In Situ Spectroelectrochemical Studies into the Formation and Stability

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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In Situ Spectroelectrochemical Studies into the Formation and Stability of Robust Diazonium-Derived Interfaces on Gold Electrodes for the Immobilization of an Oxygen-Tolerant Hydrogenase Tomos G. A. A. Harris,†,‡ Nina Heidary,†,‡,§ Jacek Kozuch,†,∥ Stefan Frielingsdorf,† Oliver Lenz,†,∞,# Maria-Andrea Mroginski,† Peter Hildebrandt,† Ingo Zebger,*,† and Anna Fischer*,‡ †

Institut für Chemie, Technische Universität Berlin, PC 14, Str. des 17. Juni 135, 10623 Berlin, Germany Institut für Anorganische und Analytische Chemie, Universität Freiburg, Albertstr. 21, 79104 Freiburg, Germany ∞ FMF - Freiburger Materialforschungszentrum, Universität Freiburg, Stefan-Meier-Straße 21, 79104 Freiburg, Germany # FIT - Freiburger Zentrum für interaktive Werkstoffe und bioinspirierte Technologien, Georges-Köhler-Allee 105, 79110 Freiburg, Germany

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‡

S Supporting Information *

ABSTRACT: Surface-enhanced infrared absorption spectroscopy is used in situ to determine the electrochemical stability of organic interfaces deposited onto the surface of nanostructured, thin-film gold electrodes via the electrochemical reduction of diazonium salts. These interfaces are shown to exhibit a wide electrochemical stability window in both acetonitrile and phosphate buffer, far surpassing the stability window of thiol-derived self-assembled monolayers. Using the same in situ technique, the application of radical scavengers during the electrochemical reduction of diazonium salts is shown to moderate interface formation. Consequently, the heterogeneous charge-transfer resistance can be reduced sufficiently to enhance the direct electron transfer between an immobilized redox-active enzyme and the electrode. This was demonstrated for the oxygen-tolerant [NiFe] hydrogenase from the “Knallgas” bacterium Ralstonia eutropha by relating its electrochemical activity for hydrogen oxidation to the interface properties. KEYWORDS: diazonium salt, hydrogenase, spectroelectrochemistry, SEIRA spectroscopy, electrochemical grafting, surface functionalization

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onto the electrode surface.1 In this context, thiols and other organosulfur compounds, which adsorb spontaneously on gold surfaces via Au−S bonds, thereby forming self-assembled monolayers (SAMs), are widely used for the surface functionalization of gold surfaces.2 However, SAMs suffer from poor chemical and electrochemical stabilities and, therefore, have limited application in electrochemical applications.3−6 To overcome this limitation, much effort has been invested into finding alternative methods for modifying gold electrode materials.7,8 One very promising method that has

INTRODUCTION In bioelectrocatalysis, the interface between the enzymatic redox catalysts and the electrode surface plays a critical role in terms of catalytic activity and stability, and hence applicability.1 Despite its higher cost, gold still finds many applications as an electrode material because of its inherent inertness, low toxicity, and applicability for a large number of spectroscopic and analytical techniques, including surface plasmon resonance, surface-enhanced infrared, and and Raman spectroscopies.2 To develop functional electrocatalytic electrodes based on enzymatic catalysts and take advantage of their particular activities and selectivities, it is necessary, in the case no mediated strategy is envisaged, to attach the enzymes in a stable and direct electron transfer (DET)-favorable configuration © XXXX American Chemical Society

Received: February 6, 2018 Accepted: June 4, 2018

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DOI: 10.1021/acsami.8b02273 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

from the interface but also to provide real-time monitoring of chemical changes happening within the interface itself. By this combined methodology, we could show that, in the case of Au, the use of radical scavengers moderates the kinetics of the grafting reaction and thereby tunes the charge-transfer resistance of the formed interface and hence the interfacial electron transfer rate between the enzyme and the electrode. The use of SEIRA spectroscopy can thus give valuable insights into the material/enzyme interfaces33 and help optimize immobilization strategies for the development of catalytic enzyme−electrode hybrids. To our knowledge, this is the first time that diazoniumderived interfaces, in terms of formation and stability, have been investigated in situ on gold by SEIRA spectroscopy and concomitantly used to achieve DET with an oxygen-tolerant [NiFe] hydrogenase without the use of SAMs.

