A Graphene Oxide and Polyelectrolyte Composed One-Way

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A Graphene Oxide and Polyelectrolyte Composed One-Way Expressway for Guiding Electron Transfer of Integrated Artificial Photosynthesis Xiaoyuan Ji, Yong Kang, Zhiguo Su, Ping Wang, Guanghui Ma, and Songping Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02902 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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ACS Sustainable Chemistry & Engineering

A Graphene Oxide and Polyelectrolyte Composed One-Way Expressway for Guiding Electron Transfer of Integrated Artificial Photosynthesis Xiaoyuan Jia,b, Yong Kanga,b, Zhiguo Sua, Ping Wanga,c, Guanghui Maa, Songping Zhanga,* a

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

Chinese Academy of Sciences, No.1 Bei-er-jie, Zhong-guan-cun, Beijing 100190, China b

University of Chinese Academy of Sciences, No.19 Yuquan Road, Shijingshan

District, Beijing, 100049, China c

Department of Bioproducts and Biosystems Engineering and Biotechnology Institute

University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108, USA

* Corresponding author at: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, No.1 Bei-er-jie, Zhong-guan-cun, Beijing 100190, China. Tel: +86 10 82544958; fax: +86 10 82544958. E-mail: [email protected]

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ABSTRACT: A novel photocatalyst/biocatalyst integrated artificial photosynthesis system (APS) based on polyurethane hollow nanofibers doped with graphene oxide (GO) and poly(allylamine hydrochloride) (PAH) were developed and employed for selective methanol conversion from CO2. The biocatalysts including formate, formaldehyde, and alcohol dehydrogenases, as well as NAD+ were in situ co-encapsulated inside the lumen of the GO-PAH-doped PU nanofibers (G-Fiber) by simply pre-dissolving them in the core-phase solution for coaxial electrospinning. While the precise assembling of the photocatalyst parts involving visible light active photosensitizer (PS) and electron mediator (M) on the surface of the G-Fiber were realized by their π-π interactions with the GO doped in the shell of fibers. By using this highly integrated APS, about 10-times higher methanol yield was accomplished as compared with the solution-based system. The significantly enhanced reaction efficiency of the G-Fiber-based APS is considered predominately due to the electron transfer “one-way expressway” composed of the doped polyelectrolyte and GO in the G-Fiber, therefore, the electron transfer distance along the PS-M-NAD+ electron transport chain could be shortened and the speed could be accelerated. As a consequence, the electron back-flow between PS and M, as well as the re-combination of excited electron and the hole of PS were eliminated. The current work will represent a new benchmark for solar-energy driven conversion of CO2 to a wide range of fuels and chemicals in an environmentally benign manner. KEYWORDS: Integrated artificial photosynthesis, graphene oxide, coaxial 2


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electrospinning, π-π interaction, methanol formation

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Introduction Graphene (GR), first reported in 2004, offers remarkable electronic, thermal, optical, and mechanical properties.1-2 In virtue of the abundance of delocalized electrons from the conjugated sp2-bonded carbon network, GR has a charge carrier mobility as high as 2.5*105 cm2 V-1 s-1. Such properties are highly desirable for use as a two-dimensional catalyst by enhancing the transport of electrons not only in photronic and photocatalytic systems,3-6 but also in some biological systems.7-8 One of the most interesting applications was the photo-catalyzed regeneration of cofactor NADH under visible light, which is a promising but challenging task in the area of biocatalysis.9-12 A significantly improved transfer of photo-excited electrons from photosensitizer (PS) to electron mediator (M) and consequent NAD+ reduction was observed by incorporating the porphyrin based PS with chemically converted GR.9-10 This observation is of great significance for designing an efficient biocatalytic artificial photosynthesis system (APS). Biocatalytic APS is an artificial process that mimics the natural photosynthesis process by reconstructing man-made PS, M, and dehydrogenases for solar synthesis of high value products with visible light-driven regeneration of cofactors as the most critical step.13-18 The practicality of the photocatalyst-enzyme coupled APS process has several challenges with the biggest challenge being the slow, astatic, and wasteful electron transfer between each of the individual components. One major cause of this problem is the lack of precise arrangement of the active components on a nanoscale. This is also one of the most fascinating aspects of natural photosynthesis.17, 19 GR not only 4


