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Electrostatic adsorption of a fluorophores-modified light-harvesting complex II on TiO2 photoanodes enhances photovoltaic performance
Journal of Power Sources 449 (2020) 227604
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Journal of Power Sources journal homepage: www.elsevier.com/locate/jpowsour
Electrostatic adsorption of a fluorophores-modified light-harvesting complex II on TiO2 photoanodes enhances photovoltaic performance Fei Li a, b, Ling Li a, b, Lishuan Wu a, b, Lin Zhang a, b, Linzhi Zhang a, b, Chunhong Yang a, b, Cheng Liu a, b, * a b
Key Laboratory of Plant Resources/Beijing Botanical Garden, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China University of Chinese Academy of Sciences, Beijing 100049, China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
� A recombinant light-harvesting complex II (rLHCII) modified by Atto 590 was prepared. � The light harvesting capacity of rLHCII is enhanced by bound Atto 590. � Atto 590 modified rLHCII exhibits a good performance in dye sensitized solar cells.
A R T I C L E I N F O
A B S T R A C T
Keywords: Sensitized solar cells Solar energy conversion Recombinant LHCII Bio-organic hybrids Biophotovoltaic cells
The major light-harvesting complex of photosystem II, or LHCII, has been utilized in photovoltaic applications because of its high pigment density. In vitro assembled recombinant LHCII is a modifiable biomimetic material for solar energy conversion. In this report, we assemble a modified recombinant LHCII from apoproteins covalently conjugated artificial fluorophores Atto 590 which absorb greatly near visible green region, where LHCII pos sesses relatively weak absorption. The absorption and fluorescence excitation spectra indicate that the modified recombinant LHCII possesses enhanced light harvesting capacity because Atto 590 molecules efficiently transfer energy to LHCII. The unmodified or modified recombinant LHCII is then adsorbed on the TiO2 electrode respectively to construct sensitized solar cells, both of which present remarkable photovoltaic enhancement. The incident photon-to-electron conversion efficiency measurements confirm the contribution of the artificial fluo rophores. The modified recombinant LHCII sensitized solar cell presents 9.1% increase in open circuit voltage and 13.6% increase in short-circuit current density, compared to the unmodified one. These results suggest that modifications of the recombinant LHCII are feasible ways to enhance its performance in biophotovoltaic cells.
1. Introduction Dye sensitized solar cells (DSSCs) have received considerable
attention due to its advantages of flexibility, low-cost, easy-fabrication [1–3]. The sensitizers, the core part of DSSCs, are responsible for light absorption and photocurrent generation, but most widely used
* Corresponding author. Key Laboratory of Plant Resources/Beijing Botanical Garden, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China E-mail address: [email protected] (C. Liu). https://doi.org/10.1016/j.jpowsour.2019.227604 Received 27 May 2019; Received in revised form 2 December 2019; Accepted 10 December 2019 Available online 14 December 2019 0378-7753/© 2019 Elsevier B.V. All rights reserved.
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Journal of Power Sources 449 (2020) 227604
sensitzers are synthetic and toxic, hindering the application of the DSSCs [4]. Recently, many attempts have utilized isolated and purified natural photosynthetic proteins as non-toxic sensitizers in DSSCs [5–9]. The distinct light-harvesting complexes, evolved to cope with the unstable and sometimes very limited light condition, have been considered as a potential pool of natural sensitizers because of their high light-harvesting capacity and environment-friendly nature. This di versity has been utilized in DSSCs when Light-harvesting 2 and 4 an tenna complexes from purple bacteria [10], Fucoxanthin-chlorophyll complexes (FCP) in diatoms [11] and phycobiliproteins of cyanobacteria [12,13] are utilized as sentitizers. The major light-harvesting complexes of photosystem II or LHCII, as the most abundant membrane protein on the thylakoid membranes, is the most potential candidate because of its high chlorophyll (Chl) density [14,15] and eighteen stromal-facing anionic residues (ten aspartic acids and eight glutamic acids) in favor of adsorption on the 3-aminopropyltriethoxysilane (APTES) modified TiO2 electrode surface [16]. It has been found that the modification or heterophase of TiO2 can effectively inhibit the recombination of photo-induced carriers at the interface and improve the photocatalytic activity [17–19]. Although isolated native LHCII has been utilized in dye-sensitized or organic solar cells [20–22], it is generally recognized that the absorption of LHCII needs improvement because LHCII absorbs mostly in visible blue and red regions. With the aim of enhancing LHCII’s absorption, researchers have successfully refolded recombinant LHCII (rLHCII) from apoproteins linked with exogenous chromophores [23–25], However, until now, there is no report about utilizing rLHCII or its modified forms as biological components assembled into bio photovoltaic cells. In this study, we built a bio-organic hybrid light-harvesting complex rLA590, in which rLHCII covalently conjugates and functionally co ordinates with artificial fluorophores Atto 590 (A590) to increase the absorption range from 520 nm to 630 nm. Furthermore, the rLHCII and rLA590 sensitized solar cells (rLSSC and rLASSC 590) were constructed respectively. The open voltage, short current density and photoelectric conversion efficiency of the recombinant LHCII sensitized solar cell is 0.571 V, 0.627 mA cm 2 and 0.202%. The photovoltaic enhancement of the modified recombinant LHCII sensitized solar cell, compared to the unmodified one, showing great potential of using modified biological components to enhance the photovoltaic performance for a wide range of biohybrid systems.
