H Yaghoubi - ACS 2014

Hybrid Wiring of the Rhodobacter sphaeroides Reaction Center forApplications in Bio-photoelectrochemical Solar CellsHouman Yaghoubi,*,†,‡,§ Zhi Li,∥ Daniel Jun,# Evan Lafalce,⊥ Xiaomei Jiang,⊥ Rudy Schlaf,∥

J. Thomas Beatty,# and Arash Takshi*,†

†Bio/Organic Electronics Lab, Department of Electrical Engineering, ‡Department of Chemistry, §Nanotechnology Research andEducation Center, ∥Surface Science Lab, Department of Electrical Engineering, and ⊥Soft Semiconducting Materials and Devices Lab,Department of Physics, University of South Florida, Tampa, Florida 33620, United States#Department of Microbiology and Immunology, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T1Z3, Canada

*S Supporting Information

ABSTRACT: The growing demand for nonfossil fuel-based energy production has drawn attention to the utilization of naturalproteins such as photosynthetic reaction center (RC) protein complexes to harvest solar energy. The current study reports on animmobilization method to bind the wild type Rhodobacter sphaeroides RC from the primary donor side onto a Au electrode usingan immobilized cytochrome c (cyt c) protein via a docking mechanism. The new structure has been assembled on a Au electrodeby layer-by-layer deposition of a carboxylic acid-terminated alkanethiol (HOOC (CH2)5S) self-assembled monolayer (SAM), andlayers of cyt c and RC. The Au|SAM|cyt c|RC working electrode was applied in a three-probe electrochemical cell where a peakcathodic photocurrent density of 0.5 μA cm−2 was achieved. Further electrochemical study of the Au|SAM|cyt c|RC structuredemonstrated ∼70% RC surface coverage. To understand the limitations in the electron transfer through the linker structure, adetailed energy study of the SAM and cyt c was performed using photochronoamperometry, ellipsometry, photoemissionspectroscopy, and cyclic voltammetry (CV). Using a simple rectangle energy barrier model, it was found that the electrode workfunction and the large barrier of the SAM are accountable for the low conductance in the devised linker structure.

1. INTRODUCTION

The increasing demand for the production of energy without adirect link to combustion of a fossil fuel and the accompanyingproduction of CO2 has brought attention to the deployment ofbiomolecules for fabrication of biophotoelectrochemical cells.The biophotoelectrochemical cell uses technologies that exploitbiomimetic means of energy conversion by utilizing plant-derived photosystems.1,2 Different types of protein complexesmay be employed to fabricate a biophotoelectrochemical cell,including reaction centers (RCs) from the Rhodobacter (R.)sphaeroides bacterium, plant photosystems, and bacteriorhodop-sin proteins.3−18 Several studies of the R. sphaeroides RC haveshown promise for the utilization of this RC in biophotoelec-trochemical cells.3,4,13−15,19−22 The RC is a transmembraneprotein that has nearly 100% quantum yield of primary chargeseparationi.e., the formation of charged primary donor (P+)

and final acceptor (QB−)and an efficient stabilization of

separated charges.23−25 Most RC-integrated photoelectrochem-ical cells fabricated to date have been comprised of a cell withisolated RCs or RCs surrounded with a light harvesting (LH)pigment−protein antenna attached to a working electrode,immersed in an electrolyte with one or more redoxmediators.3,4,14,15,19,20,22,26,27 The use of RC−LH pigment−protein by several groups has shown improved photocurrentdensities over those obtained with the RC alone.6,27,28

Although the RC’s internal quantum efficiency is very highand the use of LH ring around the RC was shown to enhancethe photon absorption,3,6 the charge transfer between RCs and

Received: July 15, 2014Revised: August 24, 2014Published: September 19, 2014

Article

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© 2014 American Chemical Society 23509 dx.doi.org/10.1021/jp507065u | J. Phys. Chem. C 2014, 118, 23509−23518

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electrodes is another feature that influences biomolecule-basedsolar energy conversion.The RC of R. sphaeroides is comprised of three protein

subunits called L, M, and H (Figure 1c). Typically,biophotoelectrochemical cells have been fabricated using RCsimmobilized from either the H-side or the P-side on the surfaceof one of the cell’s electrode.3,4,13,15,19,20,22,27,29 Uponillumination, a photocurrent can be generated by transferringone of the charges (positive or negative) from the RC to theelectrode. The opposite charges are moved to the counterelectrode via a redox mediator in the electrolyte. The electrontransfer (ET) rate between the RC and the electrode is higherwhen RCs are oriented on the electrode’s surface from the P-side due to a shorter distance between the RC P+ site and theelectrode.29 However, the charge transfer at the RC-electrodeinterface in this orientation is complicated by a bowl on the P-side that introduces a gap between the electrode and theprotein (Figure 1c). A previous work showed a higherphotocurrent can be achieved if the bowl of at least some ofthe RCs may be filled by diffusion of cytochrome c (cyt c) intothe space between a 7-His-tagged (at the C terminus of the RCM protein) RC and a nitrillotriacetic acid (NTA)-terminatedself-assembled monolayer (SAM) on a Au electrode.30 Whilethe work of Lebedev et al. pointed to the importance of thebowl problem, the approach relies on the random diffusion ofcyt c proteins rather than constructing the structure.30

