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In operando investigation of electrically coupling of photosystem 1 and photosystem 2 by means of bipolar electrochemistry Vera Essmann, Fangyuan Zhao, Volker Hartmann, Marc M Nowaczyk, Wolfgang Schuhmann, and Felipe Conzuelo Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 09 Jun 2017 Downloaded from http://pubs.acs.org on June 12, 2017
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Analytical Chemistry
In operando investigation of electrically coupling of photosystem 1 and photosystem 2 by means of bipolar electrochemistry Vera Eßmann,a Fangyuan Zhao,a Volker Hartmann,b Marc M. Nowaczyk,b Wolfgang Schuhmann,a,* and Felipe Conzueloa,* a
Analytical Chemistry ‐ Center for Electrochemical Sciences (CES), Ruhr‐Universität Bochum, Universitätsstr. 150, D‐44780 Bochum, Germany
b
Plant Biochemistry, Ruhr‐Universität Bochum, Universitätsstr. 150, D‐44780 Bochum, Germany
* Corresponding authors:
[email protected],
[email protected], Fax: +49 234 3214683
ABSTRACT: Electrochemical communication between two photobioelectrochemical half‐cells based on photosystem 1 and photosystem 2 is investigated in operando. The driving force for the electron transfer reactions is applied in a wireless mode using bipolar electrochemistry with the actual electrode potentials being self‐regulated by the redox processes. Four pa‐ rameters are assessed to understand the overall performance and elucidate the limiting reactions of the photobioelectro‐ chemical cell. In addition to the potential differences for oxidation and reduction reactions, the current flowing between the half‐cells as well as in situ collection of locally evolved O2 by photosystem 2 using a positioned SECM tip are evaluated. In this way, changes in the enzymatic performances as a result of inactivation of either of the protein complexes or variations in the external conditions are monitored.
KEYWORDS: photosystem 1, photosystem 2, bipolar electrochemistry, redox polymers, biophotovoltaics Oxygenic photosynthesis is the most relevant energy transformation process on earth. It allows the conversion of sunlight into chemical energy.1‐3 The process is per‐ formed by a complex machinery developed by nature and driven by two key protein complexes, namely photosystem 1 (PS1) and photosystem 2 (PS2). Both of them undergo charge separation upon absorption of visible light, operat‐ ing with a high quantum yield. PS2 catalyses the light dri‐ ven oxidation of water, whereas PS1 operates as an ‘electron pump that fuels the subsequent assimilation of CO2 into organic matter.4 The extraordinary efficiency and natural abundance of the photosynthetic protein complexes has raised consider‐ able interest in the realisation of sustainable solar energy devices. By integration of the photosynthetic protein com‐ plexes to electrodes, the development of semi‐artificial bi‐ ohybrid photovoltaic assemblies aims at the generation of photocurrent and light‐driven hydrogen production.3,5 Strategies for the connection of individual photosystems to various electrode surfaces have been widely reported.6‐8 As shown previously,5 higher photocurrent densities can be attained by embedding photosynthetic protein complexes into redox polymers, which simultaneously fulfil the role of a non‐leaking redox mediator and a three‐dimensional immobilisation matrix, thereby increasing the electron transfer rate and the amount of protein complexes that can be productively immobilised on the electrode surface.9,10
Of particular interest is the design of photocatalytic bioe‐ lectrochemical cells with coupled PS1 and PS2, capable of mimicking the overall photosynthetic process. Here, water as the only electron source is oxidised at the water‐oxidis‐ ing complex (WOC) in PS2, allowing electron flow all the way to the reducing end of PS1. Different architectures have been suggested for the development of efficient bio‐ photoelectrodes taking advantage of the coupling of PS1 and PS2.8 Moreover, functional photosynthetic biophoto‐ voltaic cells were described by coupling of PS1‐ and PS2‐ modified electrodes for successful autonomous solar‐to‐ chemical energy conversion.11,12 The only report to date on the study of electron transfer between isolated PS1 and PS2 has been performed by coupling of the protein complexes encapsulated in sol‐gel glasses using a 2,6‐dichloropheno‐ lindophenol (DCPIP) pool as electron carrier and monitor‐ ing the specific absorption of the chlorophyll radical cat‐ ion, P700+•.13 To design more useful and refined biophoto‐ voltaic devices, it is of importance to understand and eval‐ uate the efficiency of electron transfer between coupled photosynthetic protein complexes. In a conventional analytical systems the interfacial potential at the working electrode is controlled. In contrast, by using bipolar electrochemistry (BPE) it becomes possible to investi‐ gate the activity of both protein complexes simultaneously in a more autonomous way, because the measured potentials and currents are defined by the activities of the protein com‐ plexes instead of being controlled by an externally applied po‐