High Performance Liquid Chromatography (HPLC) (Waters, US) as described [30]. Quantitation of chlorophylls (Chls) and carotenoids (Cars) was performed according to Paulsen et al. [26].
2. Experimental section
2.7. Characterization of rLHCII and rLA590-sensitized solar cells
2.1. Preparation of bio-organic hybrid light-harvesting complexes
Photovoltaic J-V curves and synchronous photocurrent response of the rLHCII and rLA590 sensitized solar cells were performed under the irradiation of one Sun power (100 mW cm 2) using Zahner Cimps pcs (Zahner, Germany). The incident photon-to-electron conversion effi ciency (IPCE) was measured on a Newport QE/IPCE measurement sys tem QTest Station 1000AD (Crowntech, US).
2.4. Spectroscopic measurements The room-temperature absorption spectra (10 μg Chl ml 1) were recorded by Shimadzu UV-VIS 2550 spectrophotometer (Shimadzu, Japan), the fluorescence emission spectra (1 μg Chl ml 1) were recorded by Hitachi F-7000 spectro-fluorometer (Hitachi, Japan). The excitation wavelengths were 436 nm,480 nm and 600 nm. 2.5. Modification of FTO-TiO2 The designed FTO-TiO2 electrodes coated with TiO2 were fabricated by HeptaChroma SolarTech contain two layers, the first layer printed on the conductive fluorine-doped tin oxide (FTO) glass is with 20 nm par ticle size of TiO2 for 2 μm thick, and the second layer is printed with 200 nm particle size of TiO2 for 2 μm thick. The obtained FTO-TiO2 elec trodes were soaked in 10% APTES (Sigma, US) in toluene and reacted at room temperature overnight, followed by baking at 120 � C overnight [31]. The APTES modified FTO-TiO2 electrodes, APTES-TiO2, were used as the photoanodes. 2.6. Fabrication and Characterization of light-harvesting complex attachment The APTES-TiO2 electrodes were incubated in a solution containing rLHCII and rLA590 respectively in dark at 4 � C for 18 h followed by rinsing with dilution buffer (0.5 mM Tricine-NaOH pH 8.0, 0.1 M su crose, 0.1% n-Dodecyl β-D-maltoside). The active area of the cell is 0.25 cm2. The electrolyte consisted of 0.5 M Lithium iodide, 0.05 M Iodine, 0.3 M 1,2-Dimethyl-3-propylimidazolium Iodide, 0.5 M tetr-butylpyr idine and 0.1 M guanidinium thiocyanate in acetonitrile. The attach ment efficiency (ƞattach) of rLHCII and rLA590 onto the APTES-TiO2 photoanode was calculated according to the equation ƞattach ¼ [1- (Cafter/ Cbefore)] � 100% [31], where Cbefore and Cafter stand for the Chl concen tration of the rLHCII and rLA590 solution before and after incubation of APTES-TiO2 photoanode. Total Chl concentrations (Cchl)were examined according to Porra et al. [32].
The LHCII apoproteins were heterogeneously expressed and purified according to the previous protocol [26]. Atto 590 NHS ester (Sigma, USA) was added to LHCII apoproteins dissolved in reaction buffer (0.1 M sodium bicarbonate, pH 8.3, 4% lithium dodecyl sulfate (LDS)) at a ratio of LHCII apoprotein/Atto590 ¼ 1/3 (mol/mol) and incubate overnight at 4 � C in dark. The LHCII-A590 conjugates were precipitated by adding 3 volume acetone and washed with 70% ethanol. Reconstitution of rLHCII and rLA590 was performed as a previous protocol [27].
