Article pubs.acs.org/JPCC
Electron Transfer Across an Antifouling Mercapto-hepta(ethylene glycol) Self-Assembled Monolayer Thomas Doneux,*,† Alexis de Ghellinck,† Eléonore Triffaux,† Nicolas Brouette,‡,§ Michele Sferrazza,‡ and Claudine Buess-Herman*,† †
Chimie Analytique et Chimie des Interfaces, Faculté des Sciences, Université libre de Bruxelles (ULB), Boulevard du Triomphe, 2, CP 255, B-1050 Bruxelles, Belgium ‡ Département de Physique, Faculté des Sciences, Université libre de Bruxelles (ULB), CP223, Campus de la Plaine, B-1050 Bruxelles, Belgium ABSTRACT: Oligo(ethylene glycol) films are known to be very efficient at reducing the nonspecific adsorption of biomacromolecules on surfaces, but they often show a tendency to decrease drastically the rate of heterogeneous electron transfer at the modified surface, making them unsuitable for electrochemical biosensing. In this work, the heterogeneous electron transfer across the self-assembled monolayer of a short thiolated oligo(ethylene glycol) is investigated using four redox systems: [Fe(CN)6]3−/4−, [Ru(NH3)6]3+/2+, Fc(MeOH)2+/0, and [IrCl6]2−/3−. Fast electrontransfer kinetics are evidenced in all cases except the ferri/ferrocyanide couple, for which the electron transfer is completely suppressed. Interfacial characterizations by means of spectroscopic ellipsometry, electrochemical desorption experiments, and capacity measurements indicate that the film consists of a fairly hydrated single monolayer with a surface concentration of 4.1 × 10−10 mol cm−2. The peculiar behavior of [Fe(CN)6]3−/4− is discussed in terms of the hydration properties of both the monolayer and the electroactive anions. Interestingly, the self-assembled monolayer exhibits the desired antifouling properties against protein adsorption, tested with bovine serum albumin, making this system a promising platform for the development of electrochemical biosensors.
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INTRODUCTION A key challenge in the elaboration of electrochemical biosensors is the modification of electrode surfaces by organic layers that possess antifouling properties to prevent nonspecific adsorption phenomena but at the same time do not inhibit drastically electron transfer processes.1 The most promising surface modifications with respect to the antifouling criterion regard layers containing oligo- or poly(ethylene glycol) (OEG and PEG) chains2−5 and zwitterionic layers.6 Although the underlying mechanism preventing nonspecific adsorption has been the subject of intense research and for PEG different types of adsorption related to specific interactions are possible,7 the properties of water at the interface admittedly play a central role in the antifouling behavior of modified surfaces. Recent findings suggest that although both PEG and zwitterionic layers interact strongly with water8 the structure of interfacial water is not significantly different from that of bulk water.6,9 Combining the versatility and robustness of thiols-on-gold self-assembled monolayers (SAMs) with the antifouling properties of PEG, numerous studies aiming at decreasing nonspecific adsorption have employed OEG-terminated alkanethiol SAMs.2,10−13 These monolayers are built from molecules comprising, besides the thiol group, an alkane chain of 2 to 15 methylene units,12 then 3 to 45 ethylene glycol repeats.10,11 The application of such SAMs in the field of electrochemical biosensors has been rather limited because of © XXXX American Chemical Society
the detrimental effect that the layers have on heterogeneous electron-transfer kinetics, which are largely inhibited at the modified electrodes. The blocking ability of OEG-terminated alkanethiol SAMs arises, at least partly, from the significant length of the thiolated molecules composing the monolayer. Interestingly, short thiolated oligo(ethylene glycol) molecules where the OEG is directly attached to the thiol group, without any alkane bridge, have been shown to form organized SAMs with helical conformation (depending on the number of EG repeats)14 and to prevent significantly the nonspecific adsorption of proteins.13 Thus, the alkane spacer does not seem to be a prerequisite for the construction of organized, antifouling SAMs based on OEG. The properties of such short OEG SAMs toward heterogeneous electron transfer have not been deeply investigated. In the present work, the formation and characterization of a self-assembled monolayer of thiolated hepta(ethylene glycol) (t-OEG7) are presented, and its behavior against the nonspecific adsorption of proteins is evaluated. Various “outersphere” redox probes are employed to investigate the barrier Special Issue: Kohei Uosaki Festschrift Received: December 15, 2015 Revised: February 3, 2016
