pubs.acs.org/Langmuir © 2009 American Chemical Society
Optoelectronic Energy Transfer at Novel Biohybrid Interfaces Using Light Harvesting Complexes from Chloroflexus aurantiacus Arati Sridharan, Jit Muthuswamy, and Vincent B. Pizziconi* Harrington Department of Bioengineering, Ira A. Fulton School of Engineering, Arizona State University, Tempe, Arizona 85287 Received January 10, 2009. Revised Manuscript Received February 25, 2009 In nature, nanoscale supramolecular light harvesting complexes initiate the photosynthetic energy collection process at high quantum efficiencies. In this study, the distinctive antenna structure from Chloroflexus aurantiacus;the chlorosome;is assessed for potential exploitation in novel biohybrid optoelectronic devices. Electrochemical characterization of bacterial fragments containing intact chlorosomes with the photosynthetic apparatus show an increase in the charge storage density near the working electrode upon light stimulation and suggest that chlorosomes contribute approximately one-third of the overall photocurrent. Further, isolated chlorosomes (without additional photosynthetic components, e.g., reaction centers, biochemical mediators) produce a photocurrent (∼8-10 nA) under light saturation conditions. Correlative experiments indicate that the main chlorosome pigment, bacteriochlorophyll-c, contributes to the photocurrent via an oxidative mechanism. The results reported herein are the first to demonstrate that isolated chlorosomes (lipid-enclosed sacs of pigments) directly transduce light energy in an electrochemical manner, laying an alternative, biomimetic approach for designing photosensitized interfaces in biofuel cells and biomedical devices, such as bioenhanced retinal prosthetics.
Introduction In photosynthetic organisms, nanoscale light harvesting complexes (LHCs) composed of molecular pigment arrays that have high quantum efficiencies and selective tunability to specific wavelengths of light initiate the energy-harvesting process. LHCs vary widely in structure and energy collection mechanisms. Examples range from radial arrangements in certain cyanobacteria and lipid-encased pigment bodies known as chlorosomes in green bacteria to protein complexes embedded in membranes, such as LH1 peptides in plant-based photosynthetic systems and the rhodopsin proteins in mammalian eyes.1-3 From an engineering perspective, interfacing peptide-based light antennae to an external, synthetic conductor pose substantial challenges in maintaining biofunctionality since most natural downstream energy transfer events are integrally dependent on specific membrane charge-carriers.1,4 Here, we investigate the bioelectrochemical properties of photosynthetic light-harvesting structures that have a non-peptide-based design for the essential light-capturing function, specifically the chlorosome from Chloroflexus aurantiacus. Chlorosomes are photosynthetic antenna complexes lining the inner aqueous side of the cytoplasmic membrane in most green photosynthetic bacteria. In C. aurantiacus, a green nonsulfur bacterium that grows in hot springs, these supramolecular complexes are ellipsoidal sacs containing bacteriochlorophyll (BChl c)
*Corresponding author.
[email protected]. (1) Blankenship, R. E. In Molecular Mechanisms of Photosynthesis; Blackwell Science: Ames, IA, 2002; pp 63-81. (2) Nickle, B.; Robinson, P. R. Cell. Mol. Life Sci. 2007, 64, 2917–2932. (3) Bahatyrova, S.; Frese, R. N.; Siebert, C. A.; Olsen, J. D.; Werf, K. O.; Grondelle, R. v.; Niederman, R. A.; Bullough, P. A.; Otto, C.; Hunter, C. N. Nature (London) 2004, 430, 1058–1062. (4) Engel, G. S.; Calhoun, T. R.; Reed, E. L.; Ahn, T.-K.; Mancal, T.; Cheng, Y.-C.; Blankenship, R. E.; Fleming, G. R. Nature (London) 2007, 446 782–786.
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pigment aggregates enclosed in a glycolipid monolayer.5 Depending on the species of green bacteria and growth conditions, C. aurantiacus derived chlorosomes have nanoscale dimensions of 100-160 nm 20-40 nm 10-20 nm (Figure 1a).6-9 Structurally, BChl a pigment-protein complexes, known as the base plate, tether the chlorosome to the cytoplasmic membrane as shown in Figure 1a. Energetically, the BChl c oligomers within the chlorosome selectively absorb photonic energy (Figure 1b) and efficiently transfer the harvested energy to the BChl a pigments in the base plate via an exciton-diffusion mechanism.10,11 Photoenergy is subsequently captured as chemical energy in a kinetically coordinated cascade of fast chemical reactions initiated by the BChl a protein complex (P865) in a quinone-based reaction center similar to purple bacteria.12,13 There are no reports to date on using chlorosomes or its derivatives as photoelectrochemical transducers in a bioelectronic device. Previous developmental efforts using photosynthetic subcomponents (mostly reaction centers) from other species lend encouragement to the use of chlorosomes as potential biophotonic transducers. This includes spinach thylakoid-based systems, solid-state photocells using purple bacterial reaction centers, and
(5) Olson, J. M. Photochem. Photobiol. 1998, 67, 61–75. (6) Sprague, S. G.; Staehelin, L. A.; DiBartolomeis, M. J.; Fuller, R. C. J. Bacteriol. 1981, 147, 1021–1031. (7) Oelze, J.; Golecki, J. R. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer: Dordrecht, 1995; pp 259-278. (8) Martinez-Planells, A.; Arellano, J. B.; Borrego, C. A.; Lopez-Iglesias, C.; F., G.; Garcia-Gil, J. S. Photosynth. Res. 2002, 71, 83–90. (9) Montano, G. A.; Bowen, B. P.; LaBelle, J. T.; Woodbury, N. W.; Pizziconi, V. B.; Blankenship, R. E. Biophys. J. 2003, 85, 2560–2565. (10) K^e, B. In Photosynthesis: Photobiochemistry and Photobiophysics; Kluwer Academic Publishers: Boston, 2001; pp 147-178. (11) Scholes, G. D.; Rumbles, G. Nat. Mater. 2006, 5, 683–696. (12) Mix, L. J.; Haig, D.; Cavanaugh, C. M. J. Mol. Evol. 2005, 60 153–163. (13) Gupta, R. S. Photosynth. Res. 2003, 76, 173–183.