3. Results and discussion 3.1. Conjugation of A590 molecules to LHCII apoproteins
2.2. MALDI-TOF-MS analysis
Isolated native LHCII has been applied as functional components in several photovoltaic device prototypes [16,20,33,34]. Based on the fact that heterogeneously expressed LHCII apoproteins and isolated pig ments can assemble into recombinant LHCII in vitro [26], exogenous pigments can be linked to apoproteins of the recombinant LHCII to enhance its absorption characters [23,24,35]. In this study, by amide crosslinking reaction, the succinimidyl ester group of A590 (Fig. S1B) reacts with the primary amines (-NH2) group of lysine (Lys or K) residues which exist at eleven sites in LHCII apoprotein. The conjugated A590 molecules are quite stable and can make the protein band visualized without any staining process in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. S1A). The position of
LHCII-A590 conjugates were subjected to chymotrypsin (Promega, US) digestion according to the filter-aided sample preparation method [28], Mass spectra were recorded with UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonic, Germany). Determination of cross-linking sites was analyzed by bioinformatics tools ExPASy in the website. 2.3. Pigment analysis The pigments were extracted with 2-butanol [29] and analyzed by 2
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Fig. 1. Absorption spectra (Abs.) of rLA590 (solid red), rLHCII (solid black) and A590 (solid blue), and emission spectrum (Em.) of A590, dissolved in dilution buffer (dashed pink). Absorption spectra of rLA590 and rLHCII are normalized at maximum in Qy. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Photovoltaic J-V curves of rLASSC 590 (blue line) and rLSSC (pink line) under 1 sun illumination, APTES–TiO2 solar cell used as bare control (green line). FF: fill factor, η: power conversion efficiency. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
spectrum (Fig. 2). The (rLA590F600 – 0.03 � rLA590F600 – rLHCIIF600)/ (rLA590A600 – rLHCIIA600) ratio indicates that the energy transfers from A590 to Chl a with an efficiency of 42%. rLA590F600 and rLHCIIF600 represent the value of normalized 680 nm fluorescence of rLA590 and rLHCII respectively when excited at 600 nm, rLA590A600 and rLHCIIA600 represent the value of normalized absorption of rLA590 and rLHCII at 600 nm respectively, “0.03” is the proportion of autofluorescence of A590 at 680 nm in total fluorescence of rLA590 excited at 600 nm (Fig. S4). It is worth noting that the native energy transfer from Chl b to Chl a, and transfer between Chl a molecules are not affected by the introduction of the exogenous molecules, revealed by fluorescence emission spectra excited at 436 nm or 480 nm (Fig. S5). The distances between the conjugated A590 and their nearest chls are depicted as Fig. S3(B, C and D), are much shorter than the critical F€ orster distance for transferring energy to Chl a or b (4.7 nm or 4.6 nm respectively) [23]. Therefore it suggests that A590 molecules transfer energy to nearby Chl €rster mechanism. Our data cannot distinguish the direct a or b by Fo pathway from A590 to Chl a from the indirect one through Chl b, because the energy transfer from Chl b to Chl a is too fast [37].
Fig. 2. Normalized excitation fluorescence spectra of rLA590 (solid red) and rLHCII (solid blue) monitored at 680 nm compared with normalized absorbance spectra of rLA590 (solid orange) and rLHCII (solid green). These spectra are normalized at the maximum in Qy region. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3. Adsorption on APTES-TiO2 electrodes
rLA590 band is a little higher than rLHCII shown by coomassie brilliant blue (CBB) staining. MALDI-TOF-MS analyses indicate that three Lys residues (K92, K100 and K183) were found to be modified by A590 (Fig. S2). The positions of the Lys residues cross-linked with A590 were depicted in Fig. S3A. Other Lys residues might be covered by LDS or in an unavailable conformation because almost half helix of LHCII exists even solubilized by LDS molecules, which cover the hydrophobic region of apoproteins [36].
Now that rLA590 possess improved absorption compared to rLHCII, it is very interesting to study its performance in a photovoltaic cell. Since it is the first try to utilize rLHCII and modified one in biophotovoltaic devices, we screened the buffer pH and incubation time of adsorption conditions only for rLHCII and the best adsorption occurred when the buffer pH is 8.0 (Fig. S6B) and incubation time is longer than 18 h (Fig. S6A). The adsorption efficiency is about 35% by the calculation method described according to Yang et al. [31] (Fig. S7). The surface of FTO-TiO2 electrode is modified with APTES, which can increase the electrostatic adsorption of rLHCII [16]. We suggest that the small size of rLHCII monomers might also promote adsorption because of the less steric hindrance than trimers in previous reports [16,20,22,31,33].
