. These experiments proceeded by measuring the pull-off force between the tip and a plane mica surface on which the proteins have also been fixed. More details on these experiments will be published elsewhere.15

Results and Discussion In the following, we call β the relative concentration of HS-NTA in solution:

β)

[HS-NTA] [HS-NTA] + [HS-CO2H]

The kinetics of formation of the SAMs were followed by ellipsometry by measuring the thickness layer for different reaction times. Results regarding a pure HS-CO2H SAM are shown in Figure 2. As classically observed in the formation of SAMs, the increase of the thickness with time follows reasonably well a Langmuir-like law: σ ∝ 1 - exp(-t/τ), where σ is the surface coverage and τ is a typical reaction time16 (Figure 2, solid line). We have found (12) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 45. (13) Shirley, D. A. Phys. Rev. B 1972, 5, 4706. (14) Gift from H. Feracci. (15) Du Roure, O.; Feracci, H.; Silberzan, P. To be published.

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Figure 2. Evolution of the ellipsometric thickness of the HSCOOH monolayer with time. The line is a best fit with a Langmuir isotherm.

τ = 1.2 h and have considered the reaction completed after a reaction time of 12 h. The thickness of the layer is then typically 1.85 ( 0.15 nm, a value compatible with the length of the fully extended molecule. The thickness values were not significantly affected when the SAMs were performed from mixtures with HS-NTA, a sign of the good completion of the mixed SAMs. Contact angle measurements were performed with freshly drawn ultrapure water. As the contact angles on acidic surfaces are known to be sensitive to the degree of ionization of the surface,17 we have checked that the pH was consistent for all these measurements (pH ) 6.5 ( 0.2). The contact angle of water on a pure HS-NTA surface is then close to 0°. It is significantly higher (ca. 40°) on HS-CO2H surfaces, a value somewhat larger than the usually measured 0-10° on SAMs bearing carboxylic acid functions when the main chain is a simple alkane chain.8,17 We attribute this discrepancy to the more complex structure of the HS-CO2H molecules that can impose slightly different surface orientations for the carboxylic acid groups. The ionization pH of these surfaces can therefore be shifted toward higher values so that at pH ∼ 7 the carboxylic groups would not be ionized and a measurement in such conditions would exhibit a higher contact angle.17 In solution, the three pKa’s for the carboxylic acids of the NTA group are respectively 0.8, 1.9, and 2.518 versus 4.8 for a long-chain carboxylic acid. It is then expected that even with a relatively high shift in the ionization pH induced by the surface and the chain orientation, most of these carboxylic groups are ionized, therefore leading to a more hydrophilic HS-NTA surface, which is what we observe. We have taken advantage of this contrast in contact angles to quantify the surface concentrations of the two species in mixed SAMs: in that case, intermediate values were obtained (Figure 3) although no clear-cut law such as the linear Cassie’s law19 could be verified. As a reference, we have also measured contact angles on a SAM made of octadecyl mercaptan (HS-(CH2)17-CH3, Aldrich) and (16) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988, 4, 365. (17) Bain, C. D.; Whitesides, G. M. Langmuir 1989, 5, 1370. (18) Stora, T.; Hovius, R.; Dienes, Z.; Pachoud, M.; Vogel, H. Langmuir 1997, 13, 5211. (19) Cassie, A. B. D.; Baxter, S. Trans. Faraday Soc. 1944, 40, 546.

Functionalizing Surfaces with Nickel Ions

Figure 3. Advancing water contact angles vs the relative proportions of HS-NTA and HS-COOH in solution. The lines are guides for the eye.

