GlyHisGlyHis immobilization on silicon surface for copper detection

Applied Surface Science 269 (2013) 166–170

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GlyHisGlyHis immobilization on silicon surface for copper detection Sabrina Sam a,∗ , Anne Chantal Gouget-Laemmel b , Jean-Noël Chazalviel b , Franc¸ois Ozanam b , Noureddine Gabouze a a b

UDTS, 2 bd Frantz Fanon, BP 140, Alger-7 Merveilles, Algiers, Algeria Physique de la Matière Condensée, École Polytechnique, CNRS, 91128 Palaiseau, France

a r t i c l e

i n f o

Article history: Available online 6 November 2012 Keywords: Silicon functionalization Peptide immobilization Cyclic voltammetry

a b s t r a c t Hybrid nanomaterials based on organic layer covalently grafted on semi-conductor surfaces appear as promising systems for innovative applications, especially in sensor field. In this work, we focused on the functionalization of silicon surface by the peptide GlyHisGlyHis, which forms stable complexes with metal ions. This property is exploited to achieve heavy metals recognition in solution. The immobilization was achieved using multi-step reactions: GlyHisGlyHis was anchored on a previously prepared carboxyl-terminated silicon surface using N-ethyl-N -(3-dimethylaminopropyl)-carbodiimide (EDC)/Nhydroxysuccinimide (NHS) coupling agents. This scheme is compatible with the mild conditions required for preserving the probe activity of the peptide. At each step of the functionalization, the surface was monitored by infrared spectroscopy Fourier transform (FTIR) in ATR (attenuated total reflexions) geometry and by atomic force microscopy (AFM). Electrochemical behaviour of such prepared electrodes was carried out in the presence of copper ions by means of cyclic voltammetry. The recorded cyclic voltammograms showed a surface reversible process corresponding to the Cu2+ /Cu+ couple in the complex Cu–GlyHisGlyHis immobilized on the silicon surface. Copper ions concentrations down than ␮M where detected. These results demonstrate the potential role of peptide-modified silicon electrodes in developing strategies for simple and fast detection of toxic metals in solution. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Detecting trace amounts and/or removing toxic metals in water effluents is an important economical and environmental challenges [1]. It is still a real need to develop easy and fast methods allowing sensors to operate directly in the field. Electrochemical sensors based on chemical modified electrodes meet these criteria of efficiency and simplicity. Furthermore, attaching ligands having high affinity towards various metals is the key issue for developing such electrochemical sensors. Among metal cation chelating agents, oligopeptides are widely used in electroanalytical chemistry as tetradentate ligands for metal complexation [2,3]. It has been shown that the nature of the some side chains can have an even more dramatic influence on the stability of the complexes [4]. The classic and most significant example is peptides containing one or more histidine (His) entity [5,6]. The side chain imidazole ring of His has a very efficient nitrogen donor, which can form a six-membered chelate ring for coordination. Likewise, the coordination properties

∗ Corresponding author. Tel.: +213 21 43 26 30; fax: +213 21 43 26 30. E-mail addresses: [email protected] (S. Sam), [email protected] (A.C. Gouget-Laemmel), [email protected] (J.-N. Chazalviel), [email protected] (F. Ozanam), [email protected] (N. Gabouze). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2012.10.120

of a His residue within a peptide sequence depends greatly on the position of the His residue in the peptide chain [5–7]. If the His residue is the first amino acid in the peptide, there is a greater than 10-fold reduction in complex stability. So, Glycyl-Histidyl-GlycylHistidine (GlyHisGlyHis) peptide seems to be appropriate ligand to form stable complex with copper ions. However, many specific points should be dealt with: reliability of the attachment in order to obtain reproducible protocols, its efficiency in order to obtain sensitive sensors, robustness of the immobilization in order to sustain the conditions specific to the targeted test. All these constraints are often making surface chemistry the limiting factor to sensor performances. With respect to the optimization of these conditions, covalent attachment is an attractive route, since it offers the best performances in terms of robustness and can be made reproducible and with a high yield. In this framework, the advances performed in the last decade on silicon surface chemistry offer promising solutions, and potentially open the way to the use of silicon substrate as a transducer in biosensors built using an efficient and well-controlled surface chemistry [8]. For that purpose, multistep reaction schemes are often used [9], consisting in achieving a first step for the grafting on the substrate of a densely packed layer containing an appropriate amount of moieties bearing a reactive site, and a second step in which these reactive sites are reacted with the probes under mild conditions. In this paper, (GlyHisGlyHis) peptide is covalently anchored to the silicon surface using