been intensively investigated and reviewed is based on diazonium chemistry, which is applicable not only on gold but also on a broad range of other materials, including a variety of electrode surfaces such as other metals, carbons, and semiconductors.9−11 Diazonium-derived interfaces take advantage of the strong covalent carbon electrode bond formed between the diazonium-bearing organic molecule and the substrate interface, a bond that has been shown to exhibit excellent stabilities on a wide range of materials, including gold.9,12 Indeed, Au−C bonds have recently been shown to be stronger than the Au−S bonds by ca. 0.4 eV.13 Consequently, diazonium-derived interfaces exhibit improved stabilities with regard to longterm storage under laboratory atmosphere and repeated electrochemical cycling,4,14 sonication and exposure to refluxing solvents,6 and thermal treatments.5 However, a direct proof of this outstanding stability by any spectroscopic technique was not provided so far, and the true electrochemical stability window of such diazonium-derived interfaces on gold remains unknown. The potential to immobilize the enzymes on diazoniumderived interfaces via adsorption or covalent coupling has been demonstrated on carbon materials as well as on gold.15−23 One main disadvantage of diazonium chemistry is, however, the tendency to form thick multilayers because of the branching polymerization reactions, especially on gold, which often results in thick organic interfaces with insufficient conductivity and, hence, slow or suppressed heterogeneous electron transfer kinetics between the immobilized redox species and the electrode. A number of efforts have been made to achieve different outcomes in terms of interface structure, electron transfer properties, and hence applicability, including the use of cleavable protecting groups, bulky substituents, and ionic liquids.24 A promising approach is the use of radical scavengers during the interface formation, which has recently been demonstrated to result in monolayer formation on the carbon surfaces.25−27 Alternatively, in case of enzyme immobilization, the enzymes may themselves be functionalized with the diazonium groups and directly electrografted onto the electrode surface, as demonstrated for diazonium-modified horseradish peroxidase on gold electrodes.28 However, because of the lack of site-specific modification, this approach leads to a broad distribution of orientations on the electrode surface. In addition, one has to consider that the harsh modification conditions may not be suitable for many enzymes. In general, a suitable orientation of the redox-active enzyme at the electrode surface is crucial for ensuring high DET rates between the electrode and the redox center of the protein, thus eliminating the need for redox mediators.1 Herein, we present a spectroelectrochemical approach to first study the formation and electrochemical stability window of the diazonium-derived interfaces on gold electrodes, and second, tune the electron transfer properties of such interfaces using a radical scavenger to achieve DET between the electrode and a redox-active enzyme, in this instance the oxygen-tolerant membrane-bound [NiFe] hydrogenase (MBH)29 of Ralstonia eutropha. Hydrogenases such as this one are of particular interest for use in enzymatic fuel cells.30−32 Specifically, a combination of electrochemical polarization and in situ surfaceenhanced infrared absorption (SEIRA) spectroscopy is employed. The latter technique relies on a nanostructured Au film for the amplification of the spectroscopic signal and is used not only to follow the adsorption or desorption of a material

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EXPERIMENTAL SECTION

SEIRA measurements were performed with a Kretschmann attenuated total reflection (ATR)-type configuration implementing a Si prism coated with a 50 nm thick nanostructured gold film via electroless deposition, as described elsewhere.34,35 The spectra were collected between 4000 and 1000 cm−1 using a Bruker Tensor 27 spectrometer equipped with a liquid nitrogen cooled photoconductive mercury cadmium telluride detector. 4-Nitrothiophenol (4-NTP), 4-aminothiophenol (4-ATP), 4-nitrobenzene diazonium tetrafluoroborate (4-NBD), tetrabutylammonium tetrafluoroborate (TBAF), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,6-di-tert-butyl-α-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy (galvinoxyl) were sourced from Sigma-Aldrich and used as received. 4′-aminobenzanilide (Sigma-Aldrich) underwent diazotization using NOBF4,36,37 yielding 4-benzamidobenzenediazonium tetrafluoroborate (4-BABD). The product was characterized using electrospray ionization mass spectrometry (ESI-MS) (see Figure S1). The electrochemical measurements were performed using a threeelectrode setup that employed the electroless-deposited gold film as a working electrode (geometric area of 0.79 cm2), a Pt mesh as a counter electrode, and an adequate reference electrode. A schematic representation of the SEIRA spectroelectrochemical cell used in this work is depicted in Scheme S1. For measurements in aqueous solvents, a Ag/AgCl 3 M KCl (Dri-Ref, WPI) reference electrode was used, whereas for measurements in acetonitrile (MeCN, liquid chromatography−mass spectrometry grade, Sigma-Aldrich or Extra Dry AcroSeal, ACROS Organics), either a Ag/AgNO3 (0.1 M AgNO3 in MeCN) or a Ag/AgCl 3 M KCl (Leak-Free, Warner Instruments) reference electrode was used. The potentials measured relative to the Ag/AgCl reference electrode were converted to the reversible hydrogen electrode (RHE) scale using the Nernst equation (ERHE = EAg/AgCl + 0.059 pH + E°Ag/AgCl, where E°Ag/AgCl = 0.21 V). The potentials in acetonitrile were measured relative to a 1 mM ferrocene solution (0.1 M TBAF). The control of the working electrode was achieved using a Metrohm μAutolab potentiostat. Prior to surface modification, the gold films were cleaned electrochemically by repeated cycling between 0 and 1.4 V (vs Ag/AgCl 3 M KCl) at 100 mV s−1 in 0.1 M HClO4 until reproducible cyclic voltammetry (CV) traces were obtained. The real gold surface area was calculated using the Au(III) oxide reduction peak by using the formula A = Q1/Q0, where Q1 is the experimentally measured charge and Q0 is the charge required to reduce 1 cm2 of Au(III) oxide (Q0 = 400 μC/cm2).38 A surface roughness factor of 2.1 was calculated for the as-prepared gold films. All the current densities in the presented current−voltage plots are normalized to the real gold surface area and not to the geometric electrode area. The electrochemical grafting and desorption measurements were performed using 0.1 M solution of TBAF in acetonitrile. All electrochemical measurements, unless stated otherwise, were performed under an inert argon atmosphere. B