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bacteriorhodopsin protein-based logic devices.14-22 Other strategies utilize the chlorophyll pigment or its derivatives as the light harvesting component in dye-based solar cell devices.21,23,24 Chlorophyll derivatives, however, face comparatively lower lifetimes and efficiencies when incorporated in biohybrid devices. A major advantage of using chlorosomes as biophotonic transducers arises from its natural stability in aqueous environments. Additionally, the internal molecular arrangement of BChl c oligomers enables selective absorption of light with spectral peaks at ∼740 nm and a high-energy Soret at ∼460 nm as shown in Figure 1b. However, the exact architecture of the BChl c pigments (rods, lamellar plates, etc.) that lends to its high quantum efficiencies (69-92%) and stability is still under debate.25,26 Prior studies, including our earlier demonstration of covalently immobilized functional chlorosomes have reported different methods to immobilize chlorosomes on various conductive substrates.27-30 Biophotonic integration of chlorosomes in future devices may illustrate a unique method of packaging chlorophyll-like molecules to achieve stability and efficiency at biohybrid interfaces. Another advantage is that, unlike other photosynthetic apparatus, chlorosomes from C. aurantiacus are not dependent on pigment-protein interactions for the light-harvesting function.31-33 In fact, treatment with common proteases does not disrupt the light absorption characteristics of chlorosomes.34-36 Given the non-peptide-dependent light harvesting properties, aqueous stability, and high temperature tolerance due to thermophilic growth conditions (45-75 °C) of C. aurantiacus, the chlorosome seems an ideal candidate as a nanoscale biophotonic energy transducer. (14) Trammell, S. A.; Wang, L.; Zullo, J. M.; Shashidhar, R.; Lebedev, N. Biosens. Bioelectron. 2004, 19, 1649–1655. (15) Giardi, M. T.; Pace, E. Trends Biotechnol. 2005, 23, 257–263. (16) Millsaps, J. F.; Bruce, B. D.; Lee, J. W.; Greebaum, E. Photochem. Photobiol. 2001, 73, 630–635. (17) Das, R.; Kiley, P. J.; Segal, M.; Norville, J.; Yu, A. A.; Wang, L.; Trammell, S. A.; Reddick, L. E.; Kumar, R.; Stellacci, F.; Lebedev, N.; Schnur, J.; Bruce, B. D.; Zhang, S.; Baldo, M. Nano Lett. 2004, 4, 1079–1083. (18) Suemori, Y.; Nagata, M.; Nakamura, Y.; Nakagawa, K.; Okuda, A.; Inagaki, J.-i.; Shinohara, K.; Ogawa, M.; Iida, K.; Dewa, T.; Yamashita, K.; Gardiner, A.; Cogdell, R., J.; Nango, M. Photosynth. Res. 2006, 90, 17–21. (19) Li, Q.; Stuart, J. A.; Birge, R. R.; Xu, J.; Stickrath, A.; Bhattacharya, P. Biosens. Bioelectron. 2004, 19, 869–874. (20) Lam, K. B.; Johnson, E. A.; Chiao, M.; Lin, L. J. MEMS 2006, 15, 1243– 1250. (21) Huijser, A.; Marek, P. L.; Savenije, T. J.; Siebbeles, L. D. A.; Scherer, T.; Hauschild, R.; Szmytkowski, J.; Kalt, H.; Hahn, H.; Balaban, T. S. J. Phys. Chem. C 2007, 111, 11726–11733. (22) Ogawa, M.; Shinohara, K.; Nakamura, Y.; Suemori, Y.; Nagata, M.; Iida, K.; Gardiner, A. T.; Cogdell, R. J.; Nango, M. Chem. Lett. 2004, 33, 772–773. (23) Amao, Y.; Yamada, Y. Biosens. Bioelectron. 2007, 22, 1561–1565. (24) Furukawa, H.; Inoue, N.; Watanabe, T.; Kuroda, K. Langmuir 2005, 21, 3992–3997. (25) Brune, D. C.; Nozawa, T.; Blankenship, R. E. Biochemistry 1987, 26, 8644– 8652. :: (26) Psencık, J.; Ikonen, T. P.; Laurinmaki, P.; Merckel, M. C.; Butcher, S. J.; Serimaa, R. E.; Tuma, R. Biophys. J. 2004, 87, 1165–172. (27) Mimuro, M.; Hirota, M.; Nishimura, Y.; Moriyama, T.; Yamazaki, I.; Shimada, K.; Matsuura, K. Photosynth. Res. 1994, 41, 181–191. (28) Saga, Y.; Kim, T.-Y.; Hisai, T.; Tamiaki, H. Thin Solid Films 2006, 500, 278–282. (29) Shibata, Y.; Saga, Y.; Tamiaki, H.; Itoh, S. Biophys. J. 2006, 91, 3787–3796. (30) Sridharan, A.; Muthuswamy, J.; LaBelle, J. T.; Pizziconi, V. B. Langmuir 2008, 24, 8078–8089. (31) Tamiaki, H. Coor. Chem. Rev. 1996, 148, 183–197. (32) Foidl, M.; Golecki, J. R.; Oelze, J. Photosynth. Res. 1994, 41, 145–150. (33) van Rossum, B. J.; Steensgard, D. B.; Mulder, F. M.; Boender, G. J.; Schaffner, K.; Holzwarth, A. R.; de Groot, H. J. M. Biochemistry 2001, 40, 1587– 1595. (34) Balaban, T. S.; Tamiaki, H.; Holzwarth, A. R. In Supermolecular Dye Chemistry: Topics in Current Chemistry; Springer-Verlag: Berlin, 2005; Vol. 258, pp 1-38. (35) Lehmann, R. P.; Brunisholz, R. A.; Zuber, H. Photosynth. Res. 1994, 41, 165–173. (36) Sakuragi, Y.; Frigaard, N.-U.; Shimada, K.; Matsuura, K. Biochim. Biophys. Acta 1999, 1413, 172–180.