Figure 4. XPS survey spectrum from the Si/Cr/Au/SAM-Ni sample; the SAM was formed using the HS-NTA solution (β ) 1).

found the classical value of 105-108°.8 We have observed an increase of the contact angle on the pure HS-NTA monolayers with time (reaching 20-30° after a few days in the laboratory atmosphere). We have attributed this effect to the building up of a contaminant layer on top of the SAMs, and for this reason, nickel fixation was performed immediately after the formation of the monolayers. The fixation of Ni2+ ions on the SAMs did not significantly affect the measured ellipsometric thicknesses nor the contact angles. The chemical surface composition of the samples was determined by XPS analysis after incubation in the nickel solution, on the one hand to characterize the SAMs20 and, on the other hand, to study the Ni(II) fixation. A typical XPS survey spectrum obtained from a HS-NTA sample (β ) 1) after incubation in a NiSO4 solution is shown in Figure 4. Au, C, O, N, and Ni photopeaks are detected. The high intensity of the Au peaks witnesses the small thickness of the organic layer in agreement with the ellipsometric results (namely, 2 nm). For all the tested values of β, the survey spectra are similar, the only major difference being the intensity of the Ni2p photopeak. The S2p and S2s signals cannot be seen on the survey spectrum, first because of the low concentration of the S (20) Bain, C. D.; Evall, J. E.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155.

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Figure 5. XPS curve-fitted C1s spectrum from the Si/Cr/Au/ SAM-Ni sample (the same sample as Figure 4). The inset shows the HS-NTA and the HS-COOH structures; the attributions of the different carbons are indicated.

atoms and second because these atoms, being linked to the Au atoms, are buried under the organic layer. Indeed, S2p high-resolution spectra exhibit a peak located at a binding energy of 162.3 ( 0.1 eV, which is a signature of the gold-sulfur interaction.21,22 To determine if the composition of the mixed SAMs reflects the composition of the thiol grafting solution, the C1s high-resolution spectra have been studied. The C1s peak obtained for β ) 1 is shown in Figure 5. Following the interpretation of XPS studies on nylon-6,6,23 it was fitted with five contributions at binding energies of 285 ( 0.1, 285.5 ( 0.1, 286 ( 0.1, 287.4 ( 0.1, and 288.7 ( 0.1 eV. As indicated in Figure 5, peaks 1-5 are attributed to {-CH2-}, {-CH2-CO-NH-}, {-CH2-NH-CO and -CH2-CO2H}, {-CONH-}, and {-CO2H} groups, respectively (see Figure 5, insert, for the attributions of these peaks on the representations of the molecules). In Figure 6, we have plotted these contributions normalized by the total C1s peak area. We can then observe that contributions 1 (285 eV), 3 (286.2 eV), and 5 (288.7 eV) vary linearly with β whereas contributions 2 (285.5 eV) and 4 (287.4 eV) show small variations. We have also plotted on the same graph the calculated contributions based on the structures of the two thiol molecules, assuming that the surface composition of the mixed SAM is proportional to β (Figure 6, lines). Here, we had to take into account a small contribution of carbon contamination that overestimates contribution 1 by about 8%; there is no other adjustable parameter. The agreement between the experimental points and the calculation is then excellent. We have also monitored the evolution of the surface concentration of the elements Au, S, N, and O with respect to β. The Au signal normalized by the S signal shows no significant variation with β in agreement with our previous conclusion based on ellipsometry: the thickness and density of the mixed SAMs are independent of the surface relative concentrations. The ratio between the atomic percent of N and S increases linearly with β with a slope close to 1, which is expected given the structures of the (21) Zhong, C.-J.; Brush, R.; Anderegg, J.; Porter, M. Langmuir 1994, 10, 518. (22) Ishida, T.; Choi, N.; Mizutani, W.; Tokumoto, H.; Kojima, I.; Azehara, H.; Horaki, H.; Akiba, U.; Fujihira, M. Langmuir 1999, 15, 6799. (23) Beamson, G.; Briggs, D. High-resolution XPS of organic polymers: the Scienta ESCA300 database; J. Wiley & Sons: Chichester, U.K., 1992; p 196.

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Figure 6. Variation of the different contributions to the C1s peak (see Figure 5) as a function of the composition of the thiol solution used to prepare the mixed SAMs. The total peak area is normalized to 100%. The symbols are the experimental points. The lines are the calculated contributions based on the structures of the molecules and taking into account a contaminant layer that overestimates the -CH2- contribution by ∼8%.