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ACS Applied Materials & Interfaces All desorption measurements of the SEIRA spectra were recorded under a constant potential of −0.58 V versus Fc/Fc+ or 0.6 V (vs RHE) to avoid charge-induced differences in the orientation of the organic molecules at the interface. The 4-NTP and 4-ATP SAMs were deposited overnight on gold from 1 mM ethanolic (high-performance liquid chromatography grade) solutions and thoroughly rinsed with copious amounts of ethanol. The benzanilide interfaces were electrochemically deposited on gold from 1 mM 4-BABD solutions in acetonitrile by cycling two times between +0.12 and −0.88 V (vs Fc/Fc+). The nitrophenyl interfaces were electrochemically deposited from 1 mM 4-NBD solutions in 0.1 M TBAF with and without the addition of different molar equivalents of DPPH using chronoamperometry by applying a potential of −0.445 V (vs Fc/Fc+) for 60 s, followed by rinsing in copious amounts of acetonitrile, followed by ethanol. The nitrophenyl interfaces were electrochemically reduced to aminophenyl interfaces by cycling the electrode between 0 and −1.2 V (vs Ag/AgCl 3 M KCl) in a 0.1 M NaClO4 1:9 ethanol/water solution. The final spectra of the reduced interfaces were recorded in dry acetonitrile. The heterodimeric, oxygen-tolerant MBH from the “Knallgas” bacterium R. eutropha (Re) carrying a Strep-tag II peptide as an affinity tag was purified as described by Goris et al.39 MBH was adsorbed onto the modified electrode surfaces for protein film voltammetry studies by incubating the electrodes in a 1 μM solution of MBH in 10 mM potassium phosphate buffer (PB, pH 7) at 5 °C for 15 min. The electrodes were rinsed with 10 mM PB (pH 7) and bioelectrocatalysis was performed at 5 mV s−1 in 10 mM PB (pH 5.5) at 25 °C after the saturation of the buffer with O2-free Ar or O2-free H2 gas. Blank measurements of the modified electrodes without MBH were recorded in the same conditions. To ease the assignment of the measured SEIRA spectra, theoretical IR spectra of benzanilide-Au, nitrobenzene-Au, NTP-Au, aminobenzene-Au, ATP-Au, 4-mercapto-N-phenylquinone-monoimine-Au, and nitrosobenzene-Au were calculated using density functional theory (DFT) in vacuum. The thiol or benzene hydrogen was substituted by an Au atom to account for the structural changes upon binding to the Au surface. Geometry optimization and vibrational analysis were performed on the BP86 level of theory using Gaussian 09.40−42 For C, H, N, and O atoms, the 6-31g* basis set was chosen. For the heavier S, the TZVP basis set, and for Au, the LANL2DZ set (using a pseudocore potential) were employed.43−45 All geometry optimizations were performed using the keywords “opt = tight” and “int = ultrafine”. The experimental vibrational frequencies were assigned by analyzing the potential energy distribution of the normal modes obtained from the DFT calculations. All SEIRA spectra were measured in situ in acetonitrile or in PB.

Figure 1. In situ SEIRA spectra of a gold thin-film electrode incubated with 1 mM 4-BABD in acetonitrile (0.1 M TBAF) before (black) and after application (green) of two CVs between +0.12 and −0.88 V (vs Fc/Fc+), starting in the cathodic direction (see CVs in the inset). The reference spectrum was recorded for unmodified gold in acetonitrile (0.1 M TBAF). The negative bands corresponding to the displaced acetonitrile solvent molecules at the interface are asterisked.

here, to the dominant vibrational modes). As seen in Figure 1 (inset), electrochemical reduction resulted in the passivation of the electrode. This is indicated by a sharp decrease in the reductive current upon subsequent cycling and by the growth of the benzanilide band intensities along with the simultaneous disappearance of the ν(NN) band, and the two surrounding negative bands (asterisked), corresponding to the displaced acetonitrile solvent molecules at the interface. The noncovalently bound species were removed by cycling the gold electrode several times to mild anodic potentials in fresh TBAF. As it has previously been reported that diazonium salts spontaneously graft onto gold surfaces at open-circuit potential,37,46 a gold electrode was incubated with 4-BABD for 6 min (the maximum time needed to carry out the electrochemical grafting), followed by rinsing in acetonitrile, to investigate the effect of spontaneous adsorption. The SEIRA spectra of the gold electrode after rinsing indicate that most of the adsorbed species are removed (Figure S3). The band intensities for the remaining (presumably) spontaneously grafted moieties are only a fraction of those measured for an electrochemically grafted interface. For comparison, SAMs were deposited via the spontaneous adsorption of 4-ATP. The successful formation of these interfaces is seen in the SEIRA spectra (Figure 2c,d) and is indicated by the appearance of characteristic bands corresponding to the ATP moiety, as summarized in Table 1. Scheme 1 illustrates the formation of both the diazonium- and SAMderived interfaces. To assess and compare the anodic and cathodic stabilities of the diazonium-derived interfaces and the SAMs on gold in acetonitrile (0.1 M TBAF), the SEIRA spectra were recorded at a constant potential of −0.58 V (vs Fc/Fc+) after polarizing the respectively modified electrodes at different cathodic and anodic potential steps (100 mV steps) for 15 s (see Figure 2a−d). A similar methodology in terms of potential protocol has been reported in the literature for evaluating the stability of the diazonium-derived interfaces on carbon.47 The spectra were recorded at the same constant potential to minimize the baseline changes/charge-induced differences in the orientation of the molecules at the interface. The plots of the SEIRA band intensities against the applied polarization steps allow one to follow potential-induced changes in the interface structure and