S. Sam et al. / Applied Surface Science 269 (2013) 166–170

a strategy compatible with the mild conditions required for preserving the probe activity. First, alkene precursors are grafted onto the hydrogenated silicon surface using the hydrosilylation route, allowing for the formation of a carboxyl-terminated monolayer which is activated by reaction with N-hydroxysuccinimide (NHS) in the presence of a peptide-coupling carbodiimide N-ethyl-N (3-dimethylaminopropyl)-carbodiimide (EDC) and subsequently reacted with the amino linker of the peptide to form a covalent amide bond. We show that the peptide monolayer is sufficient to capture copper ions at a silicon surface and measure their amount by electrochemical measurements. 2. Experimental 2.1. SiH surface preparation Silicon samples (Siltronix, France) were cut from double-side polished float zone 1–10  cm p-type (1 1 1) silicon. They were shaped as 45◦ bevelled platelets (15 mm × 15 mm × 0.5 mm) for characterization by ATR-IR spectroscopy as described in detail elsewhere [10]. Silicon platelets were cleaned in 1/3 (v/v) H2 O2 /H2 SO4 piranha solution at 100 ◦ C and rinsed with water. They were subsequently etched in oxygen-free 40% NH4 F solution to obtain atomically flat surfaces, referred to as SiH surfaces [11].

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surface as working electrode, a platinum wire counter electrode and an Hg/Hg2 SO4 reference electrode. The electrolyte was copper free acetate at pH = 4 adjusted with HCl. The solution was degassed with argon for 15 min prior to data acquisition. Cyclic voltammetry was performed at different sweep rates between −1000 and 200 mV. 3. Results and discussion 3.1. AFM images Fig. 2 shows non contact AFM images of silicon surfaces at different steps of the chemical modification. AFM image of asprepared hydrogenated H–Si(1 1 1) surface (image a) exhibits flat terraces separated by atomic steps (3.1 A˚ height). After grafting of 10-carboxydecyl chains (image b), the surface topography is perfectly similar to that of the initial H–Si(1 1 1) surface with a the stepped structure and terraces which are still atomically flat in agreement with the formation of a homogeneous monolayer covering the terraces. After activation of the carboxyl groups, the surface topography looks unchanged and free of physisorbed contaminants (image d) in accordance with a controlled chemical conversion of acid groups to succinimidyl ester. 3.2. ATR-FTIR characterization

2.2. Organic modifications of the hydrogenated silicon surfaces

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 spectra were recorded in p polarization. The surfaces were also investigated with non contactmode atomic-force microscopy (PicoSPM from Molecular Imaging, Point Probe Si cantilevers from Nanosensors, resonance frequency 160 kHz) which provided a convenient monitoring of surface morphology and cleanliness.

The signature of acid-terminated chains grafted at silicon surface after reaction with undecylenic acid appears clearly in Fig. 3a. It consists of the intense bands at 1710 cm−1 relative to the carbonyl stretching  C O mode of the acid group [11,13] and the symmetric and antisymmetric CH2 stretching modes at 2850 and 2920 cm−1 of the methylene backbone [10,11]. The relatively lower bands are assigned to CH2 scissor deformation mode at 1465 cm−1 and C O H modes at 1285 and 1415 cm−1 [10,13]. The narrow negative band around 2083 cm−1 corresponds to the partial disappearance during the hydrosilylation reaction of the SiH bonds initially present at the hydrogenated silicon surface. After reaction with EDC/NHS, the formation of succinimidyl ester termination is evidenced in Fig. 3b by the characteristic triplet band at 1740, 1785, and 1820 cm−1 usually ascribed to the antisymmetric and symmetric  C O modes of the carbonyl groups of the succinimidyl cycle and to the  C O mode of the ester unit, respectively [14]. Other characteristic bands of the terminal succinimidyl ester group include the one corresponding to the antisymmetric and symmetric  C N C modes at 1205 and 1370 cm−1 , and  C O N mode at 1065 cm−1 were observed [15]. The strong attenuation of the acid C O band at 1710 cm−1 indicates that the major part of the acid groups was activated. The study of the spectrum in Fig. 3c confirms the covalent anchoring of GlyHisGlyHis peptide by amidation reaction. The bands corresponding to the succimimidyl ester disappear and two broad amide characteristic bands are observed at 1650 and 1550 cm−1 , commonly labelled amide I (C O) and amide II (ıNH), respectively [16,17].