Figure 7. High-resolution XPS Ni2p3/2 spectrum from the Si/ Cr/Au/SAM-Ni sample; the SAM was formed using the HSNTA solution (β ) 1).

two molecules (Figure 5). No sulfate peak due to the NiSO4 solution could be detected on the high-resolution S2p spectra (around 168 eV), indicating that the rinsing step was satisfactory in this respect. All the XPS results concur to show that the composition of the SAMs mirrors that of the grafting solution. On the survey spectrum (Figure 4), the Ni2p signal at 860 eV appears low even for a complete HS-NTA SAM as there is only one Ni per organic molecule. The Ni2p3/2 high-resolution spectrum is shown in Figure 7. The shape and binding energy of this spectrum are identical for all compositions of the mixed SAMs, that is, whatever β * 0. It consists of a main photopeak at 856.1 eV associated with a satellite peak located at a 5 eV higher binding energy. This spectrum is very similar to the one obtained from the Ni(OH)2 compound.24,25 (24) Nesbitt, H. W.; Legrand, D.; Bancroft, G. M. Phys. Chem. Miner. 2000, 27, 357. (25) Casella, I. G.; Guascito, M. R.; Sannazzaro, M. G. J. Electroanal. Chem. 1999, 462, 202.

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Figure 8. Ni2p3/2 peak area normalized by contribution 5 of the C1s peak (-COOH) as a function of the composition of the thiol solution used to form the mixed SAMs. The solid line is the calculated ratio based only on molecular structures; the other lines are best fits assuming a small quantity of nickel adsorbed nonspecifically on the layer (dotted) or interacting with the -COOH groups (dashed). The open symbols are the points after post-treatment (square, Tris buffer; diamond, EDTA solution; circle, imidazole solution; triangle, calcium solution).

Neither the Ni concentration in the solution nor the incubation time seems to be a critical parameter as comparable nickel surface concentrations were obtained for Ni2+ solutions ranging from 1 to 100 mM and for incubation times from 1 min to 1 h. The 100% HS-CO2H SAMs gave only a weak Ni signal even after a long XPS acquisition time. This result confirms that the species used for dilution is well adapted to our purpose; it covers the Au surface well but does not significantly chelate the Ni cations. SAMs made of mixtures of HS-CO2H and HS-NTA gave intermediate values. We have plotted in Figure 8 the evolution with β of the ratio χ of the Ni2p3/2 peak area to the peak area of contribution 5 (carboxylic group) of the C1s peak. This representation allows us to quantitatively compare these two elements. The calculated variation based on the molecular structures is also plotted in the same graph, assuming that the proportions of HS-CO2H and HSNTA on the surface are the same as in the grafting solution (Figure 8, solid line). Although the general trend is well accounted for, the experimental points reflect a systematic higher relative content of nickel. We can think of two interpretations for this discrepancy: either it represents a certain affinity of the nickel ions for the -CO2H moieties on top of the chelation by the NTA group or it is a fraction of nonspecifically adsorbed ions. The two interpretations differ in the expected behavior of the amount of nickel: In the first case, we expect

χ)

β(1 + 2κ1) + κ1 2β + 1

(1)

where κ1 is the amount of nickel fixed by the -CO2H groups, whereas the second situation is described by

χ)

β + κ2 2β + 1

(2)

where κ2 is the amount of nickel nonspecifically adsorbed. These two laws fit our experimental points reasonably well, although the first one gives a slightly better fit (Figure 8, dashed line) yielding 1 Ni ion fixed for 17 -CO2H groups (κ1 ∼ 6 × 10-2) on top of the chelated amount. In any case,

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Table 1. Evolution of the Surface Nickel Concentration on the Si/Cr/Au/SAM-Ni Sample (β ) 0.75) after 1 h of Immersion in Different Solutionsa relative Ni concentration no post-treatment Tris 10 mM, NaCl 100 mM Tris 10 mM, NaCl 100 mM + imidazole 200 mM Tris 10 mM, NaCl 100 mM + calcium 100 mM Tris 10 mM, NaCl 100 mM + EDTA 100 mM

1 0.82 0.79 0.73 0.82

a The nickel peak area was normalized by the C1s (-COOH) peak. By convention, the value without any post-treatment is 1.