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DOI: 10.1021/acs.jpcc.5b12260 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry C
treatment. The measurements were performed in the full spectral range at an incidence angle of 70° and at a temperature of 25 ± 0.2 °C. For the determination of the SAM thickness, the data were analyzed in the framework of a three-layer model (Au|SAM|air). The optical properties of the gold substrates (gold on glass, 11 × 11 mm from Arrandee) were measured just before the formation of the SAM. A home-built cell consisting of a base of indium oxide and the rest in Teflon for in situ studies of the solid/liquid interface was used: It allows the incident light to enter the liquid phase via a thin glass window and another glass window allows, after the reflection at the solid/liquid interface, the light to reach the detector. The refractive index of the t-OEG7 layer is obtained using the Cauchy relation n(λ) = An + Bn/λ2 + Cn/λ4, with the values An = 1.45, Bn = 0.01 μm2, and Cn = 0 μm4, used previously for thiolated poly(ethylene glycol) chains.10,16 These values assume that the hydration of the layer makes a negligible contribution to the refractive index, an assumption that was shown to be valid at relative humidity below 50%.16 The ellipsometric data were fitted with this model, the thickness of the layer being the only adjustable parameter. The refractive index of BSA was fixed at n = 1.41.17 For the electrolyte the value of pure water was considered, n = 1.33. The fit of the spectroscopic angles ψ and Δ with a fixed n allows us to obtain the pseudothickness of the protein layer. For the kinetics scan, the data of the spectrum were taken each 21.5 s. Confocal Laser Scanning Fluorescence Microscopy. Fluorescence images were recorded with a Nikon Ti-Eclipse inverted microscope, equipped with a laser emitting at 488 nm. The emitted light was collected in the epi-fluorescence mode through a filter cube comprising a 540 nm dichroic mirror and a 515−530 nm emission filter placed in front of the photomultiplier tube. A 10× magnification objective (NA = 0.30, working distance 10.4 mm) was employed. The images were processed with the software ImageJ.18
properties of the t-OEG7 SAM toward electron transfer. It is shown that all but the ferri/ferrocyanide systems exhibit fast electron-transfer kinetics and that the SAM can be, as such, considered to be a “low-impedance” film (according to the term proposed by Gooding and coworkers1). The peculiar behavior of the couple [Fe(CN)6]3−/4− is discussed in more detail.
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EXPERIMENTAL METHODS Electrochemical Measurements. Electrochemical measurements were conducted in a three-electrode cell using a saturated calomel electrode (SCE) as reference electrode, a large area platinum foil as counter electrode, and a polycrystalline gold disk (1.6 mm in diameter, from Bioanalytical Systems) as working electrode, all connected to an Autolab PGSTAT30 (Metrohm Autolab) potentiostat equipped with a ScanGen module and a frequency response analyzer. All of the potentials given in the text are reported with respect to the saturated calomel electrode. Before each measurement, the gold working electrode was polished with alumina paste (1 μm particles), sonicated in ultrapure water for 10 min, then subjected to continuous voltammetric sweeps in 0.1 M HClO4 (Merck Suprapur) between −0.4 and +1.35 V until the reproducible voltammogram characteristic of a polycrystalline gold electrode was obtained. The charge associated with the formation of one monolayer of gold oxides, recorded in the last scan, was used to estimate the real area of the electrode, taking the