The current study is focused on employing a hybridoligomer−protein linker for immobilizing the wild-type RCfrom the P-side onto an Au electrode and at the same timefilling the bowl, which resulted in higher peak photocurrentdensity compared to that in the previous work.30 This structurewas assembled through a layer-by-layer deposition of a SAMwith carboxylic acid terminal groups,31 cyt c, and RC proteins.Because the feasibility of immobilizing cyt c onto a Au electrodeusing 6-mercaptohexanoic acid had been demonstratedbefore,31,32 we utilized the same molecule to construct thehybrid SAM|cyt c linker for RCs (Figure 1a). The goal was toobtain RC immobilization which occurs via the dockinginteraction between RC and cyt c. It was assumed that thismechanism could bring the cyt c heme and the RC P cofactorinto proximity for an efficient ET. The results presented in thiswork show that the new structure binds the RC without anyneed for protein mutation. Additionally, the observed photo-current density evidenced successful docking between cyt c andRC. Furthermore, the energy structure of the hybrid linker was

studied in detail and a quantum model was proposed toestimate the electrical conductance along the linker. Aschematic of the new structure and the ET events betweenthe RC, cyt c, and the SAM-modified Au electrode areillustrated in Figure 1a, with a representation of RC−cyt cinteraction given in Figure 1b. Additionally, Figure 1c shows aschematic of the RC protein subunits and the ET pathwaythrough the cofactors. A bowl in the RC is indicated, based onthe RC−cyt c cocomplex crystal structure.33

2. EXPERIMENTAL SECTION2.1. Electrode Preparation. All materials including equine

heart cyt c were purchased from Sigma-Aldrich, except for theRC protein. Wild-type RC from Rhodobacter sphaeroides(Supporting Information (SI) Figure S1 shows the absorbancespectrum of the RC) was prepared as described,4,34,35 usingLDAO for solubilization. Planar Au working electrodes werefabricated by evaporating an adhesive Cr layer (20 nm)followed by deposition of 400 nm thick Au layers onto thecleaned glass substrates. The Au electrodes were cleaned byrinsing sequentially with acetone, methanol, isopropanol,deionized water, and dried completely under a N2 streamprior to performing experiments. The Au|SAM|cyt c|RCelectrode was fabricated by treating a cleaned Au electrode ina 10 mM 6-mercaptohexanoic acid (lower concentrations oflinkers resulted in lower photocurrents; SI Figure S2) over 5days at room temperature, rinsing the electrode in 0.1 M Tris−HCl (pH 8) buffer, followed by immersing in a 0.8 mM cyt csolution for a day at 4 °C, rinsing with buffer, and immersing ina 1.0 μM solution of RCs at 4 °C for a day. Weakly bound RCswere removed from the electrode by rinsing the electrode withbuffer. The alkanethiol self-assembly was performed assuggested by Love et al.36

2.2. Electrochemical Setup. The fabricated electrode wasused in an electrochemical cell as the working electrode. A 13cm length of Pt wire of 0.25 mm diameter was shaped to a coiland used as the counter electrode. A 60 μM solution ofcoenzyme Q2 (2, 3-dimethoxy-5-methyl-6-geranyl-1, 4-benzo-quinone; hereafter referred to as Q) in 0.1 M Tris buffer wasused as the electrolyte. It was shown by Trammell et al. that theabove-mentioned concentration is not limiting the current in acell with the RC proteins.29 For the experiments in which areference electrode was needed, a Ag/AgCl electrode was used.All the experiments were carried out at room temperature using0.1 M Tris−HCl at pH 8 as the background electrolyte. The

Figure 1. (a) ET pathway between the 6-mercaptohexanoic acid-modified Au electrode, cyt c, and the RC in the Au|SAM|cyt c|RC structure. Thephoton-generated electrons acquired at QB are transferred to the counter electrode via diffusion of QH2 (mediator). (b) The RC−cyt c cocomplex.The RC subunits are represented as ribbons and colored light blue (L), dark blue (M), and brown (H). Green ribbons show cyt c. (c) RC subunitproteins and cofactors. The dashed arrow shows the ET path from P to QB.