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RESULTS AND DISCUSSION The first part of this study will concentrate on the electrochemical stability of diazonium-derived interfaces on gold electrodes and compare them with thiol SAMs. The second part of the study will focus on the effect of radical scavenger addition on the interface deposition from diazonium salts and the influence of the interface structure/properties on the electrochemical behavior of a surface-immobilized oxygentolerant [NiFe] hydrogenase. Interface Formation and Electrochemical Stability. Upon incubation of the gold electrode with 4-BABD within the spectroelectrochemical cell, SEIRA spectroscopy shows that 4BABD species are adsorbed onto the electrode surface from the solution (Figure 1). This is indicated by the appearance of the corresponding ν(NN) stretching vibration band at 2262 cm−1 and the bands corresponding to the benzanilide moiety, as summarized in Table 1. Band assignments were made with the assistance of DFT calculations (for details, see the Supporting Information and refer, in the allocation given C


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Table 1. Major Band Assignments Made with the Assistance of DFT Calculations for Interfaces Deposited by Electrochemical Reduction of 1 mM 4-BABD or by Spontaneous Adsorption of 1 mM 4-ATP on Gold Electrodes sample

band position (cm−1)

band assignment

vibration

4-BABD

1493 1525 1598 1672 2262 1488 1593 1627 3370

δ(C−H) + ν(CC)ar δ(N−H) ν(CC)ar + δ(N−H) ν(CO) ν(NN) δ(C−H) + ν(CC)ar ν(CC)ar δ(NH2) νs(NH2)

C−H bending coupled with a CC aromatic stretching N−H bending vibration CC aromatic stretching coupled with a N−H bending CO stretching NN stretching C−H bending coupled with a CC aromatic stretching CC aromatic stretching NH2 bending NH2 symmetric stretching

4-ATP

Figure 2. In situ SEIRA spectra recorded at −0.58 V (vs Fc/Fc+) in acetonitrile (0.1 M TBAF) following the interfacial desorption induced by electrode polarization at different potential steps: behavior of a 4-BABD diazonium-derived interface after stepped polarization (15 s) at the (a) cathodic and (b) anodic potential steps, and behavior of a 4-ATP SAM after stepped polarization at the (c) cathodic and (d) anodic potentials (asterisked bands assigned to the dimerized 4′-mercapto-N-phenylquinone monoimine, NPQM species). (e) Normalized ν(CC)ar absorption band intensities at 1598 and 1593 cm−1 plotted as a function of the applied potential for spectra (a,b) and (c,d), respectively.

composition and thereby assess their electrochemical stability (see Figure S4a−d). To better compare the difference in the electrochemical stabilities of the Au−C- and Au−S-bound interfaces, the normalized SEIRA intensities of an aromatic CC stretching vibration, common to both molecular systems, are plotted as a

function of the applied potential steps (see Figure 2e). As one can see, the diazonium-derived interface is stable over a much broader potential window in acetonitrile than the SAM. The diazonium-derived interface is stable up to 0.8 V (vs Fc/Fc+), above which gold oxidation occurs, and down to −2.2 V (vs Fc/Fc+), after which a sharp decrease in band intensity is D


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from a homogeneous coupling between the reactive species in solution. It has previously been suggested that these species account for such a loss of material observed during sonication or refluxing.6 In contrast, the SAM is only stable up to 0 V (vs Fc/Fc+), at which point oxidative desorption begins to occur. Two new absorption bands (asterisked) at 1690 and 1514 cm−1 begin to appear around 0.4 V and may be attributed to a dimerized species, 4′-mercapto-N-phenylquinone monoimine (NPQM), which is known to be formed as a result of head-totail coupling between the adjacent oxidized ATP species.48−50 The calculated spectrum of this species bound to gold (Figure S5) supports this hypothesis, with a band at 1656 cm−1 for the ν(CO) stretching vibration of quinone and another at 1526 cm−1 for the ν(CC) aromatic stretching coupled with a CN stretching vibration of the imine bond. In the cathodic range, a significant desorption of the thiolate begins to occur at −1.3 V (vs Fc/Fc+) and the entire desorption process takes place over a wider potential window than that observed for the reductive desorption of the diazonium-derived interface. This

Scheme 1. Formation of Diazonium- and SAM-Derived Interfaces on Gold Formed by (a) Electrochemical Reduction of 4-BABD and (b) Spontaneous Adsorption of 4ATP

observed because of reductive desorption. The small decrease in band intensity observed between −0.6 and 0.8 V (vs Fc/Fc+) in Figure 2e may be due to the potential-induced desorption of the remaining physisorbed species that resist postmodification rinsing and electrochemical treatments; such species may arise