3.2. Spectroscopic characterization of rLA590 The pigment stochiometry analysis shows that both the chlorophyll and carotenoid content are basically same between rLA590 and rLHCII (Table S1). The absorption spectra, shown in Fig. 1, indicate that the A590 molecules dissolved in dilution buffer absorb most at 600 nm with a shoulder at 555 nm. The emission spectrum of A590 has a major peak at 624 nm. The absorption from 525 nm to 630 nm of rLA590 is increased significantly, with a peak at 598 nm. A significant spectral overlap exists between the emission spectrum of A590 and the absorp tion spectrum of Chl a and b (Fig. 1). To investigate the energy transfer efficiency in the rLA590 complex, the normalized excitation spectra emitting at 680 nm of rLA590 and rLHCII was measured and compared with their normalized absorbance
3.4. Characterization of rLHCII and rLA590 sensitized solar cells Under the optimum condition of incubation, rLHCII and rLA590 were adopted on the TiO2 nanostructure as photoanode to fabricate biophotovoltaic devices, namely rLSSC and rLASSC 590. To improve the electron transfer efficiency, 20 mM anthraquinone (AQ) was used as an electron shuttling mediator (Fig. S8) [38,39]. Fig. 3 displays the current density to photovoltage (J-V) curves, in which the rLSSC presents an 3
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Journal of Power Sources 449 (2020) 227604
Fig. 6. The performance characteristics of rLASSC 590 measured over 24 days. Values are the mean � standard deviation of three replicates. Different letters above the bars in the same color indicate significant differences (P<0.05) revealed by the Student’s t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Normalized photocurrent density of the rLASSC 590 and rLSSC under irradiation of 100 mW cm 2 “On” and “Off” refer to illuminated and dark conditions, respectively.
Besides, the IPCE spectrum from 600 nm to 685 nm is obviously enhanced in rLASSC 590 compared to rLSSC. It indicates that the ab sorption of A590 directly contributes to the photocurrent, meanwhile, it increases the light-to-charge conversion in a broad region including the absorption maxium of A590. Similar phenomena were reported when A590 molecules were covalently bound to PSI, where almost 4 times improved functionality were observed under realistic band illumination, which is far broader than the absorption peak of A590 [40]. The mechanisms of this broad region enhancement should be studied in the future. To demonstrate the robustness of rLASSC 590, we detect the photovoltaic parameters of rLASSC 590 over 24 days under open-air conditions at 4 � C. Both Jsc and η gradually decrease while the Voc and fill factor are relatively stable during the period (Fig. 6). This means that the vulnerable conductivity between hybrid material and electrode is the major limitation for the stability of these solar cells which should be focused on in the following studies. However, the rLASSC 590 still retain more than 45% in η and more than 60% in Jsc after 24 days.
Fig. 5. The IPCE spectra of rLASSC 590 (solid green) and rLSSC (solid pink). The normalized absorption spectra (Abs.) of rLHCII (dashed yellow) and rLA590 (dashed blue). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4. Conclusions
open-circuit voltage (Voc) 0.571 V and short-circuit photocurrent den sity (Jsc) 0.627 mA cm 2 compared to 0.381 V and 0.448 mA cm 2 of bare APTES-TiO2 photoanode, suggesting that rLHCII, which is easily accessible and modifiable, can achieve at least similar performance as native LHCII trimers (Table S2). The rLASSC 590 can generate an even higher Voc (0.623 V) and Jsc (0.712 mA cm 2), evidencing the hypoth esis that using modified pigment-binding complex like rLA590 can improve the performance of biophotovoltaic cells. In previous studies, we found that when a photoelectrochemical cell was fabricated with FCP, the major photosynthetic light-harvesting antenna in diatoms, very high Voc up to 0.72 V can be achieved [11]. The interactions between fucoxanthins and TiO2 photoanodes were considered very important for the formation of the charge transfer interface [11]. To identify the origin of the photocurrent generation, the synchronous response of Jsc to the illumination was recorded and illustrated in Fig. 4. All photocurrents are normalized to the average photocurrent density of the solar cell with bare APTES-TiO2 photoanode in order to correct the variation of TiO2 photoanode of the original tests (Fig. S9) [31]. The rLASSC 590 produce significantly higher stable current density than rLSSC. In both solar cells, only a very slow decline was observed after four cycles of illumination (Fig. S9). The IPCE spectra reveal the contribution of light-excited molecules in the cells. The spectra of visible region of both rLHCII- and rLA590sensitized solar cells have peaks at 450 nm in the Soret region and 643, 665 nm in the Qy region, where rLHCII absorbs most (Fig. 5).
Recombinant LHCII possesses similar pigment network as native LHCII, and it can also be subjected to designed modifications. In this study, we first present the construction of a hybrid material in which rLHCII covalently conjugates and functionally coordinates with artificial fluorophores Atto 590. The enhanced light-harvesting capacity of the material is evidenced. When the hybrid material is integrated into the sensitized biophotovoltaic cells, higher Voc, Jsc and η are presented. IPCE measurement indicates that exogenous fluorophores significantly contribute to the photovoltaic enhancement. It is the first attempt to incorporate rLHCII and its modified forms into photovoltaic researches and we demonstrate that modifications of rLHCII can be utilized to improve the performance of the biophotovoltaic cells. Acknowledgments This work was funded by the National Key R&D Program of China (Grant No. 2017YFA0503701), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB17030000), the National Natural Science Foundation of China (Grant No. 31370275, 31570236). We thank Tianning Zhang from Shanghai Institute of Technical Physics, CAS, for the assistance of IPCE measurement.