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has remarkable electronic and optical properties to improve electron transfer, but also has notable physical features, such as a two-dimensional monolayer structure and a large specific surface area. GR also served as a scaffold for the synthesis of functional hybrid materials through Van der Waals forces or π-π stacking interactions.11, 20 Compared with GR, the graphene oxide (GO), which could be regarded as GR functionalized by epoxide, hydroxyl, or carboxylic acid groups, is more noteworthy due to its tunable chemical, conductive, and optical properties,21-22 as well as higher photoinduced electron/energy transfer efficiency than GR.3, 23-27 In addition to the Van der Waals forces and π-π stacking interactions, various oxygen-rich functional groups of GO allow assembling of other active cationic components through additional robust electrostatic interactions.28 Therefore, GO could be used as an ideal host material candidate for integrating and arranging active components of APS at the nanoscale.29 In order to realize high integration and precise arrangement of individual compounds of APS with a unprecedented electron transfer and catalytic efficacy, a hybrid hollow nanofibers co-doped with GO and a cationic polyelectrolyte, poly(allylamine hydrochloride) (PAH), was designed and fabricated through co-axial electrospinning. In one of our previous works, the PAH-doped hollow nanofibers have proven to be a perfect platform for spatial assembly of enzymes and even particular small cofactors, such as NAD(H) and organic dyes.19,

30-31

To realize

incorporating of the photo-regeneration of NADH with the cascade enzymatic conversion methanol from CO2, GO-PAH-doped PU hollow nanofibers (G-Fiber) 5



were fabricated through adding GO and PAH into the shell- and core-phase solution of coaxial electrospinning, respectively, as shown in Scheme 1. Meanwhile cofactor and redox enzymes were in situ co-encapsulated in the hollow lumen of G-Fiber via pre-dissolving them in the core-phase solution. Photoactive porphyrin and Eosin Y (EY), which contain many benzene rings (Figure S1), are chosen as PSs and expected to be able to assemble to the surface of G-Fiber by π-π stacking interactions with the GO. Organometallic rhodium compounds containing multi-benzene rings (Figure S1), which are the most widely used electron mediators,32-36 could also assemble onto the surface of the G-Fiber through π-π stacking interactions or electrostatic attraction with GO. The feasibility and advantages of the integrated APS was tested by the solar-energy driven synthesis of methanol from CO2 as a representative system. Results and Discussions Preparation and Characterization of G-Fiber-based APS As illustrated in Scheme 1, G-Fiber were prepared via co-axial electrospinning through adding PAH and GO into core- and shell- phase solution, respectively. The linear PAH penetrated across the shell and distributed along the outer and inner surfaces of the G-Fiber which acted as anchors for NADH retention.30 The GO will distribute along the inner and outer surfaces of the G-Fiber and act as multi-binding sites for M (Mbpy or Mphen) and PS assembling through π-π interactions and versatile electrostatic interactions. The multienzyme system for cascade reduction of CO2 to methanol, which includes FateDH, FaldDH, ADH, and NAD+ were in situ co-immobilized in hollow chamber of G-Fiber by simply dissolving them in the 6


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core-phase solution along with PAH. Under irradiation of visible light, the photo-excited electron can be transferred to the center of M, providing high reducing equivalents for efficient photo-regeneration of NADH. Therefore, the highly integrated biocatalytic APS will enable a continuous synthesis of methanol through coupling of the photo-regeneration of NADH with the CO2 bioconversion process. Figure 1a and S2a present SEM images of the hollow nanofibers with and without GO doping. Due to the GO doping in the shell of hollow nanofibers, the surfaces of G-Fiber were significantly rougher than that of only PAH doped. However, GO did not show a noticeable influence on the diameter of the fibers which had a uniform outer diameter of approximately 800 to 1000 nm and an inner diameter of approximately 500 nm (Figure 1b and S2b). As shown in Figure S3, the color of PAH-doped hollow nanofiber membrane was very white, however, G-Fiber membrane appeared light black, which clearly demonstrated the successful GO doping. Besides, in order to further confirm the doping of GO in G-Fiber, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Raman spectra, and X-ray photoelectron spectroscopy (XPS) were adopted to analyze the composition and structure of the G-Fiber in detail. As Figure S4 shown, compared with PAH-doped hollow nanofiber, there was a characteristic weight loss stage between 200 to 300 oC which was attributed to the decomposition of GO (Figure S4b). The Raman spectra of G-Fiber also showed the characteristic absorption peak of GO at around 800 cm-1. The surface element composition of G-Fiber was also analyzed using XPS. The molecular structures of PU, PAH, and GO were shown in Figure S6. 7