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Previous strategies by this group capitalized on the fluorescent properties of electrostatically immobilized chlorosomes to enhance the spectral performance of conventional photovoltaic devices.37 In these systems, orientated chlorosomes, which absorb in the blue (470 nm) and near infrared (740 nm) region of the spectrum, transfer energy via a large Stokes’ shift to the BChl a pigment in the baseplate. This in turn fluoresces spectrally in the near-infrared region (808/810 nm) where silicon-based photosystems function most efficiently. While this earlier study used the biophotonic energy transfer properties of the chlorosome to spectrally enhance conventional photovoltaic devices, here a direct energy transfer route via a photoelectrochemical mechanism from chlorosomes to an electrode is explored. Pursuit of this energy transfer path is promising as the ground redox state of immobilized chlorosomes could be used to modulate the inherent photoelectrochemical current activity of indium tin oxide (ITO) electrodes.30 It should be noted, however, there are no known reports of photoelectrochemical energy transfer emanating directly from chlorosomes decoupled from their native reaction centers. As such, this study builds on our previous efforts where the primary aim is to demonstrate that isolated chlorosomes indeed do exhibit the ability to transfer photonic energy in an electrochemical manner. The demonstration of this heterogeneous form of energy transfer (photo- to electrochemical) may lead to new biomimetic-based hybrid device innovations. A stepwise approach is used to examine whether the light harvesting capabilities of the chlorosome can be captured as electrical energy in an electrochemical environment as shown in Figure 1c. In the first step, C. aurantiacus bacterial fragments solutions containing chlorosomes with the native photosynthetic apparatus including the reaction center are placed between graphite working and platinum counter electrodes in a customized photoelectrochemical cell. Rigorous chronoamperometry and cyclic voltammetry studies demonstrate the photoelectrochemical energy transfer capabilities of these photosensitive fragments. In the second step, purified, isolated chlorosomes (without reaction centers) are analyzed using chronoamperometry and cyclic voltammetry studies that demonstrate light-stimulated energy transfer capabilities of isolated chlorosomes. Finally, in the third step, only isolated chlorosomes closely associated with the working electrode are shown to contribute to the photocurrent. In these electrochemical cells (ECs) with isolated chlorosomes, the photocurrent that is generated is correlated with the oxidization of the BChl c aggregates that comprise the chlorosome antenna harvesting complex. Together, these results could lead to exciting bioinspired applications for chlorosomes in emerging biotechnologies such as biofuel cells and retinal prostheses among others.
Materials and Methods Growth and Isolation of Chlorosomes from C. aurantiacus. Using previously described methods, Chloroflexus aurantiacus bacteria (J-10-fl strain) were grown at of 300-500 l at 55 °C under anaerobic conditions in a modified Nitsch’s D-medium.38 Chlorosomes were purified using a similar protocol of Feick and Fuller.39 Briefly, green-colored cultures were harvested by centrifuging at 3000 g for 60 min after 7 days of growth (late exponential phase), resuspended in 2 M sodium thiocyanate buffer (1:4 (w/v)), homogenized at 4 °C, and then French-pressed (20 000 psi) to obtain fragments containing chlorosomes. The resulting mixture was ultracentrifuged at 100 000 g for 18 h at (37) LaBelle, J., T.; Pizziconi, V. B. U.S Patent 7,067,293, 2006. (38) Gerola, P. D.; Olson, J. M. Biochim. Biophys. Acta 1986, 848, 69–76. (39) Feick, R. G.; Fuller, R. C. Biochemistry 1984, 23, 3693–3700.
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Figure 1. Description of the photosynthetic apparatus as found in C. aurantiacus. (a) Chlorosomes from C. aurantiacus are flat, ellipsoidal light antenna structures found in the inner portion of the cytoplasmic membrane in the bacterium (chlorosome figure adapted from Olson, 1998). Structurally, the chlorosome body is composed of self-assembled BChl c pigment aggregates that have a spectral absorption peak at 740 nm enclosed in a lipid monolayer. Base plates are pigment/protein complexes that tether the chlorosome body to the membrane and have BChl a pigments that serve as the natural electron sink for the chlorosomal pigments. The baseplate, in turn, connects to the core antenna/ reaction center components, where photonic energy is converted to chemical energy via biochemical mediators. Note the diagram is not to scale. (b) Typical spectroscopic properties of chlorosomes (C. aurantiacus) are shown with respect to the pigments found in the photosynthetic apparatus. Characteristic absorbance peaks for the BChl c aggregates are at 740 nm and a high-energy (Soret) peak at ∼462-470 nm. The pigments in the baseplate and core antenna/reaction center typically absorb at 795 nm and at 808 and 866 nm, respectively. (c) In this study, custom electrochemical cells are used to probe charge transfer properties of chlorosomes in 3 configurations: (1) chlorosomes within bacterial fragments that contain additional components of the photosynthetic apparatus, (2) purified, isolated chlorosomes without additional photosynthetic components in solution, (3) working electrodes with weakly associated, isolated chlorosomes. Electrochemical cells (ECs) use graphite as working electrodes, platinum as counterelectrodes and a light-blocked silver/silver chloride electrode was used as reference (not shown). An external wide spectrum 150 W halogen lamp was used to stimulate the cell with chlorosome-containing solutions. Data was collected using the CHI 660a potentiostat using various electrochemical techniques. 4 °C in a 5-40% sucrose gradient. Chlorosome-containing fractions devoid of reaction centers were pooled, recentrifuged in sucrose gradients, and dialyzed 6 times into 0.2 M phosphate buffered saline (PBS) (pH 8.0) to remove any extraneous pigments and smaller soluble contaminants from the photosynthetic apparatus. Absorbance spectroscopy at 470, 740, and 795 nm was used, where the former two wavelengths are related to BChl c aggregates in the chlorosome and the latter to BChl a pigments present in the base plate. The absence of reaction centers were confirmed by the lack of peaks at 808/866 nm. The number of chlorosomes was determined using empirical relationship derived by LaBelle.40 (40) LaBelle, J. T. Dissertation, Arizona State University, 2001.