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Appendix A. Supplementary data
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Journal of Power Sources journal homepage: www.elsevier.com/locate/jpowsour
Electrostatic adsorption of a fluorophores-modified light-harvesting complex II on TiO2 photoanodes enhances photovoltaic performance Fei Li a, b, Ling Li a, b, Lishuan Wu a, b, Lin Zhang a, b, Linzhi Zhang a, b, Chunhong Yang a, b, Cheng Liu a, b, * a b
Key Laboratory of Plant Resources/Beijing Botanical Garden, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China University of Chinese Academy of Sciences, Beijing 100049, China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
� A recombinant light-harvesting complex II (rLHCII) modified by Atto 590 was prepared. � The light harvesting capacity of rLHCII is enhanced by bound Atto 590. � Atto 590 modified rLHCII exhibits a good performance in dye sensitized solar cells.
A R T I C L E I N F O
A B S T R A C T
Keywords: Sensitized solar cells Solar energy conversion Recombinant LHCII Bio-organic hybrids Biophotovoltaic cells
The major light-harvesting complex of photosystem II, or LHCII, has been utilized in photovoltaic applications because of its high pigment density. In vitro assembled recombinant LHCII is a modifiable biomimetic material for solar energy conversion. In this report, we assemble a modified recombinant LHCII from apoproteins covalently conjugated artificial fluorophores Atto 590 which absorb greatly near visible green region, where LHCII pos sesses relatively weak absorption. The absorption and fluorescence excitation spectra indicate that the modified recombinant LHCII possesses enhanced light harvesting capacity because Atto 590 molecules efficiently transfer energy to LHCII. The unmodified or modified recombinant LHCII is then adsorbed on the TiO2 electrode respectively to construct sensitized solar cells, both of which present remarkable photovoltaic enhancement. The incident photon-to-electron conversion efficiency measurements confirm the contribution of the artificial fluo rophores. The modified recombinant LHCII sensitized solar cell presents 9.1% increase in open circuit voltage and 13.6% increase in short-circuit current density, compared to the unmodified one. These results suggest that modifications of the recombinant LHCII are feasible ways to enhance its performance in biophotovoltaic cells.
1. Introduction Dye sensitized solar cells (DSSCs) have received considerable
attention due to its advantages of flexibility, low-cost, easy-fabrication [1–3]. The sensitizers, the core part of DSSCs, are responsible for light absorption and photocurrent generation, but most widely used
* Corresponding author. Key Laboratory of Plant Resources/Beijing Botanical Garden, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China E-mail address: [email protected] (C. Liu). https://doi.org/10.1016/j.jpowsour.2019.227604 Received 27 May 2019; Received in revised form 2 December 2019; Accepted 10 December 2019 Available online 14 December 2019 0378-7753/© 2019 Elsevier B.V. All rights reserved.
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sensitzers are synthetic and toxic, hindering the application of the DSSCs [4]. Recently, many attempts have utilized isolated and purified natural photosynthetic proteins as non-toxic sensitizers in DSSCs [5–9]. The distinct light-harvesting complexes, evolved to cope with the unstable and sometimes very limited light condition, have been considered as a potential pool of natural sensitizers because of their high light-harvesting capacity and environment-friendly nature. This di versity has been utilized in DSSCs when Light-harvesting 2 and 4 an tenna complexes from purple bacteria [10], Fucoxanthin-chlorophyll complexes (FCP) in diatoms [11] and phycobiliproteins of cyanobacteria [12,13] are utilized as sentitizers. The major light-harvesting complexes of photosystem II or LHCII, as the most abundant membrane protein on the thylakoid membranes, is the most potential candidate because of its high chlorophyll (Chl) density [14,15] and eighteen stromal-facing anionic residues (ten aspartic acids and eight glutamic acids) in favor of adsorption on the 3-aminopropyltriethoxysilane (APTES) modified TiO2 electrode surface [16]. It has been found that the modification or heterophase of TiO2 can effectively inhibit the recombination of photo-induced carriers at the interface and improve the photocatalytic activity [17–19]. Although isolated native LHCII has been utilized in dye-sensitized or organic solar cells [20–22], it is generally recognized that the absorption of LHCII needs improvement because LHCII absorbs mostly in visible blue and red regions. With the aim of enhancing LHCII’s absorption, researchers have successfully refolded recombinant LHCII (rLHCII) from apoproteins linked with exogenous chromophores [23–25], However, until now, there is no report about utilizing rLHCII or its modified forms as biological components assembled into bio photovoltaic cells. In this study, we built a bio-organic hybrid light-harvesting complex rLA590, in which rLHCII covalently conjugates and functionally co ordinates with artificial fluorophores Atto 590 (A590) to increase the absorption range from 520 nm to 630 nm. Furthermore, the rLHCII and rLA590 sensitized solar cells (rLSSC and rLASSC 590) were constructed respectively. The open voltage, short current density and photoelectric conversion efficiency of the recombinant LHCII sensitized solar cell is 0.571 V, 0.627 mA cm 2 and 0.202%. The photovoltaic enhancement of the modified recombinant LHCII sensitized solar cell, compared to the unmodified one, showing great potential of using modified biological components to enhance the photovoltaic performance for a wide range of biohybrid systems.