There were C, O, H, and N elements in G-Fiber theoretically, which were consistent with the XPS survey spectra of G-Fiber. Figure S7b and S7c show more detailed high-resolution XPS spectra of C1s peaks in sample PAH-doped hollow nanofibers and G-Fiber. After splitting and fitting of the raw data, the expanded peaks were deconvoluted to show different types of C1s in PAH-doped hollow nanofibers and G-Fiber more clearly. The C1s XPS spectra of PAH-doped hollow nanofibers are composed of four peaks at 288.9, 285, 286.5 and 285.7 eV, which originated from C=O, C-C, C-O, and C-N, respectively. However, for the C1s XPS spectra of G-Fiber, there is another peak at 284.5 eV, which originated from C=C of GO. So the high-resolution XPS spectra of G-Fiber also confirmed the successful doping of GO in G-Fiber. Our previous diffusion tests have demonstrated that the shell of PU hollow nanofibers have a molecular weight cut-off of approximately 20 kDa, ensuring effective retention of neutral or positively charged compounds.37-38 With doping of PAH, the electrostatic interactions between ionizable groups of PAH and negatively charged NAD(H) could retain NAD(H) inside the lumen of G-Fiber.19, 30 Therefore, the redox enzymatic system involving FateDH, FaldDH, ADH, and NAD(H) were co-encapsulated in the lumen of the hollow nanofibers by coaxial electrospinning. The retained and uniform distribution of the redox enzymatic system inside the hollow nanofibers were further confirmed by confocal laser scanning microscope (CLSM) imaging (Figure 1c, d) because of the autofluorescence property of NADH and FITC-labeled FateDH, FaldDH, ADH. Table 1 summarizes the amounts and activities 8


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of each of the encapsulated enzymes. More than 70% of their original activities were retained after co-encapsulation in the lumen of hollow nanofibers. This is mainly due to the mild preparation process and the short diffusional distance across the shell of the nanofibers. Energy dispersive spectroscopy (EDS) was employed to analyze the chemical composition of the nanofibers (Figure 1e, f). The presence of rhodium (Rh) and bromine (Br), which are the characteristic elements in M and PS (EY) respectively, confirmed the successful and uniform assembly of M and EY. Furthermore, the zeta-potential of the G-Fiber was also detected. As Figure 2 shown, the pure PU hollow nanofibers membrane has a zeta-potential of approximately zero because there is no ionizable group of PU. In contrast, for the PAH-doped hollow nanofibers a notable positive zeta-potential (135 mV) was detected. With GO doping, the positive surface zeta-potential decreased to 34 mV due to the negatively charged carboxyl groups of GO. Although the surface of G-Fiber was positively charged, the π-π and π-cation interactions between GO and M drive the assembly of positively charged M onto the surface of GO, leading to an increase in the zeta-potential. When PS, either EY or mTHPP, was further assembled on the surface of G-Fiber through similar π-π interactions and ion-exchange interactions with GO and PAH, the zeta-potential decreases correspondingly. This observation also confirmed the successful assembly of the negatively charged PS. By controlling the adsorption time and the initial concentrations of PS and M (Figure S8), a loading amount about 4 µmol-EY/g-fiber, 4 µmol-mTHPP/g-fiber, 20 µmol-Mbpy/g-fiber, and 20 µmol-Mphen/g-fiber were 9