Figure 3. In situ SEIRA spectra recorded at −0.6 V (vs RHE) in 0.1 M PB (pH 5.5) following the interfacial desorption induced by electrode polarization at different potential steps: behavior of a 4-BABD diazonium-derived interface after stepped polarization (15 s) at the (a) cathodic and (b) anodic potential steps, and behavior of a 4-ATP SAM after stepped polarization at the (c) cathodic and (d) anodic potential steps. (e) Normalized ν(CC)ar and δ(N−H) absorption band intensities at 1595 and 1532 cm−1 plotted as a function of the applied potential for spectra (a,b) and (c,d), respectively. E


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Figure 4. (a) SEIRA spectra in acetonitrile of interfaces deposited on gold electrodes by electrochemical reduction of 1 mM 4-NBD with increasing molar equivalents of added DPPH (colored lines), as well as a SAM deposited by spontaneous adsorption of 1 mM 4-NTP (black line). (b) Cyclic voltammograms of 1 mM K3Fe(CN)6 recorded at 50 mV s−1 in 0.1 M PB (pH 7) of the same interfaces, and (c) plots showing the change in the νs(NO2) band intensity, as well as the change in the peak separation ΔEp for the Fe(CN)64−/3− couple, with increasing concentrations of DPPH. The arrows refer to the y-axis of the plot.

Table 2. Major Band Assignments Made with the Assistance of DFT Calculations for Interfaces Deposited by Electrochemical Reduction of 1 mM 4-NBD or by Spontaneous Adsorption of 1 mM 4-NTP on Gold sample

band position (cm−1)

band assignment

vibration

4-NBD

1351 1526 1598 1346 1518 1572 1592

νs(NO2) νas(NO2) + ν(CC)ar ν(CC)ar + νas(NO2) νs(NO2) νas(NO2) + ν(CC)ar ν(CC)ar ν(CC)ar + νas(NO2)

symmetric NO2 stretching antisymmetric NO2 stretching coupled with a CC aromatic stretching CC aromatic stretching coupled with an antisymmetric NO2 stretching symmetric NO2 stretching antisymmetric NO2 stretching coupled with a CC aromatic stretching CC aromatic stretching CC aromatic stretching coupled with an antisymmetric NO2 stretching

4-NTP

Effect of Radical Scavenger on Interface Formation. The electrochemical reduction of 1 mM 4-NBD results in the passivation of the gold surface with a dense polymeric interface, as has previously been reported.6 The SEIRA spectra were recorded after electrochemical grafting with and without the addition of different molar equivalents of the radical scavenger DPPH (see Figure 4a). The spectrum of gold modified with 4NTP is included for comparison. The band assignments were made with the assistance of DFT calculations (see Table 2, for details, see the Supporting Information) and are in line with previous literature reports.6,22 Having a closer look, one can see the differences in the ratio between the νs(NO2) and νas(NO2) + ν(CC) ar adsorption band intensitiesI[νs(NO2)]/ I[νas(NO2) + ν(CC)ar]for each interface, as plotted in the Supporting Information Figure S7. This ratio is related to the way molecules arrange at the corresponding interface. Indeed, because of the SEIRA surface selection rules, only the modes which preferentially exhibit dipole moment changes perpendicular to the surface are enhanced. It was recently shown in the literature that the addition of a radical scavenger such as DPPH during the reduction of 4-NBD on glassy carbon (GC) inhibits the polymerization mechanism by which multilayers are usually formed and thereby promotes monolayer formation (see the Supporting Information, Scheme

may be explained by the reversible nature of the one-electron thiolate desorption/readsorption process. The exact potential at which thiolate species are generally reduced depends not only on the substrate−adsorbate interaction but also on the adsorbate−adsorbate intermolecular interactions.51 To assess and compare the anodic and cathodic stabilities of the diazonium-derived interfaces and the SAMs on gold in an aqueous electrolyte relevant for enzyme immobilization and electrochemistry (see Figure 3), SEIRA spectra were also recorded in a similar manner in 0.1 M PB (pH 5.5). A plot of the normalized SEIRA intensities against the applied potential (Figure 3e) shows that the diazonium-derived interface is once again stable over a much broader potential window than the SAM (note: the normalized intensity of the N−H bending vibration was plotted for the 4-BABD diazonium-derived interface because of the overlap of the CC stretching vibration with the broad adsorption band of water molecules at around 1640 cm−1). A partial desorption of the diazoniumderived interface is observed at around −0.1 V (vs RHE), which overlaps with the onset of the hydrogen evolution reaction on gold (see CV of the bare gold electrode in the Supporting Information Figure S6). The formation of new species, such as NPQM, is not observed under anodic aqueous conditions. F


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Scheme 2. Electrochemical Reduction of a Nitrophenyl Interface Deposited on Gold and Subsequent Surface Immobilization of the Oxygen-Tolerant [NiFe]-MBH from R. eutropha (Bearing a Strep Tag for Purification, Not Shown Here)a

a

In this scheme, the MBH is immobilized in a DET configuration.