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Ultrasonicated Bacterial Fragment Preparation. C. aurantiacus cultures were centrifuged at 3000 g, resuspended in 0.2 M phosphate buffered saline (PBS) and ultrasonicated at 25 W for ∼1-10 min at room temperature to obtain a mixture of bacterial fragments containing chlorosomes with reaction centers (∼1010-1013 chlorosome-containing fragments/mL) as confirmed by absorbance spectroscopy. The ultrasonication process was monitored by absorbance spectroscopy to ensure that no degradation of the BChl c oligomer took place, which is indicated by a spectral shift in absorbance from 740 nm (BChl c oligomer), to 670 nm (BChl c monomer). To obtain fragments of whole cells, the slopes of the absorbance spectrum between 550 and 650 nm were monitored for diminishing scattering effects. To eliminate electrogenic events occurring due to the BChl c Langmuir 2009, 25(11), 6508–6516
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Figure 2. Electrochemical characterization of bacterial fragments derived from C. aurantiacus. (a) Cyclic voltammograms (CVs) of electrochemical cells (ECs) containing ultrasonicated bacterial fragments with chlorosomes and additional components of the C. aurantiacus photosynthetic apparatus are distinctly more oxidizable at higher voltages compared to phosphate buffered saline (PBS) based ECs. (b) Comparison of charge storage densities at the electrode, calculated as the average area-under-the-CV curves (from -0.3 to 0.9 V), show that ECs with bacterial fragments containing chlorosomes and additional photosynthetic components have a higher charge storage capacity than ECs with PBS. (c) Upon light excitation of ECs containing ultrasonicated bacterial fragments with chlorosomes and additional photosynthetic components, the CV curve shows small shifts in current as indicated by arrows. The curve shown represents an average of 3 scans under dark and light conditions (∼80 mW/cm2). Inset shows that light stimulation of bacterial fragment containing ECs have a higher calculated charge storage density near the electrode. (d) Analysis of the maximum difference in current between light and dark conditions for forward (- f + bias) and reverse (+ f - bias) sweeps of the CV curve show that bacterial fragments containing chlorosomes have a larger change in photocurrent compared to PBS based ECs. The maximum current differences between the forward and reverse sweeps for each type of EC were within the margin of error. monomer or its de-metallized derivative bacteriopheophytin, since they were found to contribute to the photopotential in a different species of green bacteria,41 only freshly ultrasonicated bacterial fragment solutions of C. aurantiacus with no absorbance peak at 670 nm were used. In addition, absorbance spectra taken immediately after the electrochemical experiments, confirmed that no degradation had taken place as indicated by the lack of a 670 nm peak.
Electrochemical Setup and Chronoamperometric Studies. A custom electrochemical cell with a graphite-based working electrode and platinum counter electrode were placed 1 cm apart. A light-blocked silver/silver chloride reference in 3 M KCl (BASi, West Lafayette, IN) was placed equidistant from the other two electrodes. For isolated chlorosome-based electrochemical studies, ∼109-1012 chlorosomes/mL with graphite as the working electrode was used. Electrochemical cells (working electrode side) were stimulated with a 150 W halogen lamp (400-850 nm spectrum). Using a IL-1700 research radiometer (International Light Inc., Newburyport, MA), the light intensities were kept constant at ∼80 mW/cm2 for most studies. For dose-dependent responses, the stimulating halogen lamp was mechanically modulated from ∼30-80 mW/cm2. A CHI-660a potentiostat (CH Instruments, Austin, TX) in chronoamperometry mode was used to measure any generated photocurrent at 0 V in absence of redox mediators. Pulsatile experiments were performed at 30 s intervals. (41) Ptak, A.; Dudkowiak, A.; Frackowiak, D. J. Photochem. Photobiol., A 1998, 115, 63–68.
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Electrode Surface Characterization. Electrochemical impedance spectroscopy was performed at 5 mV from 1 to 100 000 Hz to capture differences that may have occurred upon exposure to chlorosomes. To confirm surface changes on chlorosome exposed electrodes, graphite-based working electrodes that were exposed to chlorosomes in solution for 15 min under chronoamperometric conditions at 0 V were imaged using field emission scanning electron microscopy (FESEM) at 10 kV. Imaged samples were rinsed at least six times in distilled water, dried, and placed under vacuum conditions. Cyclic Voltammetry and Determination of Charge Storage Density. Cyclic voltammetry under light and dark
conditions was performed at 50 mV/s between -0.2 and 0.8 V for isolated chlorosomes and -0.3 and 0.9 V for solutions containing bacterial fragments. Charge storage density was calculated as the average area between the two sweeps of the CV curves, which was determined using numerical integration techniques.42 The calculated total charge was then divided by the effective electrode surface area to obtain a charge storage density value. The typical working electrode surface area were estimated using the Cottrell relationship using a similar protocol to Cummings et al.43 Briefly, oxidative current at the electrodes was measured with 1 mM ferricyanide in 1 M KCl in aqueous solution (diffusion coefficient ∼6.3 10-5 cm 2 s-1) under (42) Keefer, E. W.; Botterman, B. R.; Romero, M. I.; Rossi, A. F.; Gross, G. W. Nat. Nanotechnol. 2008, 3, 434–439. (43) Cummings, E. A.; Mailley, P.; Linquette-Mailley, S.; Eggins, B. R.; McAdams, E. T.; McFadden, S. Analyst 1998, 123, 1975–1980.