obtained. NADH Regeneration and Methanol Formation The G-Fiber based integrated APS was applied to the photochemical regeneration of NADH. For comparison，the solution-based system composing of 0.25 mM of Mbpy or Mphen and 0.05 mM of EY or mTHPP, was used as control to investigate the catalytic activity for regeneration of NADH. These values correspond to the amounts that were loaded in the integrated system. According to the results shown in Figure 3, in the dark stage, there was nearly no NADH regenerated from NAD+, whereas rapid NADH regeneration from NAD+ can be seen when the reaction medium was exposed to visible light irradiation. The reaction kinetics of NADH photo-regeneration showed that the EY-Mphen combination exhibited the highest efficiency of over 80% of NAD+ being regenerated into NADH within 3 hours. Meanwhile, the mTHPP-Mphen combination presented the lowest efficiency of only approximately 50%. The difference in photocatalytic activity was mainly attributed to the difference in ability of PS to harvest solar energy in the visible light region. As Figure S8 shows, the absorbance of EY is much higher than that of mTHPP in the visible light region, which means EY has higher absorbance of solar energy than mTHPP. Both the solution-based and the integrated systems showed similar trends for the different combination of PS and M. Due to the shade generated from the nanofiber membrane, the initial reaction rate of the integrated system was slightly lower than each of the corresponding solution-based PS-M combination. However, the yield of NADH regeneration of the integrated system at equilibrium reached the same level as the 10


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solution-based system. The NADH regeneration using GO doped nanofibers with assembling Mphen in the absence of photosensitizer also has been detected. As Figure S9 shown, after reaction for 3 hours, there was only about 8% NADH regenerated from NAD+, which demonstrated the GO was not a competent photosensitizer for NADH regeneration. The synthesis of methanol from CO2 catalyzed by these four different APSs are shown in Figure 4. Batch reactions were operated with 25 mg G-Fiber based integrated APS, generating a final concentration of 0.10 mg/mL FateDH, 0.10 mg/mL FaldDH, 0.10 mg/mL ADH, 0.2 mM NAD+, 0.25 mM Mbpy or Mphen, and 0.05 mM EY or mTHPP. For comparison, the free APS with the same concentration of enzymes, NAD+, PS, and M was also applied to catalyze the reaction. The yields of methanol for both the integrated APS and free system are shown in Figure 4a-d. The bioconversion of CO2 to methanol encompassed three consecutive reduction steps: (1) CO2 to formic acid catalyzed by FateDH, (2) formic acid to formaldehyde catalyzed by FaldDH, and (3) formaldehyde to methanol catalyzed by ADH. For each of these three reduction reaction steps, NADH was the prerequisite and was consumed as a reducing agent to donate electrons. Since NAD+ was supplied as the starting coenzyme in the biocatalytic portion of the APS, the successful production of methanol proved that the photocatalytic portion well performed its role in cofactor regeneration under visible-light irradiation. Furthermore, to make sure the methanol was originated from CO2 rather than any other carbon residues in the reaction mixture, a set of control experiments was performed in the absence of CO2. This control 11



experiment failed to produce any detectable methanol, which successfully confirmed the origin of methanol to be CO2. The methanol yield at 20 h was calculated and depicted in Figure 4e. The level of methanol yields obtained with different PS-M combinations was found generally consistent with the results of NADH photo-regeneration kinetics, i.e., the more efficient the NADH photo-regeneration, the higher methanol yield with the EY-Mphen combination being the most effective. On the other hand, for each of the PS-M combinations, the system with both photo-catalytic and bio-catalytic parts in free formation exhibited the lowest methanol yield. For the system with EY as PS and Mphen as M, the methanol yield obtained with free multi-enzyme system coupled with solution-based EY and Mphen exhibited a very low methanol yield of 5.23 µM at 20 h. When immobilized multi-enzyme systems with free photocatalytic coenzyme regeneration or free multi-enzyme systems with integrated photocatalytic system for coenzyme regeneration were applied, the methanol yields reached 28.81 and 20.16 µM, respectively. Especially, for the integrated APS, the highest methanol yield of 51.83 µM was achieved. The high methanol yield accomplished by using the completely integrated APS in this study could be attributed to the following aspects. Firstly, as illustrated in one of our previous works, the PAH facilitated the shuttling of the tethered cofactor between co-encapsulated dehydrogenases within the near vicinity inside the nano-scaled lumen of hollow nanofiber, thus enabled more efficient catalysis efficacy.30 Secondly, similar to the highly efficient natural photosynthesis that is dependent on the highly integrated and precisely nano-scaled arrangement of 12