High Performance Liquid Chromatography (HPLC) (Waters, US) as described [30]. Quantitation of chlorophylls (Chls) and carotenoids (Cars) was performed according to Paulsen et al. [26].
2. Experimental section
2.7. Characterization of rLHCII and rLA590-sensitized solar cells
2.1. Preparation of bio-organic hybrid light-harvesting complexes
Photovoltaic J-V curves and synchronous photocurrent response of the rLHCII and rLA590 sensitized solar cells were performed under the irradiation of one Sun power (100 mW cm 2) using Zahner Cimps pcs (Zahner, Germany). The incident photon-to-electron conversion effi ciency (IPCE) was measured on a Newport QE/IPCE measurement sys tem QTest Station 1000AD (Crowntech, US).
2.4. Spectroscopic measurements The room-temperature absorption spectra (10 μg Chl ml 1) were recorded by Shimadzu UV-VIS 2550 spectrophotometer (Shimadzu, Japan), the fluorescence emission spectra (1 μg Chl ml 1) were recorded by Hitachi F-7000 spectro-fluorometer (Hitachi, Japan). The excitation wavelengths were 436 nm,480 nm and 600 nm. 2.5. Modification of FTO-TiO2 The designed FTO-TiO2 electrodes coated with TiO2 were fabricated by HeptaChroma SolarTech contain two layers, the first layer printed on the conductive fluorine-doped tin oxide (FTO) glass is with 20 nm par ticle size of TiO2 for 2 μm thick, and the second layer is printed with 200 nm particle size of TiO2 for 2 μm thick. The obtained FTO-TiO2 elec trodes were soaked in 10% APTES (Sigma, US) in toluene and reacted at room temperature overnight, followed by baking at 120 � C overnight [31]. The APTES modified FTO-TiO2 electrodes, APTES-TiO2, were used as the photoanodes. 2.6. Fabrication and Characterization of light-harvesting complex attachment The APTES-TiO2 electrodes were incubated in a solution containing rLHCII and rLA590 respectively in dark at 4 � C for 18 h followed by rinsing with dilution buffer (0.5 mM Tricine-NaOH pH 8.0, 0.1 M su crose, 0.1% n-Dodecyl β-D-maltoside). The active area of the cell is 0.25 cm2. The electrolyte consisted of 0.5 M Lithium iodide, 0.05 M Iodine, 0.3 M 1,2-Dimethyl-3-propylimidazolium Iodide, 0.5 M tetr-butylpyr idine and 0.1 M guanidinium thiocyanate in acetonitrile. The attach ment efficiency (ƞattach) of rLHCII and rLA590 onto the APTES-TiO2 photoanode was calculated according to the equation ƞattach ¼ [1- (Cafter/ Cbefore)] � 100% [31], where Cbefore and Cafter stand for the Chl concen tration of the rLHCII and rLA590 solution before and after incubation of APTES-TiO2 photoanode. Total Chl concentrations (Cchl)were examined according to Porra et al. [32].
The LHCII apoproteins were heterogeneously expressed and purified according to the previous protocol [26]. Atto 590 NHS ester (Sigma, USA) was added to LHCII apoproteins dissolved in reaction buffer (0.1 M sodium bicarbonate, pH 8.3, 4% lithium dodecyl sulfate (LDS)) at a ratio of LHCII apoprotein/Atto590 ¼ 1/3 (mol/mol) and incubate overnight at 4 � C in dark. The LHCII-A590 conjugates were precipitated by adding 3 volume acetone and washed with 70% ethanol. Reconstitution of rLHCII and rLA590 was performed as a previous protocol [27].