2.4. Electrochemical detection procedure

3.3. Electrochemistry

All glassware was rinsed with 6 M HNO3 , then thoroughly with MilliQ water to avoid metal ion contamination. The copper ions were first accumulated at the GlyHisGlyHis modified silicon electrode at open circuit potential by dipping the sample into 10 mL of a stirred aqueous solution of Cu2+ sulfate (10−6 M) in acetate buffer (pH = 8) for 15 min. The sample was removed from the solution, thoroughly rinsed with MilliQ water and dried under N2 stream. The electrochemical measurements were performed in copper free acetate buffer (pH = 4) with an Autolab potentiostat using a threeelectrode electrochemical cell comprising of the modified silicon

Fig. 4 shows the voltammogram recorded in a mixture of CuSO4 (10−3 M) + GlyHisGlyHis (10−3 M) in acetate buffer at pH = 3, using graphite HOPG working electrode. Two electrochemical processes were observed. The first one corresponds to the redox couple Cu2+ /Cu+ with anodic and cathodic peak potentials at EA1 = −245 mV and EC1 = −335 mV, respectively. The second process corresponds to the couple Cu+ /Cu0 with EA2 = −570 mV and EC2 = −750 mV. Detecting of cuprous ion shows that the presence of peptide makes Cu+ ions stable in solution by the formation of Cu(I)-GlyHisGlyHis complex.

Fig. 1 shows the different steps of functionalization. Grafting of carboxyl-terminated alkyl chains was achieved by immersing the freshly hydrogenated silicon sample into outgassed undecylenic acid in a Schlenk tube, which was kept under UV illumination (312 nm, 6 mW/cm2 ) for 3 h (photochemical hydrosilylation) followed by a final rinse in hot acetic acid (75 ◦ C, 30 min). The carboxyl-terminations was activated in a Schlenk tube containing a mixture of 5 mM EDC and 5 mM NHS and reacting under continuous argon bubbling for 90 min in a water bath at 15 ◦ C [12]. Subsequently, the peptide was anchored on the succinimidyl ester terminated surface via an amidation reaction in an outgassed Schlenk tube containing a solution of 10−4 M GlyHisGlyHis peptide (provided by Eurogentec, Belgium) in 1× PBS buffer at pH = 7, overnight. 2.3. Sample characterizations

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Fig. 1. Scheme of the successive modification steps.

Fig. 2. Non contact AFM images of Si surfaces at different steps of the chemical modification. (a) As-prepared hydrogenated H–Si(1 1 1) surface. (b) After grafting of 10carboxydecyl chains. (c) After activation of the carboxyl groups using 5 mM equimolar EDC/NHS concentration. Z range is 2.4 nm for both images.

Fig. 5 displays cyclic voltammograms recorded before and after copper accumulation on the GlyHisGlyHis-modified electrode. The electrode is electrochemically inactive before copper complexation and presents only a capacitive current (Fig. 5a). After copper accumulation, the voltammogram recorded in a copperfree buffer solution exhibits cathodic and anodic peaks with E1/2 = (EC + EA )/2 equal to −175 mV (Fig. 5b). The area under the anodic and the cathodic peaks are identical suggesting a reversible redox process [18]. While two redox processes were detected for Cu–GlyHisGlyHis complex in solution as seen above (Fig. 4), only one process was however observed for the same complex

immobilized on the silicon surface (Fig. 5). By analogy to the results of the study in solution, we ascribe the redox process observed to Cu2+ /Cu+ couple whose potential is close to the redox potential of the same couple in solution. Indeed, Murray has demonstrated from several examples that the redox potential of immobilized species is equal to the apparent conditional potential of the same process in solution [19]. This effect is explained by the preserving of the structural and electronic configuration of the copper complex. However, the redox reaction of the couple Cu+ /Cu0 was not detected. In fact, the deposition of metallic copper is probably blocked by the organic monolayer grafted on the silicon surface. The same phenomenon

Fig. 3. ATR-FTIR spectra. (a) After photochemical grafting of undecylenic acid. (b) After activation treatment of 90 min in an aqueous solution of 5 mM EDC and 5 mM NHS. (c) After amidation in 0.1 mM GlyHisGlyHis.

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10 20

8

Current Density (µ A/cm2 )

Courant (µA)

6

0

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4 2 0 20 mV/s 50 mV/s 100 mV/s 200 mV/s 500 mV/s 750 mV/s 1000 mV/s

-2 -4 -6 -8

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-750

-500

-250

0

-10

250

-1400 -1200 -1000

Potentiel (mV(Hg2SO4/Hg)) Fig. 4. Cyclic voltammogram in acetate buffer solution (pH = 3) containing CuSO4 (10−3 M) + GlyHisGlyHis (10−3 M). Scan rate = 20 mV/s.