we can conclude that there is a small amount of undesirable nickel adsorbed on the layer in accordance with previous observations.26 Given the homology between the structures of the two molecules, it was hoped that the final monolayer would be homogeneous. In the course of this study, we found no evidence of any phase separation between the two species. We have also checked how strong the Ni/NTA complex was regarding a buffered solution, a divalent cation (exchange experiments), and other chelatants (competitive complexation). Practically, HS-NTA/Ni substrates prepared using a β ) 0.75 thiol solution were immersed in the specified solution for 1 h immediately after fixation of nickel. The results are summarized in Table 1. Even very high concentrations of Ca2+ (100 mM), EDTA (100 mM), or imidazole (200 mM) did not result in a complete removal of the Ni2+ ions from the surface. Indeed, similar results were obtained for all three tested solutions: after the treatment, the Ni concentration on the surface was about 20-25% lower than its initial value (Table 1). These concentrations are then compatible with a desorption of the nonspecifically adsorbed Ni (κ1 ) 0 in eq 1) (Figure 8, open symbols). The large proportion of Ni not affected by the 200 mM solution of imidazole is surprising as these solutions are practically used for the elution of proteins in affinity chromatography as mentioned in the Introduction. However, the situation here is quite different: we monitor not the displacement of an anchored protein with imidazole but only the effect of this chemical on the surface concentration of nickel. This result however makes us conclude that the complex between the protein bearing the His-Tag and the nickel is more fragile than the complex between the nickel and the NTA group. This conclusion is consistent with the fact that the affinity of a given protein on the nickel-loaded resins used for purification in IMAC is very dependent on the structure of the protein itself and not only on its molecular weight. We have used the grafting strategy described in this paper to probe the adhesion properties of extracellular (26) Schmid, E. L.; Keller, T. A.; Dienes, Z.; Vogel, H. Anal. Chem. 1997, 69, 1979.

fragments of a membrane adhesion protein (E-cadherin fragments, MW ∼ 30 kDa) at the single-molecule level using the AFM. These proteins were engineered to bear an His-tag at their C-terminal. These experiments will be described in a subsequent paper. After ca. 10 min adsorption time on HS-NTA modified tips of the AFM, we could probe the adhesion of these proteins for several hours with no degradation of the performance.15 Let us emphasize here that the biological function of the protein (its adhesive properties) was preserved, demonstrating the interest of such a grafting. The good robustness of the grafting of these E-cadherin fragments correlates well with their remarkable stability on nickel-enriched resins (several months at 4 °C).27 The two situations, however, are thermodynamically different: On the resins or on other high specific surface media, one allows for the unbinding of a small fraction of the grafted proteins to yield a true thermodynamic equilibrium between a large amount of proteins bound to the surface and a smaller population of solubilized ones. On the other hand, for a plane surface in contact with a large reservoir of buffer, an equilibrium situation would require a relatively large amount of desorbed proteins to get the necessary concentration in the solution facing the surface. This would be true even for very large affinity constants and is only the consequence of a very unfavorable surfaceto-volume ratio. This is clearly not the case in the situation studied in the present paper; the grafting is unstable but is kinetically favored. Conclusion We have described the characteristics of mixed SAMs constituted of a derivative of NTA and a diluting molecule. High-resolution XPS measurements show that the SAM composition mirrors well the relative concentrations in the reactive solution. Very high concentrations of competitive chelatants or divalent ions have the same impact as pure buffer and seem to only displace the nonspecifically adsorbed nickel ions. These SAMs are thus very promising substrates for the anchoring of His-tagged proteins. This was demonstrated for a particular adhesion protein that shows a good stability and keeps its function for several hours after adsorption. Acknowledgment. We thank A. El Hajam who participated in some of the early grafting experiments, S. Thirot for synthesizing some of the molecules used in this paper, and H. Feracci for the gift of the E-cadherin fragments and for many fruitful discussions. Financial support from the Institut Curie (“programme incitatif et coope´ratif”) and from the Ministe`re de la Recherche (“action incitative concerte´e”) is gratefully acknowledged. LA020636Z (27) Feracci, H. Private communication.