3. Results and discussion 3.1. Conjugation of A590 molecules to LHCII apoproteins
2.2. MALDI-TOF-MS analysis
Isolated native LHCII has been applied as functional components in several photovoltaic device prototypes [16,20,33,34]. Based on the fact that heterogeneously expressed LHCII apoproteins and isolated pig ments can assemble into recombinant LHCII in vitro [26], exogenous pigments can be linked to apoproteins of the recombinant LHCII to enhance its absorption characters [23,24,35]. In this study, by amide crosslinking reaction, the succinimidyl ester group of A590 (Fig. S1B) reacts with the primary amines (-NH2) group of lysine (Lys or K) residues which exist at eleven sites in LHCII apoprotein. The conjugated A590 molecules are quite stable and can make the protein band visualized without any staining process in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. S1A). The position of
LHCII-A590 conjugates were subjected to chymotrypsin (Promega, US) digestion according to the filter-aided sample preparation method [28], Mass spectra were recorded with UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonic, Germany). Determination of cross-linking sites was analyzed by bioinformatics tools ExPASy in the website. 2.3. Pigment analysis The pigments were extracted with 2-butanol [29] and analyzed by 2
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Fig. 1. Absorption spectra (Abs.) of rLA590 (solid red), rLHCII (solid black) and A590 (solid blue), and emission spectrum (Em.) of A590, dissolved in dilution buffer (dashed pink). Absorption spectra of rLA590 and rLHCII are normalized at maximum in Qy. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Photovoltaic J-V curves of rLASSC 590 (blue line) and rLSSC (pink line) under 1 sun illumination, APTES–TiO2 solar cell used as bare control (green line). FF: fill factor, η: power conversion efficiency. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
spectrum (Fig. 2). The (rLA590F600 – 0.03 � rLA590F600 – rLHCIIF600)/ (rLA590A600 – rLHCIIA600) ratio indicates that the energy transfers from A590 to Chl a with an efficiency of 42%. rLA590F600 and rLHCIIF600 represent the value of normalized 680 nm fluorescence of rLA590 and rLHCII respectively when excited at 600 nm, rLA590A600 and rLHCIIA600 represent the value of normalized absorption of rLA590 and rLHCII at 600 nm respectively, “0.03” is the proportion of autofluorescence of A590 at 680 nm in total fluorescence of rLA590 excited at 600 nm (Fig. S4). It is worth noting that the native energy transfer from Chl b to Chl a, and transfer between Chl a molecules are not affected by the introduction of the exogenous molecules, revealed by fluorescence emission spectra excited at 436 nm or 480 nm (Fig. S5). The distances between the conjugated A590 and their nearest chls are depicted as Fig. S3(B, C and D), are much shorter than the critical F€ orster distance for transferring energy to Chl a or b (4.7 nm or 4.6 nm respectively) [23]. Therefore it suggests that A590 molecules transfer energy to nearby Chl €rster mechanism. Our data cannot distinguish the direct a or b by Fo pathway from A590 to Chl a from the indirect one through Chl b, because the energy transfer from Chl b to Chl a is too fast [37].
Fig. 2. Normalized excitation fluorescence spectra of rLA590 (solid red) and rLHCII (solid blue) monitored at 680 nm compared with normalized absorbance spectra of rLA590 (solid orange) and rLHCII (solid green). These spectra are normalized at the maximum in Qy region. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3. Adsorption on APTES-TiO2 electrodes
rLA590 band is a little higher than rLHCII shown by coomassie brilliant blue (CBB) staining. MALDI-TOF-MS analyses indicate that three Lys residues (K92, K100 and K183) were found to be modified by A590 (Fig. S2). The positions of the Lys residues cross-linked with A590 were depicted in Fig. S3A. Other Lys residues might be covered by LDS or in an unavailable conformation because almost half helix of LHCII exists even solubilized by LDS molecules, which cover the hydrophobic region of apoproteins [36].
Now that rLA590 possess improved absorption compared to rLHCII, it is very interesting to study its performance in a photovoltaic cell. Since it is the first try to utilize rLHCII and modified one in biophotovoltaic devices, we screened the buffer pH and incubation time of adsorption conditions only for rLHCII and the best adsorption occurred when the buffer pH is 8.0 (Fig. S6B) and incubation time is longer than 18 h (Fig. S6A). The adsorption efficiency is about 35% by the calculation method described according to Yang et al. [31] (Fig. S7). The surface of FTO-TiO2 electrode is modified with APTES, which can increase the electrostatic adsorption of rLHCII [16]. We suggest that the small size of rLHCII monomers might also promote adsorption because of the less steric hindrance than trimers in previous reports [16,20,22,31,33].