The Journal of Physical Chemistry C Article

dx.doi.org/10.1021/jp507065u | J. Phys. Chem. C 2014, 118, 23509−2351823510

current polarity convention was set in a fashion that definedcathodic current as negative. Each cell was kept in the dark untilthe open-circuit potential (OCP) stabilized. For the photo-current measurements, the same potential was applied to thecell by the potentiostat such that the current in the dark waszero.4,11 The cells were illuminated with a commercial solarsimulator (RST300S (AM 1.0), Radiant Source Technology) atan incident light intensity of 80 mW cm−2 at the electrode’ssurface. The solar light source uses a XL3000 PerkinElmerFiber Optic Illumination (FOI) system that employs a 300-WCermax Xenon light. Photocurrents and photovoltages wererecorded using a VersaSTAT 4 (Princeton Applied Research)potentiostat in both three and two electrode setups. The threeelectrode measurements were performed to accurately studythe reactions only on the surface of the working electrode (thepotential changes of the working electrode are measuredindependent of changes that may occur at the counterelectrode). Hence, the surface area of Pt counter electrodewould not be a rate-limiting factor.2.3. External Quantum Efficiency (EQE) Measure-

ments. Light from a Tungsten Halogen lamp (Oriel 6334NS24 V250W) was focused onto the entrance slit of amonochromator (Cornerstone 260 1/4M) using a pair ofparabolic mirrors. The dispersed light passing through the exitslit (slit width: 5 nm) was subsequently focused onto the deviceusing a convex lens. The photocurrent was measured from 590to 950 nm in steps of 6 nm in the three-electrode cell. At eachwavelength step, the photocurrent was monitored for twocomplete cycles consisting of 20 s of illumination followed by20 s in the dark, where the light was blocked by a computercontrolled shutter at the exit slit of the monochromator. Theincident power was measured by a thermopile detector (Oriel71945) connected to a multimeter (Keithley2000). The EQEwas measured as the ratio of collected electrons to incidentphotons.37,38 The equation for calculation is thus:

ω= ×

·ℏ·

Je I

EQE 100(1)

where J is the current density in A cm−2, e is the electron chargein C, I is the incident photon power density in W cm−2, and ℏωis the energy per photon in Joules, all at the wavelength λ (seeSI for further details).2.4. Ellipsometry. The thickness measurements were

performed using a Rudolf Research Type ellipsometer AutoEL(wavelength of 6328 Å (He−Ne laser)) at an incident angle of70° for carboxylic acid-terminated SAMs; a Sopra spectroscopicellipsometer ES 4G (multilayer optical spectrometric scanner)at an incident angle of 70.1° was used for cyt c and RC layers.The refractive index and the coefficient of absorption values forthe Au substrates were measured to be 0.1508 and 3.3280,respectively. The ellipsometric data were analyzed assuming anindex of refraction of 1.4846 for the SAM monolayer, assuggested by the supplier (Sigma-Aldrich).2.5. Photoemission Spectroscopy. For photoemission

spectroscopy, all samples were prepared in a glovebox whichwas fitted to the fast load lock of a multifunctionalcharacterization system.39 This commercial multichambersystem (SPECS, Berlin, Germany) consists of two preparationchambers and one analysis chamber outfitted for X-rayphotoemission spectroscopy (XPS). The base vacuum level ofthis system is 2 × 10−10 mbar. An Mg Kα X-ray emission sourcewith incident energy of 1253.6 eV and 20 mA emission currentwas used for the core level XPS. Low intensity XPS (LIXPS)

measurements were performed prior to XPS in standby modewith 0.1 mA emission current. The corresponding significantlylower amount of photon flux was generated and used tomeasure the sample work function (WF) free of chargingartifacts.40 The ultraviolet photoemission spectroscopy (UPS)measurement was carried out with a SPECS UVS10/35 UVsource by discharging highly pure helium gas (99.99%). The HeI line was generated by controlling the discharging voltage in arange of 600 to 750 V. The data analysis was performed usingIgor Pro software. The energy gap of the linker molecule wasestimated from the optical absorption spectrum using aThermo Scientific (Evolution 201) UV−vis spectrophotometer.Further experimental details are given in the SI.

3. RESULTS3.1. Photochronoamperometry Analysis. The photo-

current density of the Au|SAM|cyt c|RC structure was measuredin both three and two electrode setups with only one diffusibleredox mediator (Q) in the electrolyte. As shown in Figure 2a, in

a three-electrode experiment, a cathodic photocurrent wasachieved upon illumination. Immobilizing RCs using hybridSAM|cyt c linker resulted in peak current density of up to 0.5μA cm−2, which is at least 3 times of that using randomdiffusion of cyt c proteins in a previous work.30 Thephotocurrent density stabilized at −185 nA cm−2 after 400 swhile the working electrode was held at the dark OCP of +0.05V vs normal hydrogen electrode (NHE). There was an initialspike of photocurrent at the onset of illumination similar towhat has been observed in a recent work.27 It was reasoned thatthis initial spike was originated from a kinetic limitation at theRC’s primary acceptor side (QB) due to the different rates of P