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tential. With bipolar electrochemistry, oxidation and reduc‐ tion reactions can be carried out at the opposite ends of an electrically conductive object that is not wired to a potenti‐ ostat. Many publications report on the use of BPE for various purposes such as for wireless sensors,14 locomotion of small objects,15 or material gradients.16,17 In a closed bipolar system, as used in this study, the two opposite ends of a conducting object of any shape (e.g. an ITO‐stripe,18 two RDEs,19 etc.) are placed in two different compartments.17,20 In order to apply an electric field, a pair of so‐called feeder electrodes is used, lo‐ cating one of them in each of these two compartments. In this way, the conductive object, being the only electrical connec‐ tion between the compartments, is polarised and thus be‐ comes a bipolar electrode (BE). The BE potential is floating to a value in between the solution potentials in the two compart‐ ments. As a result, potential differences of opposite direction are induced at the BE extremities. With a sufficiently high voltage applied to the feeder electrodes, the potential drop across the BE (∆EBE) drives oxidation and reduction reactions at the BE. The required ∆EBE is approximately equal to the dif‐ ference of the formal potentials measured for each reaction in a conventional three‐electrode setup. Also here, the redox re‐ action which is proceeding at a lower rate is limiting the cou‐ pled reaction at the opposite BE extremity.21 A closed bipolar system has the advantage that the bipolar and the feeder elec‐ trode currents are equal due to elimination of ionic current between the poles. This effect has mostly been exploited in the field of analytical BPE for quantification of analytes such as H2O2,18 for characterisation of catalysts19 or for the determina‐ tion of solution conductivity.22 The behaviour of closed bipo‐ lar systems, e.g. as a function of the sizes of the BE poles, has been investigated previously.21,23
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Materials. Au surfaces were prepared by vapour deposi‐ tion in a metal vaporisation setup (Leybold Univex 300) de‐ positing 50 Å of titanium as an adhesion layer and a 1000 Å thick Au layer on Si(100) wafers (Wacker). The wafers were cut precisely to (8 × 5) mm² rectangles by laser cutting at the chair of Electronic Materials and Nanoelectronics (Ruhr‐Universität Bochum). Organic contaminants were removed by immersion in a freshly prepared piranha solu‐ tion (H2SO4 from Sigma‐Aldrich; H2O2 from VWR, 3:1 (v/v)) for 10 min (Caution! Piranha solution is highly corro‐ sive and a powerful oxidising agent. This solution must be handled with extreme care). Afterwards, the surface was rinsed thoroughly with ultrapure water. A thin strip of cop‐ per tape was attached to the edge of the Au surface and insulated, leaving (5 × 5) mm² of Au surface electrochemi‐ cally accessible. For the cathodic extremity of the bipolar electrode, the same procedure was repeated using two rec‐ tangles to increase the electrochemically accessible surface area to (10 × 5) mm². The synthesis and purification of the redox polymer poly(1‐vinylimidazole‐co‐allylamine)‐Os(bpy)2Cl has been shown previously.9,24 Isolated PS1 trimers from the thermo‐ philic cyanobacterium T. elongatus were prepared follow‐ ing a previous report.10 PS2 was isolated from T. elongatus according to a previously reported procedure.25 For control measurements the protein complexes were inactivated by thermal treatment using a silicone oil bath at 120 °C for 1 h. A 50 mM sodium citrate buffer (pH 4) containing 100 mM KCl, 10 mM CaCl2, 10 mM MgCl2, and 2 mM 1,1’‐dimethyl‐ 4,4’‐bipyridinium (methyl viologen, MV2+, Sigma‐Aldrich) was used for PS1 and inactivated PS1 (iPS1) samples. For PS2 and inactivated PS2 (iPS2) samples, 50 mM 2‐(N‐morpho‐ lino)ethanesulfonic acid (MES, Biomol) buffer (pH 6.5) containing 50 mM KCl, 15 mM MgCl2, and 15 mM CaCl2 was used. All solutions were prepared with ultrapure water (ρ = 18 MΩ cm, SG Water). Sample preparation. For sample preparation, 2.5 µL of a mixture of the redox polymer (5 µg µL‐1), poly(ethylene glycol) diglycidyl ether (PEGDGE, 0.02 µg µL‐1, Polyscien‐ ces), and isolated photosystem (PS1 or PS2, 1 µg µL‐1) were dropped onto the surface of the Au wafer. In case of PS1 or iPS1, the sample was incubated overnight in the dark at 4 °C. Prior to measurements the Au surfaces modified with PS1 or iPS1 were incubated with 50 mM Tris‐HCl buffer so‐ lution (pH 9, containing 100 mM KCl, 10 mM MgCl2, and 10 mM CaCl2) for 30 min to induce polymer collapse and crosslinking.26 PS2 or iPS2 samples were incubated for 1 h in the dark at 4 °C.