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chronoamperometric conditions for 20 s at 0.7 V with 2 s quiet time at 0 V. The effective electrode area for 3 samples was 2 determined √ to be 0.67 ( 0.09 cm from the calculated slope of I = 1/ t where I represent current and t represents time in the Cottrell equation.
Results Photostimulation of C. aurantiacus Bacterial Fragments Increases Charge Storage Densities. Comparative cyclic voltammetry (CV) showed that electrochemical cells (ECs) with bacterial fragments (Figure 1c, #1) were asymmetric with an increased oxidation peak, indicated by significantly larger current at higher voltages (Figure 2a). Calculated as an average area under the CV curve, the charge storage density of these ECs were evaluated and plotted in Figure 2b. The charge storage density metric is typically used to characterize the near surface charge properties of a given microelectrode for establishing safe biological tissue stimulation parameters.42 The use of this metric was extended here to not only better understand the complex electrochemical charge transfer properties of the chlorosome light antenna structure, but also establish comparable parameters for electronic assessment of the biohybrid interface in a given electrochemical system. Under dark conditions, the baseline charge storage density for ECs containing bacterial fragments with intact chlorosomes and other photosynthetic components was significantly higher than that of PBS. This indicated the presence of highly charged entities, like chlorosomes (essentially sacs of reduced dye molecules) and other photosynthetic components near the electrode. Under light stimulation, the charge storage densities showed a moderate increase, indicating a photoinduced charge accumulation in the vicinity of the electrode (Figure 2c, inset). Further, the light-todark current differences were more prominent at oxidative voltages as seen in Figure 2c. In addition, ECs with bacterial fragments had a significantly higher photoinduced current change for both the forward and reverse scans of the CV curves, suggesting that these photosensitive bacterial fragments were primed to photo-oxidize in an aqueous environment (Figure 2d). The maximum light-to-dark difference in current between the forward and reverse scans was similar regardless of the EC configuration. Chlorosomes within Bacterial Fragments Contribute Indirectly to the Photoelectrochemical Response. Light stimulation of ECs containing intact chlorosomes and other components typically showed a steady linear increase in current (0.3 nA/s to 0.8 nA/s) under chronoamperometric studies (Figure 3a). Cessation of light abruptly decreased the rate of change in current. The fast current spikes observed in the upper curve in Figure 3a upon light cessation were likely due to artifacts from the light source. The photoinduced current response was repeatable, although the exact magnitude depended on multiple parameters such as bacterial fragment concentrations and growth conditions (data not shown). Experimental controls, i.e., ECs with PBS solution, indicated no changes upon light excitation (Figure 3a, inset), suggesting that the observed photocurrent was due to the presence of photoactive components in the bacterial fragment solutions. Continuous light excitation of C. aurantiacus bacterial fragments showed a photosaturation response (Figure 3b). The maximum photocurrent (∼27 nA for the highest concentration (∼3 g/mL)) was linearly dependent on the starting concentration of the ultrasonicated bacteria (Figure 3b, left inset). After the light was turned off, the current decayed akin to the Cottrell relationship, where electrochemical current was dominated by 6512
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Figure 3. Chronoamperometric characterization of ultrasonicated bacterial fragments containing chlorosomes and components of the C. aurantiacus photosynthetic apparatus (a) A representative EC containing ultrasonicated bacterial fragments in solution (∼3 g/mL at 500 l growth conditions) was excited by a bright white light (∼80 mW/cm2) for 30 s at 0 V. Arrows indicate light stimulation. Photocurrents increase linearly with time with typical slopes of 0.37 nA/s, 0.32 nA/s, 0.45 nA/s, and 0.77 nA/s labeled 1-4, respectively. The current spikes seen in the upper curve were artifacts emanating from the mechanical switches of the light source instrument. Controls with just PBS electrolyte showed no change in current due to light. (Inset). (b) A representative EC with bacterial fragment solutions was subjected to sustained light exposure at 30 s until a maximum photocurrent (∼27 nA) could be discerned at ∼50 s after the start of the light excitation. (Inset) Upon stimulation with blue light using a photonic band-pass filter (400-550 nm), a photocurrent response is observed for ECs containing bacterial fragments containing the photosynthetic apparatus including the chlorosome, which has a strong absorption peak at ∼462-470 nm.