+

reduction and QB− oxidation, which results in buildup of

negative charges within the protein.27 Accordingly, theoveroxidation of the redox mediator equilibrates the chargeaccumulation inside RC.27

To confirm the contribution of the protein complexes to thephotocurrent generation, control experiments were performedon a cell containing a Au|SAM|cyt c working electrode and Q asthe charge carrier, but without any RC protein component. Thenegligible photocurrent density in the Au|SAM|cyt c structure(black line in Figure 2a) demonstrates that the photocurrent

Figure 2. (a) Time dependence photoresponse of a fabricatedbiophotoelectrochemical cell under 80 mW cm−2 illumination withcoenzyme Q as the single diffusible redox mediator. In the graph thearrows indicate light ON (↑) and OFF (↓), with the current obtainedfrom the configurations shown as nA cm−2 according to the key. (b)EQE of the photocurrent, per incident photon, generated on the Au|SAM|cyt c|RC electrode (red dots), compared to the absorptionspectrum of the RC (bold black line).



stems from the charge generation in the RC. The cathodicphotocurrent in the Au|SAM|cyt c|RC structure implies ETfrom the Au electrode to the RC, which likely suggests theprotein orientation with the primary donor (P-side) facing theelectrode. Hence, the majority of cyt c molecules likely boundto the P-side of the RC protein complex. Adding more cyt c tothe electrolyte did not enhance the photocurrent, whichsupports the interpretation that the majority of the RCs weredocked onto SAM-bound cyt c proteins. The photochronoam-perometry study of the two electrode setup resulted in a shortcircuit steady-state photocurrent density (JSC) of −156 nA cm−2

and a steady-state open circuit voltage (VOC) of ∼90 mV undercontinuous illumination (SI Figure S3). As shown in thefollowing text, the energy barrier at the SAM is one of thelimiting factors for an efficient charge transfer and energyconversion. However, the very low photocurrent (15 nA cm−2)from an electrode without any SAM (Au|cyt c|RC in Figure 2a)shows the importance of the linker molecule in a successful useof the incubated cyt c for the RC immobilization. The lowphotocurrent may be due to variable orientation of cyt c uponadsorption on Au and/or cyt c conformational changes, proteinunfolding, and even denaturation on this bare metal electrode,as it has been shown in several studies.41−43

To further verify that the observed photocurrent in the Au|SAM|cyt c|RC cell stems from the photon absorption andcharge generation by RCs, a photocurrent action spectrum wasobtained across 590 to 950 nm and the EQE was estimated, aswell (see Experimental). Figure 2b shows a substantial matchbetween the RC absorption spectrum and the efficiency ofphotocurrent generation across this wavelength range. Thedistinctive triplets of RC cofactor absorptions are clearlypresent in the EQE spectrum.The stability of the Au|SAM|cyt c|RC structure was studied

further by measuring the photocurrent density of a single cellover a course of 5 days. As shown in Figure 3, the magnitude of

the photocurrent density dropped from 185 nA cm−2 for a freshelectrode to 102 nA cm−2 after 4 days of storage in aerobiccondition. The result suggests that the rate of reduction in thephotocurrent density is faster in the first couple of days. Thiscould be due to the degradation of fraction RC complexes, aswas shown in an earlier study by Ciesielski et al. forphotosystem I (PSI)-based photoelectrochemical cells.11

Extended device lifetime by appropriate sealing and oxygenremoval can be achieved as it has been shown earlier.11

Additionally, these results show that, despite the lack of a

covalent bond between RC and cyt c, the protein−proteininteraction is strong enough to hold more than half of the RCsafter 4 days, in aerobic conditions, while the protein complexeskept their integrity and functionality.Since the thickness and the energy levels of the SAM and the

cyt c layer affect the electron transfer rate through the hybridlinker, further studies were carried out to characterize eachlayer. The thickness of the layers was measured using theellipsometry technique. The energy levels of the SAM werestudied using the photoemission spectroscopy and the UV−visabsorption methods. Additionally, cyclic voltammetry (CV)was used to investigate the energy level of cyt c as well as toestimate the success rate of the cyt c and the RCimmobilization. The results from these measurements wereused to confirm the successful construction of the Au|SAM|cytc|RC structure using the layer-by-layer assembly.