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the active molecules, the efficient NADH regeneration was certainly favored by the highly integrated APS factors. Last but not least, the doped GO not only provides anchoring sites for assembling of PS (EY or mTHPP) and M (Mbpy or Mphen) through π-π and π-cation interactions. Their conjugated sp2-bonded carbon network also provides abundant delocalized electrons and excellent electrical conductivity to facilitate the dispersion and transfer of the photo-excited electrons from the PS to the M (Figure S10). The electron accepted by M will then be transferred to NAD+ for regeneration of NADH. Therefore, the GO acted as an “expressway” to enhance the transport of excited electrons through the PS-M-NAD(H) electron transfer chain. Hence, the electron back-flow between PS and M, as well as the re-combination of electrons and holes of PS, were eliminated. In this regard, the GO “expressway” was also a “one-way” that guides more efficient electron transfer by following the PS-M-NAD(H) electron transfer chain. The same trend regarding the methanol yield was shown from the other three PS-M combinations. Figure 5a showed the operational stability of integrated APS. The reaction reacted for 20 h, then we ceased the reaction by simply taking out the membrane using tweezers. By defining the methanol yield obtained at 20 h in the first cycle as 100%, approximately 95% of the original activity of integrated APS using Mphen as the electron mediator, with either EY or mTHPP as photosensitizer, remained after 10 cycles of reuse, which lasted a total of approximately 200 hours. However, for the other APS using Mbpy as their M, the integrated system retained only 40% of their original activities after 10 cycles of reuse. This remarkable difference in stability of 13



each of these four APSs was mainly due to the difference in strengths of π-π interactions between GO and M. As reported in previous studies,36 the number of aromatic rings in the π-electron system of adsorption molecules is the most dominant factor influencing the strength of a π-π interaction. The PSs all possess many benzene rings ensuring a sufficiently strong π-π interaction with GO. Compared to Mbpy, which only has two benzene rings, there are three continuous benzene rings in Mphen. Therefore, the π-π interaction between Mphen and GO would be stronger than that of Mbpy. As a consequence, the systems using Mphen showed significantly higher operational stability, as demonstrated by the retention of its original activity was as high as 95% after 10 cycles of reuse. This excellent stability observed in the integrated EY-Mphen system also indicated an enhanced stability of photosensitizer EY by forming strong π-π interaction with the GO as illustrated in Figure S10, since the solution-based EY was well known for its instability.34,39 In order to further demonstrate the improvement in stability of EY, EY-Mphen integrated on G-Fiber was immersed in a buffer solution and exposed to 450 W Xenon lamp irradiation. At specific time intervals, certain amounts of the G-Fiber membrane were taken out to investigate changes in its activity namely methanol formation. As shown in Figure S11, the activity of EY-Mphen integrated on G-Fiber remained over 95% after 200 h of continuous irradiation. This result provides more direct evidence exhibiting the excellent stability of EY in the integrated system. On the other hand, the low stability of the EY-Mbpy system suggest again that the strength of the GO-M interactions played a more dominate role in the overall stability of the integrated APSs with 14


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different combination of PS and M. Moreover, in control experiments using pure PU hollow nanofibers or nanofiber doped with PAH only for PSs and M attachment, their loading amount was negligible. This further proved the necessity of π-π interaction driven assembling of PSs and M onto the G-Fiber. The integrated APS with EY-Mphen combination also showed excellent thermal stability. As shown in Figure 5b, about 50% of activity remained for a 10 h period of storage at 60°C, which was approximately a 40-fold increase compared with that of the free APS. The high thermal stabilities of the G-Fiber-based APS may be due to the nano-confining effect of hollow nanofibers enhancing the stability of enzymes and NAD(H). Photochemical and Electrochemical Analysis of the G-Fiber based APS According to the photo-induced electron transfer mechanism involved in the biocatalyzed APS, immediate transferring of the electron from PS to M would be crucial for the subsequent regeneration of NADH by receiving electrons from M. The differential effect of PS and M on photochemical NADH regeneration could be due to an energetic relationship between PS and M. Therefore, we examined the electrochemical properties of PS and M in detail by cyclic voltammetry (CV). The reduction potential at a cathodic peak current of M (Mbpy and Mphen) and PS (EY and mTHPP) was observed at around -0.77 V, -0.72, -0.91 V, and -0.79 (Figure 6 and Table S1), respectively. Significant anodic shift of PS (EY and mTHPP) reduction, or in other words, a cathodic shift of M reduction, was observed when these two components were mixed together in buffer solution (Figure 6). This indicates electron 15