diffusion-controlled charge-transfer kinetics of the bulk concentration. Further, blue light stimulation (400-550 nm) of C. aurantiacus bacterial fragments generated a similar linear increase in photocurrent with a maximum photocurrent of ∼9 nA, as observed earlier under full spectrum stimulation (Figure 3b, inset). Considering the strong chlorosome pigment absorption peak at ∼470 nm, the photocurrent data here suggested that chlorosomes contributed to the total photocurrent response indirectly via its associated reaction center and photosynthetic apparatus. Light Modulates Charge Storage Densities of Purified Chlorosomes. Under dark conditions (after extensive purification), increasing concentrations of isolated chlorosomes showed proportionally increasing oxidation/reduction currents, indicating that ECs with chlorosomes (Figure 1c, #2) were susceptible to oxidation (Figure 4a). The oxidation/reduction peaks exhibited sharp, rapid changes in current, suggestive of fast interfacial charge transfer. The charge storage densities demonstrated increasing log-linear relationship with chlorosome concentration (r2 = 0.92), indicating a concentration dependent increase Langmuir 2009, 25(11), 6508–6516
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Figure 4. Electrochemical characterization of isolated chlorosomes in solution. (a) CVs of increasing concentrations of isolated chlorosomes in solution have larger oxidation peaks at incremently smaller biases. Inset shows that the average charge storage density, calculated as the average area-under-the-CV from -0.2 V to 0.8 V, increases with concentration. (b) The relative change in charge storage densities are calculated for ECs containing 1010 chlorosomes/mL in solution under dark, low, and high light conditions (n = 3 for each condition). The charge density stored near the electrode increases with light intensity. Inset shows representative CV curves obtained under dark, low light intensities (∼30 mW/cm2) and high light conditions (∼80 mW/cm2). (c) Nyquist plots of ECs containing different concentrations of isolated chlorosomes in solution show an increasing slope indicative of increasing capacitative behavior of the EC at lower concentrations of chlorosomes. Inset shows that this trend is consistent at impedance values obtained at higher frequencies. Impedance spectroscopy was performed from 1 to 100 000 Hz at 5 mV amplitude at 0 V. (d) Impedance spectroscopy of ECs under high light stimulation shows a slight shift in the curve indicative of lower interfacial charge transfer resistance. Filled data points represent light-stimulation conditions and unfilled data points represent dark conditions. (e) A representative EC containing 1012 chlorosomes/mL is stimulated with light (∼80 mW/cm2) at 30 s under chronoamperometric conditions (0 V). An increase in photocurrent is observed, which reaches a maximum after ∼10 s.
in the number of charged particles near the electrode surface (Figure 4a, inset). Charge storage densities for control ECs with PBS (3.5 10-7 ( 0.02 10-7 C cm2) was not significantly different from those measured from ECs with chlorosome concentration of ∼109 chlorosomes/mL (∼2 μM), indicating the detection limit for this EC configuration. Increasing the light intensity from 30 mW/cm2 to 80 mW/cm2 approximately doubled the peak oxidation currents (Figure 4b) and resulted in more than 3-fold increase in charge storage density as shown in Figure 4b (inset). This suggested that there was an apparent dose-dependent relationship between light intensity and number of oxidized, isolated chlorosomes in solution. Light Stimulation Decreases Charge-Transfer Resistance at the Biohybrid Interface. Electrochemical impedance spectroscopy was performed on incremental concentrations of Langmuir 2009, 25(11), 6508–6516
isolated chlorosomes in solution. With decreasing concentrations of isolated chlorosomes, a gradual increase in slope was observed in Nyquist plots as shown in Figure 4c indicating a more capacitive interface at the electrode even at high frequencies. When stimulated with light for a given concentration, the impedance spectra shifted to the left without a change in slope, suggesting a decrease in the interfacial charge transfer resistance with light stimulation. Despite inherent, systemic variations across samples of same chlorosome concentration, the effect of light stimulation for several different concentrations demonstrated a similar trend (Figure 4d). Isolated Chlorosomes Contribute Approximately OneThird of Overall Photocurrent Induced by Photosynthetic Apparatus. Isolated chlorosomes generated a current in response to light stimulation that opposed the direction of the DOI: 10.1021/la900112p
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baseline dark current as shown in chronoamperometric studies in Figure 4e. The photocurrent reached a maximum (∼10 nA) before decaying to baseline levels over a period of 40 s of continuous light excitation. The maximum photocurrent observed with isolated chlorosomes was approximately one-third of the overall photocurrent achieved in ECs with bacterial fragments. This was similar in magnitude to the photocurrent obtained in ECs with bacterial fragment solutions under blue light stimulation (Figure 3b).Therefore, chlorosomes within bacterial fragments appear to be functionally equivalent to isolated chlorosomes. However, the rise time to achieve maximum photocurrent in ECs with isolated chlorosomes was ∼10 s compared to ∼50 s in ECs with bacterial fragments as shown in Figure 3b. Lower rise times in photocurrents for isolated chlorosomes may be due to a faster charge transfer response from uncoupled, isolated chlorosomes. Depending on growth conditions for a given batch of bacteria, subsequent experiments indicated that the average risetime for ECs with isolated chlorosomes was always less than ECs containing bacterial fragments grown under similar conditions. Electrochemical Oxidation of Isolated Chlorosomes Contributes to Photoelectronic Response. A dose-dependent decrease in absorbance at 740 nm (characteristic for the BChl c light harvesting pigment) with increasing light intensity under chronoamperometric conditions indicated that an increasing fraction of the BChl c pigments in the chlorosome was oxidized (as measured by the proportional decrease in absorbance at 740 nm), supporting a photoelectrochemical energy transfer mechanism (Figure 5a). No denaturation had occurred as indicated by