3.2. Ellipsometry Analysis. The Au|SAM|cyt c|RCstructure was further studied by measuring the thickness ofthe SAM, SAM|cyt c, and SAM|cyt c|RC layers usingellipsometry. This was performed to confirm that the thicknessof each layer is in agreement with the size of employedmolecules. As shown in Figure 4, the SAM prepared from an

ethanolic solution of 10 mM 6-mercaptohexanoic acid showeda ∼14 Å thick layer. Assuming a 30° tilt for the alkanethiolchain,32 the measured thickness is slightly larger than thetheoretical thickness expected for a close-packed monolayeroriented to the surface. This has been explained by coverage ofa high free energy surface (i.e., Au) with reversibly physisorbedlayers of water, hydrocarbons, and other organic compoundsunder laboratory ambient conditions.44 As shown in Figure 4,the thickness of the SAM increased by ∼33 Å after cyt cimmobilization on top, which is in a good agreement with thesize of cyt c reported by other groups.45−47 Upon deposition ofthe RCs the thickness increased from ∼47 Å to ∼120 Å,indicating that a monolayer of RCs (thickness ∼70 Å) hadattached on top of the Au|SAM|cyt c.

3.3. Photoemission Spectroscopy Analysis. In order toevaluate how the energy levels of the SAM affect the ETbetween the Au electrode and the RC, the highest-occupied-molecular-orbital (HOMO) and the lowest-unoccupied-molec-ular-orbital (LUMO) of the SAM were measured using LIXPS,UPS, and UV−vis absorption spectroscopy methods. Addition-ally, XPS data provided further evidence for the successfulattachment of the desired molecules (see Figures S4−S5 for

Figure 3. Change in the steady-state photocurrent density of a cell,measured over a course of 5 days (one test each day).

Figure 4. Ellipsometric estimation of the thickness of sample 1 (SAMof 6-mercaptohexanoic acid), sample 2 (SAM|cyt c), and sample 3(SAM|cyt c|RC).



further details). Figure 5 shows the UP spectra measured beforeand after deposition of the SAM during this experiment. Thecenter panel (Figure 5b) shows the complete spectra, and theside panels show the secondary edge normalized (Figure 5a) aswell as the valence bands region after background subtraction(Figure 5c).The secondary edge spectral cutoffs acquired via LIXPS

(Figure 5a) allowed for the determination of the WF of the Auand the Au|SAM substrates. The WF was calculated bysubtracting the cutoff binding energy value from the excitationenergy (21.2182 eV) and taking the analyzer broadening of 0.1eV into account. Figure 5b shows the complete set ofnormalized UPS. The main emission features include theFermi level, the valence bands/HOMO (VB/HOMO) densityof states, and the secondary edge. The magnified VB/HOMOspectra with background removed are shown in Figure 5c.Before deposition of a SAM, the valence bands and the Fermilevel of the Au substrate can be clearly observed. After thedeposition of a SAM, these features are suppressed andreplaced by features corresponding to the emissions from theSAM. The valence bands maximum (VBM) of the Au electrodecoated with a SAM relative to Au alone are shown in themagnified VB/HOMO spectra.In order to estimate the optical band/HOMO−LUMO gap

in the linker molecule, the absorption spectrum of the linkersolution (10 mM in ethanol) was measured. As shown in Figure6, the absorption threshold starts around 300 nm whichcorresponds to an energy gap of 4.13 eV. From the LIXPS andUPS results the HOMO level is measured to be 7.2 eV belowthe vacuum level. Hence, the absorption results indicate aLUMO of 3.07 eV below the vacuum level. As explained in theDiscussion section, the energy levels in the SAM can be used todraw an energy diagram across the Au|SAM|cyt c|RC to assessthe limitations in the ET.3.4. Electrochemistry Analysis. To estimate the electro-

chemical midpoint potentials (i.e., energy levels) and thesurface coverage of the proteins, electrochemical CV wasperformed for each layer. Figure 7a shows the CVs of Au, Au|SAM, Au|SAM|cyt c, and Au|SAM|cyt c|RC electrodes at a scanrate of 0.05 V·s−1 at room temperature. As curve ii shows, the

Au electrode primed with a layer of 6-mercaptohexanoic acidSAM exhibited no electroactivity in the potential range from 0.1to 0.6 V in the 0.1 M Tris−HCl (pH 8.0) backgroundelectrolyte. Previous studies have mainly reported the CVs ofcarboxylic acid-terminated SAM electrodes for positivevoltages.32 When the scan range was extended below 0.0 V vsNHE (i.e., the case here) the CV of the SAM showed a pair ofpeaks corresponding to protonation and deprotonation of thesurface COOH groups.48

After immobilization of cyt c, direct electrochemistry ofsurface-bound cyt c was achieved (Figure 7, curve (iii), and apair of quasi-reversible redox peaks due to the one-electronoxidation and reduction of a heme FeIII/FeII couple wasapparent at ∼0.23 V vs NHE (Figure 7b). The surface formalpotential of cyt c is nearly identical to the values previouslyreported for cyt c bound to physiological membranes.49 Fromthe average of the reduction and oxidation peak potentials, theredox potential of cyt c was measured to be ∼0.23 V vs NHE(4.73 eV below vacuum level).The CV result from RCs in the Au|SAM|cyt c|RC structure in

the presence of quinone (Q) in the electrolyte is presented inFigure 7(c). Because the immobilized proteins are present as a

Figure 5. LIXPS and UP spectra before (red) and after (black) the deposition of SAM on a clean Au substrate. (a) The normalized secondary edgesmeasured with LIXPS before UPS. (b) The complete normalized UP spectra. (c) The evolution of the VB emission features through the depositionprocess (black, SAM; red, Au).