2 for an illustration of this effect).24,25 The monolayer formation, if occurring in the case presented here, would therefore result in an increase in the I[νs(NO2)]/I[νas(NO2) + ν(CC)ar] ratio, assuming the orientation of the molecules in one 4-NBD-derived monolayer is homogeneous. This is exemplified by the spectra of the 4-NTP SAM on our gold electrode, which exhibits a ratio of 4.7 corresponding to a highly ordered monolayer, in line with the literature reports.52 Conversely, for the diazonium salt-derived interfaces on gold, the ratio remains at 1.3 for the lower concentrations of DPPH, which is close to the ratio of 0.93 obtained for a bulk sample of 4-NBD (see Figure S8), and increases to 1.7 for higher concentrations (0.5 and 1 molar equiv) of DPPH. One can thereby conclude that no uniform preferential orientation of the molecules in the deposited interfaces is formed on gold upon the addition of DPPH and that the interfaces remain polymeric in nature. Meanwhile, the overall band intensities in the SEIRA spectra decrease proportionally as the concentration of DPPH increases, indicating a decrease in the amount of the material. Theoretically, for monolayer formation to occur during the grafting procedure, the rate of phenyl radical coupling to the electrode surface needs to be greater than the rate of coupling with the scavenger, while the rate of phenyl radical coupling to already grafted moieties needs to be smaller, which thereby results in the suppression of polymerization and multilayer formation.25 In the case of gold, our results indicate that the rate of coupling with the radical scavenger is greater than the rate of coupling to the gold surface, leading to less material being deposited in a less dense, albeit still polymeric, interface. The CVs of a ferricyanide electrochemical probe measured for the diazonium-derived interfaces deposited in the presence of various DPPH concentrations support this finding (see Figure 4b). The peak-to-peak separation (ΔEp) of the oxidation/reduction features of the Fe(CN)64−/3− (Fe2+/3+) couple decreases with the DPPH concentration, indicating a substantial decrease in the charge-transfer resistance RCT of the interface, until at 1 equiv of DPPH the electrochemical response is almost the same as that for an unmodified electrode. This is in sharp contrast to the behavior observed on GC where RCT reaches a steady-state minimum with an increase in the DPPH concentration, which coincides with the formation of an approximate monolayer coverage.25 A CV of a gold electrode modified with a 4-NTP SAM is included for comparison, where ΔEp of 0.29 V is obtained. To assess the reproducibility of the grafting procedure in the presence of DPPH, multiple electrodes were modified using 0.375 molar

equiv of DPPH, and CVs in the presence of Fe(CN)64−/3− were measured. The obtained ΔEp were in the same range (ΔEp = 169 ± 7 mV), indicating that the outcome of grafting in the presence of DPPH is highly reproducible (see Figure S9). Even though a monolayer coverage with a uniform orientation could not be achieved on gold in the present work, this method of using a radical scavenger leads to reproducible, less dense interfaces with decreased interfacial charge-transfer resistance, a result that should be beneficial for electrochemical applications. Trials using the less reactive radical molecule galvinoxyl showed a similar behavior, and comparable results could be achieved using higher radical scavenger concentrations (Figure S10). In conclusion, the addition of radical scavengers can reduce RCT sufficiently to allow the use of the modified/functionalized interfaces for the immobilization and utilization of electroactive species, while still taking advantage of the interfaces’ superior stability. Formation of Amino-Functionalized Interface and Immobilization of Hydrogenase. As previously mentioned, diazonium-derived interfaces have been used to immobilize enzymes on a range of electrode materials, including gold.15,17−21 We already showed that it is possible to immobilize the oxygen-tolerant Re MBH onto aminoterminated SAMs on gold via electrostatic interactions, resulting in a monolayer coverage, while preserving the enzyme conformation (secondary structure). It could be shown that two orientations prevailedone leading to DET and another leading to mediated electron transfer (MET).53 To introduce amino-functionalities and facilitate the immobilization of MBH, the nitrophenyl interfaces deposited in the presence of varying molar equivalents of DPPH (see Figure 4a) were electrochemically reduced to aminophenyl interfaces in protic conditions via a reaction that has been widely reported.22,54,55 A successful reduction from −NO2 to −NH2 was thereby revealed by SEIRA by comparing the spectra of the nitrophenyl interfaces before and after electrochemical reduction, as exemplarily shown for the nitrophenyl interface deposited with 0.375 equiv of DPPH (see Figure S11). The band assignments were made with the assistance of DFT calculations (for details, see the Supporting Information). Although the ν(CC)ar band of the aromatic ring at the 1600 cm−1 remains unchanged, a pronounced decrease in the band intensities of the νs(NO2) and νas(NO2) modes, in line with previous reports for the reduction of the nitrophenyl interfaces on gold,22 is observed, along with the appearance of a broad shoulder around 1626 cm−1 corresponding to the δ(NH2) G


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Figure 5. (a) SEIRA spectra in acetonitrile of the amino-functionalized interfaces deposited by the electrochemical reduction of 1 mM 4-NBD with increasing molar equivalents of DPPH added and subsequently electrochemically reduced in 0.1 M NaClO4, 1:9 ethanol/water solution and (b) SEIRA spectra in 10 mM PB buffer of MBH, adsorbed onto the same amino-functionalized interfaces, after subsequent rinsing with buffer (inset: shows a characteristic active site band of the enzyme, namely the ν(CO) stretching vibration of the Fe-coordinated carbon monoxide ligand, which can be detected at 1948 cm−1 for the so-called “Nir-B” state of the oxidized enzyme). (c) Plots showing the change in the ν(CC)ar band intensities, as well as the amide I and II band intensities, for all spectra with increasing concentration of DPPH, and (d) plot of the corresponding amide I to amide II band intensity ratios. The arrows refer to the related y-axis of the plot.