subsequent analysis of absorbance at 670 nm, which is indicative of degradation of the BChl c pigment aggregate to a monomer. Therefore, the data presented in Figure 5a showed photoinduced energy transfer from isolated chlorosomes correlated with oxidation of the main light harvesting pigment without additional components of the C. aurantiacus photosynthetic apparatus. This further supported the hypothesis that isolated chlorosomes directly contributed to photoinduced electrogenic response. In addition to evidence of BChl c pigment oxidation, the BChl a pigment from the base plate region (characterized by a 795 nm peak) is also shown to oxidize in light intensity dose dependent manner (Figure 5b). In comparing the ratios of the changes in absorbance normalized to light intensity, it is seen that for every unit change in the 795 nm peak, there is approximately a 3-fold change in 740 nm peak (BChl c). This suggests that at least some portion of the bioelectronic energy transfer that is observed involves the base plate region of the chlorosome body. Chlorosomes Close to the Electrode Contribute to the Photocurrent. The small photocurrent generated by ECs containing isolated chlorosomes suggested that the photoelectrochemical signal was obtained from photoactive elements in close proximity to the electrode, i.e., via adsorption or other weakly associated mechanisms. In Figure 6a, electrodes that were exposed to isolated chlorosomes under chronoamperometric conditions, subsequently rinsed in distilled water, dried, and imaged using field emission electron microscopy (FESEM) showed that isolated chlorosomes appeared to be either adsorbed or weakly associated with the electrode. Control electrodes treated similarly with PBS showed no presence of any chlorosome particle association. The average electrochemical impedance spectra (shown as a Nyquist plot in Figure 6b) of similarly treated electrodes that were measured in PBS (Figure1c, #3) showed a shift toward the left indicating a decrease in the interfacial charge transfer resistance upon 6514
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Figure 5. Evidence of bacteriochlorophyll-c (BChl c) and BChl a oxidation after light exposure under chronoamperometric conditions. ECs containing 1010 chlorosomes/mL in solution were subjected to dark, low (∼30 mW/cm2), or high (∼80 mW/cm2) light intensities under chronoamperometric conditions of 0 V for 15 min. (a) Analysis of the absorbance spectra of these solutions indicate that the absorbance peak of main chlorosomal pigment (BChl c), which absorbs at 740 nm, decreases with increasing light intensities, indicative of increasing levels of oxidation of the pigment. There was no increase in absorbance at 670 nm, which is indicative of degradation of the Bchl-c aggregate typically found in the chlorosome ultrastructure to a monomer. (b) Changes in absorbance at 795, which is indicative of the BChl a pigment in the base plate region of the chlorosomes, suggests that pigment oxidation increases with higher levels of light intensity, similar to the BChl c pigment aggregates.
exposure to varying concentrations of isolated chlorosomes. In fact, chlorosome-associated electrodes exhibited a photocurrent akin to that of an EC with isolated chlorosomes in solution (Figure 6c). The photocurrent generated by chlorosomeexposed electrodes decreased with time (∼28 h), implied that an apparent physical desorption had occurred (Figure 6c, inset).
Discussion This study provided electrochemical evidence that naturally derived, nanoscale biophotonic constructs, such as chlorosomes from C. aurantiacus, were capable of photoinduced energy transfer in an electrochemical environment. Although not previously demonstrated for green nonsulfur bacteria, a photoelectrochemical response from bacterial fragments with chlorosomes was expected since the native system utilized an electrochemical system to harness energy. The significant photoinduced increase in the collective charge stored near the electrode demonstrated that photosensitive C. aurantiacus bacterial fragments containing intact chlorosomes and additional photosynthetic components were electrogenic. Additionally, the saturating current response, as seen in Figure 3b, showed that light-stimulated energy transfer in bacterial fragment solutions was likely limited by the finite availability of native cell energy mediators. Therefore, a portion of the photocurrent seen in the experimental data could be attributed to the photoactivity of the reaction center and downstream membrane based charge carriers. This was supported by several groups who had previously observed a photovoltage in Langmuir 2009, 25(11), 6508–6516
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Figure 6. Characterization of isolated-chlorosome associated electrodes. (a) Field emission scanning electron microscopy (FESEM) of graphite electrodes that are exposed to chlorosomes under chronoamperometric condtions of 0 V and stimulated with light (∼80 mW/cm2) for 15 min is shown. The electrodes were rinsed in distilled water to remove any unassociated chlorosomes. (b) Nyquist plots of typical chlorosome-exposed electrodes at 0 V, 5 mV amplitude signal from 1 to 100 000 Hz are shown. The graphite electrodes that are exposed to different concentrations of chlorosomes under chronoamperometric conditions show a shift in impedance as seen in the representative Nyquist plots, suggesting that chlorosomes that are closely associated to the electrode have changed the electrode surface properties. (c) Chlorosome-exposed electrodes stimulated with high light intensities show a reproducible photocurrent compared to ECs with just PBS. Graphite electrodes exposed to chlorosomes in solution and rinsed in distilled water have been subsequently characterized in ECs with PBS as the main electrolyte solution. The estimated areas-under-the-curve found using the integrative trapezoidal rule for both the light pulse stimulations in the representative curve are found to be similar (2.3 10-7 coulombs and 2.0 10-7 coulombs, respectively). The photocurrent is found to steadily decrease over ∼28 h. (Inset) (d) A potential mechanism to explain the photoelectric activity of ECs with chlorosomes shows that upon light stimulation, chlorosomes in near proximity to the electrode undergo a charge transfer event with the working electrode, resulting in an increasing current. W indicates the working electrode, while C indicates the counterelectrode.