Figure 6. UV−vis absorption spectrum of the linker solution (10 mM6-mercaptohexanoic acid).



monolayer, the concentration of RC is very low, and the redoxpeaks in the CV graph are relatively small. Nevertheless, the RCP peaks at ∼0.4 and 0.6 V vs NHE were observed, whichconfirms the successful RC immobilization (see SI Figure S6for the pronounced oxidation and reduction peaks of the RCprimary donor in the presence of excess RCs). The midpointpotential of RC was estimated to be ca. +0.45 (vs NHE), whichis similar to that reported in previous works.3,4,20 This confirmsthat in this structure the RCs are still redox-active, upondocking to cyt c. As explained in the Discussion section, thearea under the anodic peak (Figure 7c, inset) has been used toestimate the total amount of exchanged charges in the redoxreaction of RCs, from which the RC density and surfacecoverage were estimated.

4. DISCUSSION

The cathodic photocurrent in Figure 2 indicates the directionof ET from the Au electrode to the RC protein via the SAM|cytc structure, and accordingly confirms the anticipated proteinorientation with the P-side facing the electrode. Although thephotocurrent result shows the feasibility of immobilizing RCsthrough cyt c proteins, the overall photocurrent depends on thenumber of the immobilized RCs and the ET rate through theSAM|cyt c linker. Assuming a high degree of surface coverageby the SAM,31 the density of immobilized RCs is limited by thefrequency of binding to cyt c. Using the results from theelectrochemical experiments, we describe in the following textan estimation of the surface coverage of the cyt c and the RClayers. Also, the ET rate is estimated from the transientphotocurrent response.For surface coverage estimation of active cyt c heme proteins,

the total charge was calculated using the CV peak integration(Figure 7b) after background subtraction.32 For the Au|SAM|cyt c structures, the electroactive surface density of cyt c wasdetermined by automatic CV peak integration, usingVersaSTAT 4 software. Integrating the area under the peakof photocurrent density vs potential gives the charge density(Qtotal). Using Faraday’s Law, the electroactive surfaceconcentration of cyt c can be then estimated using eq 2:20,32

Γ =Q

nFtotal

(2)

where Γ is the electroactive surface density of cyt c, n is thenumber of unit charges in the redox reaction of the protein (n =1 in this case), and F is the faradaic constant (96485 C·mol−1).Considering the surface area of the electrode, the surfacedensity of ∼14 × 10−12 mole·cm−2 was estimated forimmobilized cyt c, which is consistent with a previous report.32

Bearing in mind the approximate diameter of a cyt c molecule,3.3 nm,45 the calculated value of the cyt c surface concentrationcorresponds to a ∼70% surface coverage of the electrode.The CV peak integration technique was also applied to

estimate the surface coverage of RC using the CV plot in Figure7c. The measured charge density of ∼581 nC (Figure 7c, inset)corresponds to a surface density of RC of 6.02 × 10−12 mol·cm−2. Considering the estimated density of cyt c on the surface(14 × 10−12 mol·cm−2) the number of attached RCs is almosthalf of the cyt c proteins on the electrode. Despite the lowernumber of RCs, based on the protein diameter (∼5 nm) theRC surface coverage is estimated to be ∼70%, which is in thesame range as cyt c. Previous research has shown thatelectrostatic interactions between acidic amino acids on theRC periplasmic surface (P-side) and the basic amino acidresidues, primarily lysines, surrounding the cyt c hemecontribute to interprotein docking and stability of the RC−cyt c cocomplex;33,50 Our unpublished results shows that R.sphaeroides cyt c2 and horse heart cyt c are ∼30% structurallysimilar. Additionally, cyt c was found to bind to the proximalposition faster, as well as with a higher affinity of the oxidizedform to the RC.51 In the current study, the photocurrentdensity of the Au|SAM|cyt c|RC structure confirms the bindingof cyt c and RC complexes. Additionally, the photocurrentdirection proves that cyt c binds to the P-side of the RC proteincomplex. This binding configuration here can be compared tothe mechanism known for the in vivo RC and cyt c proteinsbond, as proposed by others.52−54