reference value of 400 μC cm−2 for polycrystalline gold surfaces.15 The supporting electrolyte was a 0.10 M sodium phosphate buffer (0.061 M Na2HPO4 + 0.039 M NaH2PO4, both of analytical grade from Merck). The differential capacity−potential curves were obtained by impedance measurements, performed at a perturbation frequency f = 37 Hz and an amplitude of 10 mV (rms). A low frequency was chosen to maximize the capacitive contribution to the total impedance over the contribution from the noncompensated resistance of the cell. The differential capacity was calculated as Cd = 1/(ωZ″), with Z″ being the imaginary part of the total impedance and ω = 2πf being the angular frequency of the perturbation, under the assumption of a simple RC circuit to describe the electrochemical cell. The αmethoxy-ω-mercapto-hepta(ethylene glycol) (t-OEG7, Iris Biotech, >95%) was dissolved in 1 M sodium phosphate buffer at a 0.1 mM concentration. The electroactive reagents Ru(NH3)6Cl3 (Alfa Aesar, metal content >32.1%), K3Fe(CN)6 (Merck, pro analysi), K4Fe(CN)6·3 H2O (Merck, pro analysi), K2IrCl6 (Alfa Aesar, metal content >39%), and Fc(MeOH)2 (ferrocene 1-1′-dimethanol, Aldrich, >98%) were used without further purification. Bovine serum albumin (BSA, SigmaAldrich, 99%) and FITC-BSA (Sigma-Aldrich, product number A9771) were dissolved at 50 μg mL−1 concentrations in 0.1 M sodium phosphate buffer, pH 7.4. All of the solutions were prepared with ultrapure water (Milli-Q system from Millipore). The SAMs were formed by simple immersion of the freshly cleaned (polishing with 1 μm alumina paste then sonication in ultrapure water for 10 min) electrodes into the 0.1 mM t-OEG7 solution for 2 h. The electrochemical experiments were reproduced several times on freshly prepared SAMs. The Figures present individual but representative voltammograms. Ellipsometry. The thickness of the t-OEG7 SAM and the amounts of adsorbed proteins were determined by spectroscopic ellipsometry. The ellipsometer (Horiba Jobin Yvon MM16) is equipped with a tungsten-halogen lamp and a blue LED, providing a continuous illumination between 430 and 850 nm. The software DeltaPsi2 was used for data acquisition and
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RESULTS AND DISCUSSION Interfacial Characterizations of the SAM. The formation of the SAM of t-OEG7 was evidenced by electrochemical measurements. Figure 1a presents five consecutive voltammograms recorded with a freshly prepared SAM in the potential range −0.40 to +1.10 V. An anodic wave starts around +1.00 V in the first sweep in the positive direction, followed by a cathodic peak centered at +0.56 V on the reverse scan. Upon continuous scanning, both peaks shift negatively and their intensities evolve until the voltammogram displays the characteristic features recorded for the bare gold electrode in the sodium phosphate electrolyte. Such a behavior indicates that the SAM is progressively removed from the surface through a mechanism involving the oxidation of the thiol moiety and of the gold surface, in agreement with the oxidative desorption behavior reported for numerous thiolated molecules self-assembled on gold surfaces.19,20 The SAM can also be removed at negative potentials, as shown in Figure 1b, which presents five consecutive voltammograms recorded between +0.60 and −0.80 V. A pronounced cathodic peak is observed in the first cycle at −0.70 V. This peak shifts to less negative potentials and decreases in intensity in the subsequent scans. Meanwhile, the capacitive current increases at each cycle and a pair of capacitive peaks appears at +0.40 V. This voltammetric behavior is consistent with the occurrence of a reductive desorption process, whereby the B
DOI: 10.1021/acs.jpcc.5b12260 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry C