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C. aurantiacus membrane preparations due to the reaction center itself 44 and to the presence of multiple charge carriers like menaquinone and cytochrome 554 complexes.45,46 However, the contribution of the chlorosome light antenna structure cannot be neglected since they are expected to contribute indirectly via the reaction center and downstream charge carriers. The slow and steady increase in photocurrent supported this assertion, since the multistep process of charge migration from the pigments in the chlorosome to downstream carriers and eventually charge injection into the electrode would occur on a longer time scale. The comparatively lower rise time for ECs with isolated chlorosomes further supported this observation, since removal of additional rate-limiting electrochemical reactions from the native machinery would be expected to increase the charge transfer rate under favorable conditions. Similar conclusions for chlorosomecontaining bacterial fragments from green sulfur bacteria (Prosthecochloris aestuarii) were reached by Ptak et.al. 41 A strongly supported key finding was that extensively purified, isolated chlorosomes, which are LHCs uncoupled from the photosynthetic apparatus, directly induced charge transfer under light stimulation. Charge storage density measurements and absorbance spectroscopy indicated that the main pigment within the chlorosome body underwent oxidation. It could be speculated that the lipid monolayer surrounding the pigment aggregate in the chlorosome body was leaky enough for charge transfer to occur via charges present in the electrolyte or charge transfer occurred through the pigments present in the baseplate region of the chlorosome body (Figure 6d). Previous work suggested that the lipid monolayer may be leaky when the BChl c pigments within the chlorosome body were oxidized in the presence of strong oxidants.30 Experimental data suggested that only those chlorosomes closely associated with the working electrode contributed to the photoinduced current in the electrochemical system. Utilizing the charge storage density metric (also known as charge capacity or charge injection capacity) made it possible to compare the heterogeneous biohybrid interface across a variety of electrochemical systems. Systems used in this study (chlorosomes in bacterial fragments, colloidal solutions, and adsorbed systems) all increased the charge stored near the electrode when photostimulated. Calculated as an area under the light-stimulated portion of the amperometric curve (Figure 6c), the actual charge accumulated during photostimulation (∼2.1 10-7 coulombs) was approximately half of that of the EC with isolated chlorosomes in solution (∼5.5 10-7 coulombs, Figure 4e). This indicated that photoinduced charge transfer for isolated chlorosomes in the bulk solution originated largely from those chlorosomes directly adsorbed to the electrode and those weakly associated in the thin double layer surrounding the electrode. The process of rinsing may have removed very weakly associated chlorosomes. This is the first report to date showing photocurrent activity directly emanating from isolated chlorosomes in an electrochemical environment. While previous work showed a homogeneous energy transfer mechanism where photonic energy is absorbed and emitted as fluorescence energy, here a heterogeneous photoelectrochemical energy transfer mechanism is demonstrated. (44) Schimdt, K. A.; Trissl, H.-W. Photosynth. Res. 1998, 58, 43–55. (45) Mulkidjanian, A.; Venturoli, G.; Hochkoeppler, A.; Zannoni, D.; Melandri, B. A.; Drachev, L. Photosynth. Res. 1994, 41, 135–143. (46) Mulkidjanian, A. Y.; Hochkoeppler, A.; Zannoni, D.; Drachev, L. A.; Melandri, B. A.; Venturoli, G. Photosynth. Res. 1998, 56, 75–82.
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In addition, although the ground redox state of the pigments of immobilized chlorosomes in indium tin oxide (ITO) based ECs previously were shown to modulate the inherent ITO electrode photocurrent activity in an electrochemical environment, direct photoinduced electrochemical activity due to chlorosomes was not observed.30 Here, chlorosomes with reduced BChl c pigments directly contribute to the photocurrent and photoactivity of the electrochemical cells. Experimental data showed that chlorosomes photoinduced ∼8-10 nA or ∼5-6 1010 electrons. FESEM images suggested that an incomplete chlorosome monolayer (∼10 chlorosomes/ μm2) were generated, implying a yield of ∼75 photoinduced electron transfer events per chlorosome may have been attained. However, if every pigment molecule in the chlorosome contributed, a complete chlorosome monolayer in this system would generate a maximal estimate of ∼96 000-210 000 electrons per chlorosome or ∼300-700 mA. This calculation was based on the reported estimate of ∼9600-21 000 Bchl-c molecules per chlorosome47 and assuming that a bacteriochlorophyll molecule absorbs similarly to a chlorophyll molecule (∼10 photons/s). Optimization strategies currently being pursued include maximization of electrode surface area, increased chlorosome particle loading on the electrode via controlled adsorption methods and amplification of the overall photocurrent using effective redox couples.
Future Perspectives Demonstration of bioelectronic energy transfer from chlorosomes may facilitate innovations in renewable bioenergy applications and wavelength-selective laboratory-on-a-chip biotechnologies that may utilize ultra-small-scale biohybrid chlorosome-based detectors. For instance, a large dynamic range from dark to near sunlight levels as shown for chlorosomes would be beneficial to solar cell technologies. The nanoscale particle size of chlorosome could improve resolution and color discrimination in emerging retinal prosthetic devices.48 Alternatively, selective electroporation of cells using microelectrode arrays as previously demonstrated49,50 with chlorosome derivatives may allow therapeutic regeneration of retinal cells for retinopathy or color blindness. Therefore, the non-peptide dependent chlorosome light antenna structure may represent a unique biomimetic solution to package stable pigment complexes for multiple applications. Acknowledgment. We acknowledge the National Science Foundation (NSF) funded Integrated Graduate Education and Research Training (IGERT) grant in Optical Biomolecular Devices: From Natural Paradigms to Practical Applications (NSF0114434) for generous graduate support (PI: Dr. Neal Woodbury, Department of Chemistry & Biochemistry, Biodesign Insitute, ASU). This study was also partly funded by the Graduate and Professional Students Association (GPSA) dissertation grant (2005-2006) and the generous support of the Faculty Emerti Association at Arizona State University (ASU). We thank Dr. David Lowry in the SOLs EM Core Laboratories at ASU for his help in obtaining images of the chlorosomes using FESEM. (47) Saga, Y.; Shibata, Y.; Itoh, S.; Tamiaki, H. J. Phys. Chem B 2007, 111, 12605–12609. (48) Lakhanpal, R. R.; Yanai, D.; Weiland, J. D.; Fujii, G. Y.; Caffey, S.; Greenberg, R. J.; Eugene de Juan, J.; Humayun, M. S. Curr. Opin. Ophthalmol. 2003, 14, 122–127. (49) Jain, T.; Muthuswamy, J. Lab Chip 2007, 7, 1004–1011. (50) Jain, T.; Muthuswamy, J. IEEE Trans. Biomed. Eng. 2008, 55, 827–832.
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