possible because of the absorption properties of the metalbound CN− and CO ligands, which advantageously give rise to bands in a spectral region free from any other absorptions.58 The appearance of two broad vibrational bands at 1658 and 1550 cm−1 in the SEIRA spectra (see Figure 5b) corresponding to the amide I and amide II modes of the protein backbone proves the successful binding of the MBH to the aminofunctionalized diazonium-modified electrode surface. The relatively low band intensities of the enzyme amide I and amide II modes compared to those of the deposited interface can be explained by the fact that the surface enhancement effect decays with a distance dependence of d−6.59 Comparing the dimensions of the interface (approximately 1−2 nm6) and the dimension of MBH (8 nm29), it is clear that the interface vibrations will be more enhanced than the enzyme-related vibrations. The appearance of the band at 1948 cm−1 related to the ν(CO) stretching vibration of the CO ligand, which is coordinated to the [NiFe] active site and characteristic for the highest oxidized, so-called “Nir-B” state, indicates that the enzyme’s active-site integrity is preserved upon surface immobilization.35,60,61 These results are in close agreement with the previous SEIRA study by Heidary et al. reporting the electrostatic immobilization of MBH onto amino-1-hexanethiol gold-modified electrodes under the same conditions.53 This clearly underlines the “binding” similarity between the diazonium-derived reduced and thiol-derived amino-terminated interfaces, a result that again emphasizes the successful −NO2 reduction. The pKa of the amino-1-hexanethiol SAM was determined close to 6, and hence the interface is assumed to be 8% protonated at pH 7.62 For the amino-functionalized surfaces derived from the aliphatic amine-containing 4-aminobenzyldiazonium and 4-(2-aminoethyl)benzenediazonium salts, the pKa

band. Similar results are obtained for the other nitrophenyl interfaces deposited in the presence of various DPPH amounts (data not shown). One has to note that the νs(NO2) adsorption at 1350 cm−1 does not vanish completely after reduction, indicating the presence of residual −NO2 moieties within the interface, a phenomenon previously reported.54−56 In line with this conclusion, we can see a shoulder close to the δ(C−H) + ν(CC)ar band located at 1520 cm−1, which may correspond to the ν(NO) stretching vibration of the incompletely reduced nitrosophenyl species (a calculated spectrum for this species is shown in Figure S12). Nevertheless, the contribution of the residual −NO2 moieties is negligible (Figure S11). Upon inspection of the SEIRA spectra of all reduced interfaces (Figure 5a), one can see that the SEIRA spectra of the electrochemically reduced interfaces are similar to those of a SAM deposited from the spontaneous adsorption of 4-ATP (Figure S2d,e), highlighting the similar surface functionalities present. In addition, the band intensities (of all bands) of the reduced nitrophenyl interfaces decrease proportionally with the amount of DPPH added during the nitrophenyl interface formation (Figure 5a). As such, the density of −NH 2 functionalities present in the interfaces decreases with the increasing amount of DPPH used. To evaluate the suitability of the deposited interfaces for enzyme immobilization and bioelectrocatalysis, we studied the electrostatically controlled adsorption of MBH onto the aminofunctionalized surfaces from a bulk solution at pH 7 (10 mM PB) using SEIRA spectroelectrochemistry. SEIRA spectroscopy has previously been shown to be a powerful tool for the investigation of protein immobilization on electrode surfaces.33,57 Additionally, it is an invaluable tool for elucidating the underlying molecular catalytic mechanisms by directly probing the active-site states.58 In the case of hydrogenases, this is H


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Figure 6. Protein film voltammograms recorded in 10 mM PB buffer at pH 5.5 saturated with H2 with a scan rate of 5 mV s−1 of MBH adsorbed onto amino-functionalized interfaces (a) before and (b) after the addition of the redox mediator methylene blue. (c) Plots showing the change in the maximum electrocatalytic current for H2 oxidation reaction because of DET and the corresponding MBH amide I and II band intensities with the increasing concentration of DPPH added during the interface formation. The arrows refer to the y-axis of the plot.

values have been reported around 10.0 and 10.5, respectively,63 whereas a pKa of 6.9 was determined for 4-ATP SAMs on gold.64 We thus assume that the pKa values of our aminophenyl surfaces are at least greater than 6, and therefore a significant proportion of the surface will be protonated leading to an electrostatic interaction with the MBH. A decrease in MBH adsorption is observed with decreasing aminophenyl density (i.e., with increasing amounts of DPPH added during the nitrophenyl interface formation), as indicated by a decrease in the amide band intensity (plotted in Figure 5c). It is tentatively proposed that this is a result of decreasing electrostratic attraction because of a decrease in the density of the protonated aminophenyl moieties at the interface. A plot of the amide I to amide II band intensity ratio as a function of added DPPH (Figure 5d) shows that while a small decrease is observed at 0.25 equiv DPPH, overall the ratios remain constant, suggesting that the orientation of the MBH stays the same for all interfaces. Cyclic voltammograms of the functionalized gold electrodes incubated with MBH were conducted in a H2-saturated, aqueous PB buffer at pH 5.5 and are shown in Figure 6a. A pH of 5.5 has previously been shown to give the highest activity.65 The sigmoidal shape of the CV traces indicates the electrocatalytic oxidation of H2 into protons (H+) and electrons as a result of DET between the catalytic redox center of the MBH and the electrode. The interface remains stable in the potential range probed. Overpotentials similar to those reported by Heidary et al. for the same hydrogenase immobilized on amino-terminated SAM-modified gold electrodes are observed.53 In that study, a maximum peak current of 1.8 μA/cm2 was obtained, whereas in the present study, currents between 1.3 and 5.4 μA/cm2 are obtained. These differences certainly result from the difference in surface protonation (e.g., as a result of different pKa values), in