The ET rate between RC and the Au electrode in the Au|SAM|cyt c|RC structure was estimated through the photo-current transition at the onset of illumination as suggested byTrammell et al.29 Figure 8 shows the photocurrent transition(1.4 s) for the RC-modified Au electrode with RC proteinssitting on cyt c-terminated SAMs. Assuming the transientphotocurrent follows an exponential profile, the current density,J, was fitted to J = Jpeak(1 − e−kt), where Jpeak = −450 nA cm−2 is

Figure 7. (a) CVs of a Au electrode (i, black trace), a Au|SAM electrode (ii, red trace), Au|SAM|cyt c (iii, blue trace), and Au|SAM|cyt c|RC (iv,orange trace). The scan rate was 0.05 V·s−1, and the background electrolyte was 0.1 M Tris buffer. (b) Close-up view of the green rectangle in panela, which shows a pair of quasi-reversible redox peaks attributed to the heme FeIII/FeII couple in cyt c, at ∼0.23 V vs NHE (blue trace), wherein thecomparative CV for iv is shown in orange. (c) CV of the Au|SAM|cyt c|RC electrode (scan rate of 0.05 V·s−1) in the presence of Q, which showsoxidation−reduction peaks of the RC primary donor (P). The inset shows the anodic peak scaled and colored to emphasize the area of interest. Thesurface under the peak (purple) was used to estimate the density of immobilized RCs.



the peak current density, k is the ET rate, and t is time (t = 0 isthe onset of the illumination). Based on the fitting curve inFigure 8, the ET rate between the RC and the Au electrode wasestimated to be k = 7.1 s−1. The relatively low ET rate in theAu|SAM|cyt c|RC structure can be explained by an energydiagram of the different layers, shown in Figure 9. The

electrochemical midpoint potentials of the cyt c heme and P+ inRC were measured at 0.23 and 0.45 V vs NHE, respectively(Figure 7). The energy levels inside the RC have been studiedin detail by others and explained by Blankenship.55 Themidpoint potential of Q at pH 8 was measured in our earlierwork as ∼0.042 V vs NHE.4 The vacuum potential of Pt andthe electrochemical potential of the QB site within the RC werealso presented in a previous report.3 The HOMO and LUMOlevels for the SAM were obtained from UPS, LIXPS, and UV−vis absorption results (Figures 5 and 6).The observed cathodic photocurrent implies ET from the Au

to P+ in the RC, while the energy diagram in Figure 9 showsthat the ET is hindered by the energy barrier of the SAM andthe unfavorable energy difference between Au and cyt c.Although the Au Fermi level is below the cyt c energy level, thedensity of electrons above the Fermi level is not zero at roomtemperature. Additionally, WF measurement of the sputtered

Au was performed in vacuum. The WF of a surface can bestrongly affected by the condition of the surface. In the event ofsurface reactions (such as oxidation or reduction), the WF canchange considerably. Hence, it is reasonable to assume thatthere are electrons with enough energy to tunnel through theSAM to the cyt c.Considering the energy barrier (ΔE1) of 1.93 eV (the

difference between EF (Au) and ELUMO (SAM)) and thetunneling length (a) of 3.0 nm (Figure 4, sample 2 - thedistance from the electrode to the middle of cyt c where hemeis located), the wave function for an electron tunneling throughthe SAM experiences attenuation. Using a simple square barriermodel for the SAM, the one-dimensional conductance, G,through the barrier can be found from eq 3:56

= ΔGeh

T E2

( )2

2 (3)

where e = 1.6 × 10−19 C is the charge of one electron, h = 6.626× 10−34 J·s is Planck’s constant, and T(ΔE2) is the tunnelingtransmission coefficient between two energy states across thebarrier with an energy difference of ΔE2. For a large barrier, thetransmission coefficient is estimated by eq 4:56

πΔ ≈

ΔΔ

−Δ⎛

⎝⎜⎜

⎞⎠⎟⎟T E

EE

m E

ha( )

16exp

4 22

2

1

e 1

(4)

where me = 9.11 × 10−31 kg is the electron mass. From eq 2 and3, the one-dimensional conductance of the barrier is estimatedto be G = 4.65 × 10−23 Ω−1, which is very low. It should benoted that G is not the conductance of the SAM, but is theconductance along a single linker molecule (in one dimension).As shown in Figure 2, the SAM has a crucial role in the proteinimmobilization since without the linker no photocurrent wasobserved (mainly due to the cyt c adsorption orientation). Inorder to eliminate the charge transfer barrier, a conjugatedlinker molecule with a HOMO level slightly higher than theenergy level in cyt c could be used. In this case, the ET wouldoccur by the charge hopping through the hybrid conjugatedmolecule-cyt c linker, instead of tunneling. Additionally, a lowWF material for the working electrode would greatly increasethe ET rate. A detailed study of such approaches is underway inour laboratory, and the results will be reported in forthcomingpapers.The proposed docking mechanism, described above, may

also be exploited for other types of carotenoid-based proteinssuch as RC-light harvesting (LH) complexes and possibly PSIfor intriguing applications in photovoltaic, photonic, optoelec-tronic devices, and biosensors.9,27,57−60