was observed on modified gold electrode with an organic monolayer on which metal deposition was inhibited [20,21]. Fig. 6 shows cyclic voltammograms recorded at different potential sweep rate (20–1000 mV/s). As predicted by the theory [18], these voltammograms present the feature of immobilized species on the surface. The reduction and oxidation peaks are symmetric and the peak separation (Ep = Epa − Epc ) is quasi null and is still constant with increasing the sweep rate. In addition, a linear dependence of the peak current on the sweep rate was obtained as expected for a surface-immobilized redox couple (Fig. 7) in opposite to the case of electrochemical process in solution (controlled by the diffusion of species to the electrode) where the peak current is proportional to v1/2 [22]. The electrochemical charge measured under the oxidation (or reduction) peak yields the amount of Cu ions chelated to the peptide-modified electrode by integrating the peak (Faraday ∗ = Q/nFA, Q = S/v, where  is the surface concentration low): Ox (mol cm−2 ), Q is the charge amount, n is the number of exchanged

5 4

-400

-200

0

200

electrons, F is the Faraday constant (96,485 C), A is the electrode geometrical area (0.125 cm2 ), S is the area under the oxidation or reduction peak and v is the potential sweep rate (V/s). The calculated value is ∼5·10−11 mol cm−2 or ∼3·1013 mol cm−2 . This result corresponds to one monolayer grafted on an electrode surface [23]. This value is identical for all the sweep rates, result which stands for the reversibility of the electrochemical process. The concentration of the complexed copper ions on surface was also calculated by using the Laviron formula [24] of a fast redox process on surface: ∗ , where j is the current density, R is the perfect jp = (n2 F 2 /4RT )vOx p gas constant and T is the temperature. A value of ∼5·10−12 mol cm−2 was found. The expected low value is due to the enlargement of the peak compared to as predicted in the theory which predicts that the value of the peak width at half maximum is of 90 mV for redox surface immobilized species with one electron reversible process [24]. Peak broadness is due to a non-equivalent distribution of the redox sites leading to small differences in redox potential from a site to another [25].

a b

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2

6 2

1 0 -1 -2 -3 -4 -5 -1000

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Fig. 6. Cyclic voltammetry of a GlyHisGlyHis-modified silicon surface performed in copper free acetate buffer (pH = 4) after copper accumulation at different scan rates = 20, 50, 100, 200, 500, 750, 1000 mV/s.

Peak Current Density (µA/cm )

Current Density (µ A cm-2)

3

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Potential (mV (Hg 2SO4/Hg))

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4 2 0 -2 -4 -6

Potential (mV (Hg 2SO4 /Hg)) Fig. 5. Cyclic voltammetry of a GlyHisGlyHis-modified silicon surface performed in copper free acetate buffer (pH = 4). (a) Before copper accumulation and (b) after copper accumulation in stirred aqueous solution of Cu2+ sulfate (10−6 M) in acetate buffer (pH = 8) for 15 min. Scan rate = 50 mV/s.

0

200

400 600 800 Scan Rate (mV/s)

1000

1200

Fig. 7. Plot of anodic and cathodic peak intensity as a function of the scan rate.

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4. Conclusion GlyHisGlyHis peptide was covalently grafted on silicon surface using multi-step chemistry. Infrared spectroscopy confirmed the efficiency of the process at each stage of surface modification. The cyclic voltammogram exhibits cathodic and anodic peaks attributed to the reversible process of Cu2+ /Cu+ couple of the copper chelated by the GlyHisGlyHis peptide immobilized on silicon surface. GlyHisGlyHis peptide stabilizes the monovalent copper Cu+ by the formation of stable complex. Moreover, the linear plot of the intensity of potential peaks versus the sweep rates shows that the observed process is effectively due to species grafted on the silicon surface. Cyclic voltammetry displayed the ability of the GlyHisGlyHis-modified silicon surface to complex Cu2+ ions from solution. This result would then demonstrate the role of peptide monolayer in metal detection strategies. References [1] P. Viel, L. Dubois, J. Lyskawa, M. Sallé, S. Palacin, Applied Surface Science 253 (2007) 3263. [2] H. Kozlowski, W. Bal, M. Dyba, T. Kowalik-Jankowska, Coordination Chemistry Reviews 184 (1999) 319. [3] H. Sigel, R.B. Martin, Chemical Reviews 82 (1982) 384. [4] J. Brasun, A. Matera, S. Oldziej, J. Sxiatek-Kozlowska, L. Messori, C. Gabbiani, M. Orfei, M. Ginanneschi, Journal of Inorganic Biochemistry 101 (2007) 452. [5] N.I. Jakab, B. Gyurcsik, T. Kortvélyesi, I. Vosekalna, J. Jensen, E. Larsen, Journal of Inorganic Biochemistry 101 (2007) 1376. [6] R.P. Bonomo, F. Bonsignore, E. Conte, G. Impellizzeri, G. Pappalardo, R. Purrello, E. Rizzarelli, Chemical Society, Dalton Transactions 8 (1993) 1295.

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