Here, we demonstrate coupling of two light‐induced re‐ dox reactions at a BE in a closed bipolar system to assess the electrochemical behaviour of both half‐cells individu‐ ally while they are in electrical contact with each other. The BE was modified with isolated PS1 and PS2, both em‐ bedded in the redox polymer poly(1‐vinylimidazole‐co‐al‐ lylamine)‐Os(bpy)2Cl, at the cathodic and anodic pole, re‐ spectively. This electrode architecture has previously pro‐ ven to exhibit high photocurrents with both protein com‐ plexes, hence ensuring controlled conditions for the eval‐ uation of electrode potentials and electron transfer.9,10,24 Constant operation of the system was ensured by applying an electric field via the feeder electrodes. The response upon illumination was studied. By recording the resulting changes in the overall system current through the feeder electrodes, the O2 reduction current at a SECM tip posi‐ tioned above the PS2‐modified end of the BE, and the two interfacial potential differences at the BE poles, infor‐ mation on the activity of both protein complexes can be obtained simultaneously and in operando. Since the BE ex‐ tremities are located in different compartments, working conditions for the two biomolecules can be individually ad‐ justed. Moreover, this study provides interesting insights into the working principle of closed bipolar systems.
Experimental setup. Two petri dishes (ø = 87 mm) were used as the two compartments of the closed bipolar setup (see Scheme 1). In each of them a stainless steel plate was placed at the outer rim serving as feeder electrode. For application of the driving voltage, the two plates were connected to a power supply (33120 A, arbitrary waveform generator, HP). The cur‐ rent between the feeder electrodes was measured with a mul‐ timeter (CEM).
EXPERIMENTAL SECTION
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RESULTS AND DISCUSSION Bipolar electrochemistry for in operando measurements A closed bipolar system was used (Scheme 1). The two ex‐ tremities of the BE are located in two different compartments, which are only electrically connected by the BE. The anodic (BEa) and cathodic (BEc) poles of the BE are two Au wafers modified with PS2 and PS1, respectively, embedded in the re‐ dox polymer poly(1‐vinylimidazole‐co‐allylamine)‐Os(bpy)2Cl (Os‐P). Application of a driving voltage between the feeder electrodes supports electron transfer between the two BE ex‐ tremities modified with the protein complexes. Since the re‐ dox potential of the redox polymer is 0.19 V vs. Ag/AgCl (3 M KCl), the interfacial potential difference at the two poles, ∆EBEa‐Sol and ∆EBEc‐Sol, needs to be adjusted to drive the oxida‐ tion and reduction reactions at the BE (see Scheme S‐1). In ad‐ dition to regulating the applied driving voltage, the potential differences can be fine‐tuned via the size of the poles, in this case the dimensions of the Au wafers.27 A suitable range for the interfacial potential differences avoiding side reactions like extensive O2 reduction is achieved by an applied voltage of 0.6 V and using a cathodic pole with twice the size of the anodic pole.
Scheme 1. Schematic of the closed bipolar electrochemistry setup. A stainless steel sheet was placed in each cell compart‐ ment as feeder electrode for application of the driving voltage. The cathodic BE pole is modified with PS1 embedded within an Os‐complex modified redox polymer (visualised by the brown spot) and has twice the surface area than the anodic pole, which is modified with PS2 embedded in the same Os‐complex modified polymer (brown spot). Both poles are illuminated simultaneously. Interfacial potential differences are measured with two reference electrodes positioned close to the BE poles. O2 is collected in‐situ at a Pt‐microelectrode positioned 5 µm above the PS2‐modified surface. Additionally, the system current is monitored.