interface thickness (around 1 nm for a SAM, whereas up to 2.5 nm can be obtained for the polymeric interfaces6), and finally differences in the electron-transfer resistance. The lower currents of around 1.3/1.4 μA/cm2 observed for the thick interfaces obtained without or with the addition of 0.25 M equiv of DPPH reflect the higher charge-transfer resistance of these interfaces, despite the relatively higher adsorption of the MBH catalyst (as revealed by SEIRA spectroscopy). Typically, tunneling can take place across distances of 2−3 nm.66,67 An increase in current to 5.6 μA/cm2 with 0.375 equiv of DPPH is observed, despite a decrease in MBH adsorption (as revealed by SEIRA spectroscopy), and indicates either a decrease in the charge-transfer resistance of the interface, or a decrease in the distance between the distal cluster of the electron transfer conduit of the adsorbed MBH and the electrode surface, or a combination of both. The current decreases to 3 μA/cm2 with 0.5 equiv of DPPH, despite the fall in the charge-transfer resistance of the interface, which corresponds to a decrease in MBH adsorption (again, as revealed by SEIRA spectroscopy). The change in current and MBH adsorption with DPPH concentration is plotted in Figure 6c. The results shown here suggest that there is a trade-off between the coverage of the electrode surface (and hence the adsorption of MBH) and the charge-transfer resistance of the interface (and hence the electroaccessibility of the adsorbed MBH). The addition of the redox mediator methylene blue results in an increase in current as a result of MET, as shown for the interfaces deposited with 0 and 0.375 equiv of DPPH in Figure 6b. The proportional increase in current is much greater than that observed on SAM-coated gold electrodes and might be attributed to the polymeric nature of the interface, which results in a substantial number of enzyme molecules immobilized further from the electrode surface.53 I


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Author Contributions

CONCLUSION In situ SEIRA spectroscopy was used to provide an insight into the formation and structure of the interfaces deposited on gold electrodes via electrochemical reduction of diazonium salts, while spectroelectrochemical investigations showed that the electrochemical stability window of such interfaces is significantly wider than the thiol-derived SAMs in both acetonitrile and water. This emphasizes their potential for use in electrocatalysis or in sensors. A radical scavenger was deployed during the interface deposition from a 4-nitrobenzene diazonium salt, resulting in the moderation of interfacial growth and a concomitant decrease in the charge-transfer resistance of the interface. After subsequent conversion into biocompatible amino-functionalized interfaces, this led to an increase in DET between the electrode and an immobilized oxygen-tolerant hydrogenase that could be correlated to the concentration of the scavenger added during the interface deposition and the structure/properties of the interface. Thus, this study not only highlights the potential of the diazonium-derived interfaces for electrochemical devices, but also underlines the potential of in situ spectroelectrochemical methods in the rational design and optimization of interfaces for such devices. Indeed, we have successfully used this approach in ongoing works to immobilize the catalytic species on nanostructured oxide electrodes, which have a great potential for use as functional devices.

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

This work was funded by DFG, the cluster of excellence UniCat (EXC 314), the Berlin International Graduate School of Natural Sciences and Engineering BIG-NSE, as well as the University of Freiburg and the BMBF (FKZ 01FP13033F). J.K. acknowledges a DFG Forschungsstipendium (KO 5464/1-1). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS DET, direct electron transfer; DFT, density functional theory; MET, mediated electron transfer; MBH, membrane-bound hydrogenase; PB, phosphate buffer; SAM, self-assembled monolayer; SEIRA, surface-enhanced infrared adsorption

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b02273. Experimental procedures; scheme of the spectroelectrochemical cell used; scheme of polymeric and monolayer structures on electrode surfaces; ESI-MS spectra; IR adsorption spectra, including the spectra of interfaces deposited spontaneously in the absence of an applied potential; interfaces deposited with the scavenger galvinoxyl; nitrophenyl interface before and after electrochemical reduction; ATR-IR spectrum of 4-NBD and 4NTP; IR spectra calculated from DFT and the optimized geometries of these species; plot of I[νs(NO2)]/ I[νas(NO2) + ν(CC)ar] for each interface; CV of the unmodified gold film electrode at pH 7; and CVs of three electrodes modified with 4-NBD and 0.375 mM DPPH (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (I.Z.). *E-mail: anna.fi[email protected] (A.F.). ORCID

Stefan Frielingsdorf: 0000-0002-4141-7836 Maria-Andrea Mroginski: 0000-0002-7497-5631 Peter Hildebrandt: 0000-0003-1030-5900 Ingo Zebger: 0000-0002-6354-3585 Anna Fischer: 0000-0003-4567-3009 Present Addresses §

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom (N.H.). ∥ Stanford University, Department of Chemistry, Stanford, California 94305-5012, United States (J.K.). J


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In Situ Spectroelectrochemical Studies into the Formation and Stability

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