t-OEG7-covered electrode. In the potential range between +0.60 and −0.50 V, the SAM modified electrode exhibits an almost constant differential capacity with a low value, in contrast with the differential capacity recorded at the bare gold electrode, whose value is much larger and varies significantly in this potential window. This capacitive behavior of the bare gold is associated with the adsorption/desorption of the electrolyte anions as the potential is made more positive/negative, a process that is strongly inhibited in the presence of the SAM. At potentials more negative than −0.50 V, the differential capacity recorded at the t-OEG7-modified electrode increases markedly, giving rise to an important pseudocapacitive peak consistent with the occurrence of the reductive desorption phenomenon. Spectroscopic ellipsometry measurements were performed to determine the thickness of the t-OEG7 SAM. For the “dry” monolayer (measured in air), a value d = (16 ± 3) Å was obtained from the average of five independent measurements made at different locations on six samples (i.e., 30 measurements). For the “wet” monolayer (measured in water), it was not possible to properly fit the data within the framework of a Au|SAM|water model, likely because the t-OEG7 SAM is hydrated, which decreases its refractive index and reduces the optical sharpness of the SAM|water interface.27,28 The thickness of the SAM is slightly smaller than the values reported reported by Heeb et al.29 (20.6 ± 0.4 Å) and Malysheva et al.30 (24.1 ± 0.5 Å) for the same system, although in their case the selfassembly proceeded from an ethanolic solution. The former authors assigned their value to a predominantly helical conformation of the t-OEG7 chain, for which a theoretical value of 19.5 Å is predicted (7 units × 2.78 Å per ethylene glycol unit), against 24.9 Å (7 × 3.56 Å per ethylene glycol unit) for an all-trans conformation. From the thickness of the layer, it is possible to estimate the average area per molecule in the filled monolayer from the following relation31
Figure 1. Electrochemical characterization of the t-OEG7 SAM in 0.1 M sodium phosphate buffer (pH 7.4). (a) Five consecutive cyclic voltammograms recorded at 50 mV s−1, showing the oxidative desorption of the t-OEG7 SAM. (b) Five consecutive cyclic voltammograms recorded at 50 mV s−1, showing the reductive desorption of the t-OEG7 SAM. (c) Capacity curves of the bare gold (red open squares) and of the t-OEG7 SAM (black full circles).
cathodic peak can be assigned to the following electrochemical reaction19−26 RS − Au + 1e− ⇌ RS− + Au
am =
(1)
M ρdNA
(2)
where d is the thickness of the layer, M is the molar mass of t-OEG7 (356.5 g mol−1), ρ is its mass density (1.0 g cm−3)31 and NA is the Avogadro constant (6.02 × 1023 mol−1). The obtained value of 37 Å2 is in fairly good agreement with the value determined from the reductive desorption experiments. This experimentally determined molecular area can be compared with theoretical values based on the molecular dimensions of t-OEG7. A close-packed layer in all-trans conformation would correspond to a molecular area of 17.1 Å2 for an “upright” orientation11 and ca. 111 Å2 for a “flat-lying” orientation. For a helical conformation, the molecular areas would be 21.3 and ca. 96 Å2, respectively.11 The packing density of our monolayer is thus fairly high, but still far from that expected for an ordered, close-packed monolayer. With the knowledge of the SAM thickness, the differential capacity results previously obtained can be interpreted in terms of a Helmholtz capacitor εε Cd = r 0 (3) d
All of the adsorbed molecules are reduced during the first sweep in the negative direction and start to diffuse toward the bulk of the electrolyte solution. When the potential is swept back to less negative potentials, a part of the desorbed molecules can be redeposited onto the electrode through the reverse reaction. As a result, the total amount of t-OEG7 diminishes at each cycle and the capacitive current varies accordingly. The peaks appearing at +0.40 V are associated with the adsorption/desorption of the electrolyte anions on the electrode surface, a process that is inhibited in the presence of the intact, freshly prepared SAM. Equation 1 indicates that the reductive desorption involves one electron per adsorbed thiolated molecule, and thus the initial amount of t-OEG7 in the SAM can be estimated from the charge evolved in the cathodic process during the first scan. By integration of the cathodic peak, we obtained a charge σ ≈ 40 μC cm−2, which corresponds to a surface concentration of 4.1 × 10−10 mol cm−2. This value represents an average area per molecule in the filled monolayer am ≈ 40 Å2, in good agreement with the results of spectroscopic ellipsometry (vide infra). The reductive desorption behavior is further confirmed by the differential capacity measurements presented in Figure 1c. The Cd−E curve of bare gold is compared with that of a