The system allows us to investigate the electrical com‐ munication between PS1 and PS2 embedded in the redox polymer in operando by monitoring four parameters sim‐ ultaneously, i.e. the system current between the feeder electrodes isys, the interfacial potential differences at the two poles ∆EBEc‐Sol and ∆EBEa‐Sol, and the O2 reduction cur‐ rent at a microelectrode positioned close above the PS2‐ modified area of the BE for the collection of O2 evolved upon water splitting. isys directly reflects the electron trans‐ fer between PS2/Os‐P and PS1/Os‐P induced upon irradia‐ tion, corresponding to electron flow from water as electron source at the PS2 side, to the reducing end of PS1 where MV2+ acts as a fast redox mediator for the final reduction of O2 in solution. Since the potential of the bipolar elec‐ trode is floating between that of the solution potentials in the two compartments, the maximum possible current flow is adjusted and the final electrode potentials are de‐ fined by the performance of the photosystem protein com‐ plexes upon irradiation. Consequently, a change in work‐ ing conditions influencing the activity of PS1 and PS2, such as light intensity, solution composition or availability of electron donors and acceptors, is mirrored in the recorded values. As proved experimentally (data not shown), an ad‐ ditional system current originates from the common ground of the potentiostat and the power supply, however with no influence on the recorded parameters more than an offset in isys.
While the PS1‐modified twin Au wafer was attached to the bottom of one petri dish at the rim opposite of the feeder an‐ ode, the PS2‐modified wafer was positioned opposite of the feeder cathode in the other petri dish. The PS1 compartment was filled with citrate buffer pH 4.0 containing 2 mM MV2+ and the PS2 compartment was filled with MES buffer of pH 6.5. The copper tape strips of both poles were connected. A Pt‐microelectrode (ø = 25 µm) was positioned directly above the PS2‐spot at a distance of about 5 µm and connected as working electrode to a potentiostat (IPS). A Ag/AgCl (3 M KCl) electrode was used as reference electrode and positioned close to the wafer surface. A Pt‐wire served as counter elec‐ trode, which was positioned more distantly. An additional pair of Ag/AgCl (3 M KCl) electrodes were placed close to each pole of the BE and connected via a multimeter (CEM) to the BE. Irradiation of the samples was performed with an LC8 type 03 lamp (Hamamatsu Photonics) using visible light at an inci‐ dent power of 55 mW cm‐2.
BPE measurements. An experimental sequence was defined for the bipolar experiments scheduling the times at which the voltage and light was switched on and off (Ta‐ ble S‐1). Briefly, the system was first kept at OCP for about 3 min. Prior to a sequence of light and dark periods, a volt‐ age of 0.6 V was applied to the feeder electrodes. Subse‐ quently, the response in the absence of the electric field was investigated followed by recording the response over a long‐term irradiation period of approximately 10 min. Dur‐ ing the entire procedure, the interfacial potential differ‐ ence at both BE poles, the feeder current, and the SECM tip current for the collection of evolved O2 (Etip = −0.6 V) were monitored.