Carotenoid-based proteins have shown promise for photo-voltaic applications. Several studies have focused on theassembly of PSI onto various substrates such as P-dope siliconand graphene oxide, which resulted in enhanced currentdensities over 100 μA cm−2.10,61 Other studies on integration oforiented RC-LH1 complexes, mostly on transparent conductiveoxide electrodes, showed several μA current densities,19,27 withan exception of a recent work on a densely packed monolayerof RC-LH1 complexes on a Au electrode using Langmuir−Blodgett technology, which resulted in a 45 μA cm−2 currentdensity.62 Beside carotenoid-based photosystems, bacteriorho-dopsin as a robust light-driven proton pump has found variousapplications in solar energy conversion,17,18,63 optoelec-tronics,64 and organic field effect transistors.65 A recent studyon fabricating a photovoltaic cell using aqueous bacteriorho-

Figure 8. Photocurrent transition (inset, close-up view of the dashedrectangle) at the onset of illumination for Au|SAM|cyt c|RC electrode(onset of illumination at 0.0 s, and cessation of illumination asindicated by the upward and downward pointing arrows).

Figure 9. Energy diagram of the bioelectrochemical cell with the Au|SAM|cyt c|RC structure and the proposed mechanism for operation ofthe cells with Q as the single diffusible redox mediator. RC complexesare modeled as oriented with the P-side toward the Au electrode.Arrows indicate the route of ET from the Au to cyt c, into the P-side ofthe RC, and through Q to the Pt electrode. The energy level at eachlayer is relative to the vacuum level. The corresponding electro-chemical potentials can be found from the normal hydrogen electrode(NHE) axis at the right.



dopsin generated a photoelectric response of ∼33 μA cm−2.66

Overall, the tendency toward biomimetic devices and the needfor the production of clean energy by mimicking nature bringsthe light-capturing proteins applications in bioelectronic devicesto the forefront of cutting-edge research. The overall effort willadvance the application of biological materials in electronicdevices with a far reaching impact in the fields of solar cells,biosensors, and bionanotechnology.

5. CONCLUSIONSWe have demonstrated the feasibility of utilizing a protein−protein interaction to immobilize the R. sphaeroides wild typeRC protein from the P-side through a hybrid carboxylic acid-terminated alkanethiol SAM|cyt c linker. The new linkerstructure has demonstrated the ability to control the orientationof the RC with reasonable stability in bonding to the RCprotein complex. A simple layer-by-layer deposition/incubationmethod was applied to build a Au|SAM|cyt c|RC structurewhich resulted in 70% of the surface coverage by RC proteins.The ET rate between the Au electrode and RCs was found tobe k ≈ 7.1 s−1. Our detailed energy study of the layers showed alarge energy barrier at the SAM which limits the ET rate. Theseresults provide key information about how specific surfacemodification of an electrode may control the performance ofRC complexes in systems suitable for the development of solarenergy converters, and other types of photon energy-harvestingbiomaterials. This work provides an experimental andtheoretical baseline for future work.

■ ASSOCIATED CONTENT*S Supporting InformationAdditional information including further experimental details,absorbance spectra of RCs of R. sphaeroides at roomtemperature, the effect of linker solution concentration onthe photocurrent density for the Au|SAM|cyt c|RC cell, the two-electrode photocurrent and photovoltage measurements of theAu|SAM|cyt c|RC structure, XPS spectra of O1s, N1s, C1s, andS2p core level emissions for Au, Au|SAM, Au|SAM|cyt c, andAu|SAM|cyt c|RC samples, Gaussian−Lorentzian fit to the RCN 1s emission line measured on the Au|SAM|cyt c|RCelectrode, CV of the Au|SAM|cyt c|RC electrode in thepresence of Q with excessive RCs, which shows pronouncedoxidation and reduction peaks for the RC primary donor (P),and effects of cycling the applied potential on the mean steadystate current densities, obtained from the Au|SAM|cyt c|RCelectrode. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: [email protected]; Tel.: +1-813-409-8192.*E-mail: [email protected] Tel.: +1-813-421-3037.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors wish to acknowledge the University of SouthFlorida for the financial support of this work. This work wassupported by the National Science Foundation through NSF1400017. The authors also wish to acknowledge Ms. CaitrinEaton for valuable assistance with data processing.

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■ NOTE ADDED AFTER ASAP PUBLICATIONDue to a production error, this paper was published on theWeb on October 2, 2014, with none of the author correctionsimplemented. The corrected version was reposted on October3, 2014.



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