3.2. Spectroscopic characterization of rLA590 The pigment stochiometry analysis shows that both the chlorophyll and carotenoid content are basically same between rLA590 and rLHCII (Table S1). The absorption spectra, shown in Fig. 1, indicate that the A590 molecules dissolved in dilution buffer absorb most at 600 nm with a shoulder at 555 nm. The emission spectrum of A590 has a major peak at 624 nm. The absorption from 525 nm to 630 nm of rLA590 is increased significantly, with a peak at 598 nm. A significant spectral overlap exists between the emission spectrum of A590 and the absorp tion spectrum of Chl a and b (Fig. 1). To investigate the energy transfer efficiency in the rLA590 complex, the normalized excitation spectra emitting at 680 nm of rLA590 and rLHCII was measured and compared with their normalized absorbance
3.4. Characterization of rLHCII and rLA590 sensitized solar cells Under the optimum condition of incubation, rLHCII and rLA590 were adopted on the TiO2 nanostructure as photoanode to fabricate biophotovoltaic devices, namely rLSSC and rLASSC 590. To improve the electron transfer efficiency, 20 mM anthraquinone (AQ) was used as an electron shuttling mediator (Fig. S8) [38,39]. Fig. 3 displays the current density to photovoltage (J-V) curves, in which the rLSSC presents an 3
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Fig. 6. The performance characteristics of rLASSC 590 measured over 24 days. Values are the mean � standard deviation of three replicates. Different letters above the bars in the same color indicate significant differences (P<0.05) revealed by the Student’s t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Normalized photocurrent density of the rLASSC 590 and rLSSC under irradiation of 100 mW cm 2 “On” and “Off” refer to illuminated and dark conditions, respectively.
Besides, the IPCE spectrum from 600 nm to 685 nm is obviously enhanced in rLASSC 590 compared to rLSSC. It indicates that the ab sorption of A590 directly contributes to the photocurrent, meanwhile, it increases the light-to-charge conversion in a broad region including the absorption maxium of A590. Similar phenomena were reported when A590 molecules were covalently bound to PSI, where almost 4 times improved functionality were observed under realistic band illumination, which is far broader than the absorption peak of A590 [40]. The mechanisms of this broad region enhancement should be studied in the future. To demonstrate the robustness of rLASSC 590, we detect the photovoltaic parameters of rLASSC 590 over 24 days under open-air conditions at 4 � C. Both Jsc and η gradually decrease while the Voc and fill factor are relatively stable during the period (Fig. 6). This means that the vulnerable conductivity between hybrid material and electrode is the major limitation for the stability of these solar cells which should be focused on in the following studies. However, the rLASSC 590 still retain more than 45% in η and more than 60% in Jsc after 24 days.
Fig. 5. The IPCE spectra of rLASSC 590 (solid green) and rLSSC (solid pink). The normalized absorption spectra (Abs.) of rLHCII (dashed yellow) and rLA590 (dashed blue). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4. Conclusions
open-circuit voltage (Voc) 0.571 V and short-circuit photocurrent den sity (Jsc) 0.627 mA cm 2 compared to 0.381 V and 0.448 mA cm 2 of bare APTES-TiO2 photoanode, suggesting that rLHCII, which is easily accessible and modifiable, can achieve at least similar performance as native LHCII trimers (Table S2). The rLASSC 590 can generate an even higher Voc (0.623 V) and Jsc (0.712 mA cm 2), evidencing the hypoth esis that using modified pigment-binding complex like rLA590 can improve the performance of biophotovoltaic cells. In previous studies, we found that when a photoelectrochemical cell was fabricated with FCP, the major photosynthetic light-harvesting antenna in diatoms, very high Voc up to 0.72 V can be achieved [11]. The interactions between fucoxanthins and TiO2 photoanodes were considered very important for the formation of the charge transfer interface [11]. To identify the origin of the photocurrent generation, the synchronous response of Jsc to the illumination was recorded and illustrated in Fig. 4. All photocurrents are normalized to the average photocurrent density of the solar cell with bare APTES-TiO2 photoanode in order to correct the variation of TiO2 photoanode of the original tests (Fig. S9) [31]. The rLASSC 590 produce significantly higher stable current density than rLSSC. In both solar cells, only a very slow decline was observed after four cycles of illumination (Fig. S9). The IPCE spectra reveal the contribution of light-excited molecules in the cells. The spectra of visible region of both rLHCII- and rLA590sensitized solar cells have peaks at 450 nm in the Soret region and 643, 665 nm in the Qy region, where rLHCII absorbs most (Fig. 5).
Recombinant LHCII possesses similar pigment network as native LHCII, and it can also be subjected to designed modifications. In this study, we first present the construction of a hybrid material in which rLHCII covalently conjugates and functionally coordinates with artificial fluorophores Atto 590. The enhanced light-harvesting capacity of the material is evidenced. When the hybrid material is integrated into the sensitized biophotovoltaic cells, higher Voc, Jsc and η are presented. IPCE measurement indicates that exogenous fluorophores significantly contribute to the photovoltaic enhancement. It is the first attempt to incorporate rLHCII and its modified forms into photovoltaic researches and we demonstrate that modifications of rLHCII can be utilized to improve the performance of the biophotovoltaic cells. Acknowledgments This work was funded by the National Key R&D Program of China (Grant No. 2017YFA0503701), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB17030000), the National Natural Science Foundation of China (Grant No. 31370275, 31570236). We thank Tianning Zhang from Shanghai Institute of Technical Physics, CAS, for the assistance of IPCE measurement.
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Appendix A. Supplementary data
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