Electrical communication between PS1 and PS2 A series of different light and potential conditions was defined to investigate the electrochemical communication between PS1 and PS2 (Table S‐1). The first half of the exper‐ imental sequence provides extensive information on the extent of light‐induced electron transfer and its reasons. In addition, the working stability of both protein complexes is studied in the second half. For sake of simplicity, the data for the second part is shown in the Supporting Information
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(Figure S‐1 and S‐2). The responses obtained for fully active PS1 and PS2 at BEc and BEa, respectively, are presented in Figure 1. Initially, the system was kept at OCP. After the power supply is switched on, the applied electric field causes an increased system current (Figure 1a). According to ∆EBEa‐Sol and ∆EBEc‐Sol (Figure 1c, d), the initial peak in isys can be ascribed to the oxidation and reduction of the Os‐ complex within the redox polymer. Illumination of the samples after approximately 7 min enables the photosyn‐ thetic processes according to Scheme S‐1. This is reflected by rapid changes in all four monitored parameters. The system current increases by about 1.5 µA and slowly de‐ creases as inactivation of the protein complexes takes place over time. As a result of the light‐activated water splitting process at PS2 also the SECM tip current increases signifi‐ cantly (Figure 1b). The additional current through the sys‐ tem causes a decrease in the overall potential drop across the BE, ∆EBE (∆EBEa‐Sol − ∆EBEc‐Sol). Importantly, the cathodic interfacial potential difference ∆EBEc‐Sol decreases while the anodic potential difference ∆EBEa‐Sol slightly increases. We suggest that this indicates which redox process requires a higher driving force. Since the overall oxidation and reduc‐ tion reactions at the BE proceed at the same rate, the slower reaction does not only limit the opposite bipolar re‐ action but also the overall system current. Consequently, the whole equilibrium of potentials including ∆EBEc‐Sol and ∆EBEa‐Sol is continuously adjusted to achieve the maximal possible current. From the resulting potential changes upon the decrease in charge transfer resistance through il‐ lumination, it is possible to determine the limiting reaction and presumably also to estimate the degree of limitation. If, for example, the reduction reaction requires a lower driving force to reach the maximal oxidation current, ∆EBEc‐Sol would be decreased while ∆EBEa‐Sol would remain constant or increase. This hypothesis is evaluated by the implemented control measurements (see below). The modulation of potential differences in the coupling of PS1 and PS2 upon illumination indicates the anodic reaction to be limiting, which is attributed to the sensitivity of PS2 leading to faster inactivation. After 10 min, Vapp was switched off for almost 1 min. Without the external driving force, no redox reactions are enabled so that no system cur‐ rent and no increase in tip current were recorded. For a second and long‐term illumination period (Figure S‐1), the same trends as before are observed. Irradiation for over 10 min under Vapp was performed to monitor the op‐ erational stability of the isolated protein complexes. Again, a peak in isys is recorded at similar potential differences as before. As isys decreases, ∆EBEa‐Sol and ∆EBEc‐Sol are first shifted to more positive potentials meaning that PS2 re‐ quires a higher driving force to deliver the current while PS1 is able to attain it at lower potential differences. After 12 min, ∆EBEa‐Sol and ∆EBEc‐Sol reach a maximum shift and a peak is recorded in iSECM. After this point, ∆EBEa‐Sol de‐ creases while ∆EBEc‐Sol remains unchanged as the loss in
Figure 1. Data recorded in the bipolar setup for the evalua‐ tion of electrical communication between redox‐polymer em‐ bedded PS1 and PS2. Sample illumination is indicated by the bright yellow background and the application of 0.6 V bet‐ ween the feeder electrodes is indicated by the shaded boxes at the bottom and top of the graphs. a) Current between the feeder electrodes isys, b) current at the SECM tip iSECM, c) in‐ terfacial potential difference between BEa and the solution ∆EBEa‐Sol, and d) interfacial potential difference between BEc and the solution ∆EBEc‐Sol.
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Analytical Chemistry
activity of the immobilised PS2 becomes significant. Consequently, less electrons can be delivered causing the overall driving force ∆EBE to decrease. As soon as the light is switched off, isys declines by approximately 0.8 µA indi‐ cating residual activity. This is accompanied by a drop of the reduction current at the SECM tip back to the initial value and a small increase in ∆EBE as the charge‐transfer resistance increases again. As photosynthetic redox pro‐ cesses do not contribute to the current anymore, the po‐ tentials shift to enable other redox reactions. In this way, oxidation reactions occurring at lower anodic potential dif‐ ferences are balanced by reduction reactions, such as O2 reduction, that require a higher cathodic potential than the photosynthetic reaction before. Investigation of limited electrochemical communication To prove that the observed responses originate from the light‐induced charge separation performed by active pro‐ tein complexes and reflect the electrical communication between the isolated protein complexes, a control meas‐ urement was performed with immobilised but inactivated protein complexes (iPS1 and iPS2). The application of Vapp resulted in a much lower background current (Figure 2, red trace). In accordance to this, also ∆EBEa‐Sol and ∆EBEc‐Sol are significantly lower than for the active biomolecules. Illumination of the samples did not cause significant changes in any of the parameters, because no photores‐ ponse was induced. For the SECM tip current, a decrease was recorded, which can be explained by the previously de‐ scribed light‐induced O2 consumption by iPS2.28‐30 During the long‐term illumination period no significant changes were recorded for all four parameters either. Only the O2 reduction current slowly decreased as the O2 concentra‐ tion was diminished with time. In order to demonstrate that the individual protein com‐ plex activity can be deduced from the course of potential differences upon illumination and over time, and to de‐ monstrate the system’s working principle, several control measurements have been carried out in which one of the reactions was impaired. First, inactivated PS2 was com‐ bined with active PS1 (Figure 2, green trace). Upon appli‐ cation of the driving voltage at the feeder electrodes and in dark conditions, all four parameters show very similar val‐ ues to the measurement with inactivated protein com‐ plexes. Under illumination, the system current increased minimally suggesting that iPS2 is limiting the bipolar cur‐ rent considerably. As a result, ∆EBEa‐Sol and ∆EBEc‐Sol are shifted to more positive values to reach the maximal cur‐ rent possible. The cathodic potential difference is signifi‐ cantly decreased to adjust to the lower current that the an‐ odic side is able to deliver. And again, the SECM tip current is decreased under light due to O2 photoconsumption. For the long‐term irradiation period under Vapp, the same re‐ sponses are observed. As the light is switched off, all four parameters adjust to almost the initial values and ∆EBE slightly increases as the current decreases.