where ε0 is the permittivity of vacuum (8.85 × 10−12 F m−1), εr is the relative permittivity of the SAM, and d is its thickness. Using Cd = 10.0 μF cm−2, extracted from the data of Figure 1c, C
DOI: 10.1021/acs.jpcc.5b12260 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry C and d = (16 ± 3) Å, as determined by ellipsometry, the relative permittivity of the SAM can be estimated at εr = 18 ± 3. Such value seems reasonable in comparison with the static permittivity data of neat oligo(ethylene glycols) published by Schrödle et al.32 (15.40 for six ethylene glycol units, 14.77 for PEG300 and 12.96 for PEG400) and taking into account that the film is significantly hydrated in contact with the aqueous electrolyte. Nonfouling Properties of the t-OEG7 SAM. The properties of the SAM against protein adsorption were evaluated using BSA, a frequently employed protein. The adsorption of the proteins on bare gold and on t-OEG7modified gold substrates was monitored in real time by spectroscopic ellipsometry. The refractive index was fixed, and the pseudothickness of the protein layer was fitted.33 By applying the De Feijter equation,34 it is possible to derive the amount of protein adsorbed per unit area, Γ (mg m−2) n protein − nelectrolyte Γ= d dn dc
estimate of the overall adsorption properties rather than a very accurate determination of the adsorbed amounts. This was confirmed by confocal laser scanning fluorescence microscopy, using fluorescently labeled FITC-BSA. A SAM tOEG7 was formed on a gold-coated glass plate, which was subsequently immersed in a 50 μg mL−1 solution of fluorescently labeled BSA for 3 h. Figure 3 presents two fluorescence images corresponding to two zones of the same plate, one that has been protected by the t-OEG7 SAM (Figure 3a) and the other that has not (Figure 3b).
(4)
where d is the thickness derived from the fitted data, nprotein and nelectrolyte are the refractive indices of the protein and of the electrolyte, respectively, and dn/dc is the refractive index increment of the protein. For BSA in 0.1 M phosphate buffer, dn/dc is equal to 0.19 mL mg−1.34,35 Figure 2 presents the time evolution of the amount of adsorbed BSA upon immersion of the bare and SAM-modified
Figure 3. Fluorescence images (256 × 256 pixels, 10× magnification) acquired on a Au|t-OEG7 (a) and on a bare gold (b) samples immersed in 50 μg mL−1 of fluorescently labeled BSA for 3 h. The acquisition parameters were exactly identical for both images.
Heterogeneous Electron Transfer. The impact of the t-OEG7 SAM on the heterogeneous electron transfer was evaluated with d ifferent redox couples, namely, [Fe(CN) 6 ] 3−/4− , [Ru(NH 3 ) 6 ] 3+/2+ , Fc(MeOH) 2 +/0 , and [IrCl6]2−/3−. All of these couples are known to exhibit fast (reversible) electron-transfer kinetics at bare gold electrodes and are frequently used to assess the electron-transfer properties of modified electrodes. Figure 4 presents the cyclic voltammograms recorded at the bare and t-OEG7-coated electrodes in the presence of 1 mM [Fe(CN)6]3− + 1 mM [Fe(CN)6]4−. While the characteristic voltammogram of a reversible electrochemical system is observed for the bare gold electrode, the electron transfer is completely suppressed at the t-OEG7
Figure 2. Amount of adsorbed BSA versus time for bare gold (black trace) and t-OEG7-modified gold (red trace), as determined from spectroscopic ellipsometry measurements performed in 0.1 M phosphate buffer solutions containing 50 μg mL−1 of protein.
gold substrates in the protein solutions. The results show clearly that the adsorption of the protein is significantly reduced, although not completely suppressed, in the presence of the t-OEG7 SAM, in agreement with previous reports on the antifouling properties of oligo(ethylene glycol) monolayers.1,2,13 This behavior was independently verified by quartz crystal microbalance measurements, which also showed that the adsorption of BSA was largely inhibited (