Figure 2. Control measurements performed in the bipolar setup for: iPS1+iPS2 (red), PS1+iPS2 (green) and in the absence of MV2+ as redox mediator (blue). Sample illumination is indi‐ cated by the bright yellow background and the application of 0.6 V between the feeder electrodes is indicated by the shaded boxes at the bottom and top of the graph. a) Current between the feeder electrodes isys, b) current at the SECM tip iSECM, c) interfacial potential difference between BEa and the solution ∆EBEa‐Sol, and d) interfacial potential difference between BEc and the solution ∆EBEc‐Sol.
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Second, the cathodic process was impaired by excluding the redox mediator MV2+ (Figure 2, blue trace). In the ab‐ sence of the redox mediator in solution O2 is directly re‐ duced at the FB site of PS1 but at a considerably slower rate. Therefore, a lower current is expected in this case although PS2 is assumed to be able to deliver a similar current as in the first measurement. When Vapp was applied, a similar background isys and ∆EBEc‐Sol value as in the measurements with iPS2 and both inactivated protein complexes were recorded. However, ∆EBEa‐Sol levelled off at a similar value than that observed for the PS1/PS2 measurement. Upon il‐ lumination, the system current shows an increase of 0.5 µA, which lies in between the responses of the meas‐ urements with active protein complexes and with inactive PS2. The production of O2 monitored by the SECM tip re‐ flects an initially high reaction rate of PS2 that is immedi‐ ately limited by the capability of the cathode side to accept electrons. This aligns with the overall potential drop across the BE. Under light it is ∆EBEa‐Sol that decreases to deliver only the current that the impaired reduction reaction can attain at a slightly higher potential than under dark condi‐ tions. The same behaviour is observed during the long illu‐ mination period where ∆EBEa‐Sol increases slowly, which can be ascribed to continuous inactivation of PS2 over time. The response of all parameters shows that the electrical communication between the active protein complexes takes place but is restricted by the electron transfer which is limited at the reducing end of PS1.
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We have shown that electrical communication between immobilised PS1 and PS2 can be monitored and the protein complex activity can be analysed for each BE pole individ‐ ually while the photoelectrochemical cell is in operation. In case of high activity, high currents at high interfacial po‐ tential differences are observed. Here, the decrease of the charge transfer resistance upon illumination leads to a de‐ crease in ∆EBE as isys increases by the photocurrent. Because the potentials within the system are in equilibrium to ena‐ ble the maximal possible current, the changes in ∆EBE‐Sol indicate which reaction limits the overall performance. In this way, the potential difference for the non‐limiting reac‐ tion always decreases, while the potential difference for the limiting reaction increases or remains constant.
CONCLUSIONS We have presented a first electrochemical study on the electrical communication between PS1 and PS2 both em‐ bedded and wired to the underlying electrode using the same Os‐complex modified redox polymer using a closed setup for bipolar electrochemistry. Extensive information on the performance of the photobioelectrochemical cell was obtained from four different parameters recorded sim‐ ultaneously. From the system current, isys, the in situ col‐ lection of O2 evolved at PS2, and the interfacial potential differences at the BE poles, the protein complex limiting the photocurrent can be derived. The experiments can be seen as a combination of galvanostatic and potentiostatic measurements. As the potentials within the system are in equilibrium, the measured potential differences are con‐ tinuously adjusted in dependence on the activity of the protein complexes to achieve the maximal possible cur‐ rent. The proposed system allows to change and evaluate experimental conditions for each photosystem‐modified electrode individually. In this way, the effect of different pH values, temperatures, electron acceptor or inhibitor concentrations may be analysed. It is important to notice that no membrane or salt bridge is required to connect the two half‐cells as the bipolar reactions are counterbalanced by the feeder electrode reactions.
In addition, a control measurement was carried out in which only PS1 has been inactivated (Figure S‐1, purple trace). As soon as the light is switched on, isys increases by approximately 0.16 µA. In accordance with the ∆EBE value, this current as well as the SECM tip current are lower than the values recorded in the absence of MV2+. Also here PS1 is limiting so that ∆EBEa‐Sol decreases while ∆EBEc‐Sol for the slower reaction slightly increases to reach the maximal cur‐ rent the system can achieve. At the end of the long illumi‐ nation phase, the currents and potential differences barely change indicating that no photoactivity is left. At last, a control measurement was performed, in which both half‐cells were working at the same pH value (Fig‐ ure S‐1, orange trace). By using the same pH value as for the PS2 half‐cell, the additional pH‐related potential dif‐ ference that may influence the system’s response is elimi‐ nated. Because the optimal pH value for the PS1/Os‐P as‐ sembly is 4.0,26 a lower activity at the cathodic side is ex‐ pected under these conditions. In this measurement, the light‐induced system current is similar to the one obtained for the measurement performed in the absence of MV2+ in the buffer solution used for PS1. From the change in poten‐ tial differences with light, it can be seen that a pH value of 6.5 does not impair the performance of PS1, but only de‐ creases ∆EBE. While ∆EBEa‐Sol remains at the same value or increases slightly under illumination, ∆EBEc‐Sol decreases just as observed with the fully active photosystems. The lower driving force only decreases the maximum possible current. Thus, also the SECM tip current is lower than for the PS1/PS2 measurement.
ASSOCIATED CONTENT Supporting Information. A scheme showing the standard potentials of the components involved in the system is pre‐ sented as well as a table with the detailed experimental proce‐ dure for the bipolar electrochemistry measurements. Moreo‐ ver, additional experimental data completing those shown in the manuscript and additional control experiments are in‐ cluded. This material is available free of charge via the Inter‐ net at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * E‐mail:
[email protected], phone: +49 234 322 5474. * E‐mail:
[email protected], phone: + 49 234 322 6200. Fax: + 49 234 321 4683.
Author Contributions
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All measurements were conducted by F.C. and V.E. using sam‐ ples prepared by F.Z. The manuscript was written by V.E. with contributions of F.C. and F.Z., and in discussion with W.S. V.H. and M.M.N. contributed isolated PS1 and PS2. All authors have given approval to the final version of the manuscript.
(13) Kopnov, F.; Cohen‐Ofri, I.; Noy, D. Angew. Chem. Int. Ed. 2011, 50, 12347–12350. (14) Zhang, X.; Zhai, Q.; Xing, H.; Li, J.; Wang, E. ACS Sens. 2017, 2, 320–326. (15) Bouffier, L.; Ravaine, V.; Sojic, N.; Kuhn, A. Curr. Opin. Colloid Interface Sci. 2016, 21, 57–64.
Notes The authors declare no competing financial interest.
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ACKNOWLEDGMENT This work was supported by the Deutsche Forschungsgemein‐ schaft (DFG) in the framework of the Cluster of Excellence Re‐ solv (EXC1069) and the Deutsch‐Israelische Projektkoopera‐ tion (DIP) in the framework of the project “Nanoengineered Optoelectronics with Biomaterials and Bioinspired Assem‐ blies”. The authors are grateful to the chair of Physical Chem‐ istry I, the chair of MEMS Materials, and Mr. H. Austenfeld from the chair of Electronic Materials and Nanoelectronics at the Ruhr‐Universität Bochum for their help with the Au wafer fabrication. Moreover, S. Alsaoub and Dr. A. Ruff are acknowl‐ edged for the synthesis of the redox polymer, C. König for as‐ sistance with the preparation of the photsynthetic protein complexes, and Dr. N. Plumeré for valuable scientific discus‐ sion. V.E. and F.Z. express their gratitude to the German Na‐ tional Academic Foundation and to the China Scholarship Council (CSC), respectively, for their support.
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