transfer from PS to M. Such reduction potential shifts were also observed on all PS and M combinations, as well as on the corresponding integrated PS and M (Figure 6 and Table S1). Interestingly, although the shifts in integrated PS and M reduction potential were all approximately the same, the cathodic peak current at their shifted reduction potential differed. For the integrated PS and M, a remarkable increase in the cathodic peak current was observed, indicating an enhanced rate and efficacy for electron transfer from PS to M. With the presence of NAD+, the current at the reduction peak of the PS and M complex increased substantially, demonstrating that the PS and M complex was able to catalyze the reduction of NAD+. As expected, the integrated system revealed a more significant increase in the reduction peak current with NAD+. The integration of PS and M based on G-Fiber may be responsible for the catalytic activity of the PS and M complex. For example, the electron transfer distance from PSs to NAD+ was shortened largely through assembling PSs and M onto fiber surfaces and encapsulating NAD+ in their lumens. On the other hand, the doped GO would not only provide binding sites for PS and M assembly through π-π interactions, but would also act as charge-relay material to facilitate charge transfer and prevent the back transfer and re-combination of the photo-excited electrons. Moreover, the increase in the reduction peak current with NAD+ using EY as the PS were much higher than that of mTHPP. This also explains the differences in NADH regeneration and methanol formation with different PSs. The solid UV-visible diffuse reflectance spectra of the GO-nanofibers with integrated EY-Mbpy, EY-Mphen, mTHPP-Mbpy, or mTHPP-Mphen are presented in 16


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Figure 7a. The absorbance threshold (λg) of each of these catalytic systems are approximately at 700, 710 nm, 630 and 650 nm; their corresponding band gaps (Eg) therefore are estimated to be 1.77, 1.75, 1.97, and 1.90 V, respectively, from the formula Eg=1240/λg40. Linear sweep voltammograms experiments were used to determine the lowest unoccupied molecular orbital (LUMO) value of each integrated APS. According to Figure 7b, the LUMO values of each G-Fiber-based catalytic systems are -0.87 V (EY-Mbpy), -0.88 V (EY-Mphen), -0.83 (mTHPP-Mbpy), and -0.82 V (mTHPP-Mphen). The highest occupied molecular orbital (HOMO) of each integrated APS therefore can be calculated from the differences between the band gaps (Eg) and LUMO. The corresponding HOMO values are 0.90, 0.87, 1.14 and 1.08 V, respectively. Based on these results, the energy band and electron movement of the whole photocatalytic system was schematically illustrated in Figure 7c. This helps to further understand the catalytic mechanism for the NADH photo-regeneration and methanol formation. Considering the oxidization potential and reduction potential of TEOA and NAD+ was about 0.8 and -0.3 V, there is a match of energy bands among TEOA, integrated PS-M, and NAD+, i.e. the oxidization potential of TEOA is higher than the HOMO of the PS-M system, and reduction potential of NAD+ is lower than the LUMO of the PS-M system.31, 37 Therefore, the photo-excited electrons from the HOMO of the integrated APS could be directly transferred to the LUMO of the PS-M system and eventually transferred to NAD+ for its reduction. Meanwhile, the holes in the HOMO of integrated APS can be consumed by the sacrificial agents (TEOA). As shown in Figure 7c, although four integrated APS have similar LUMO, the HOMO of 17



the EY-based system was closer to the oxidization potential of TEOA, which means the transfer of the electron from TEOA to EY will be easier than that of mTHPP-based system. Moreover, as mentioned before, the EY also presents much higher absorbance than that of mTHPP in the visible light region (Figure S8). Therefore, the APSs using EY as the photosensitizer are generally more efficient than that using mTHPP. In order to further investigated the electron transfer relationship among PSs, electron mediator, and GO on the surface of the nanofibers, the energy labels of PSs (EY and mTHPP), electron mediator (Mbpy and Mphen), and the GO have been detected by solid UV-visible diffuse reflectance spectra and linear sweep voltammograms respectively. As shown in Figure S12a, the λg of EY, mTHPP, and GO are approximately at 590, 570, and 515 nm; their corresponding Eg therefore are estimated to be 2.10, 2.18, and 2.41 V, respectively, from the formula Eg=1240/λg40. According to Figure S12b, the LUMO values of EY, mTHPP, and GO are -1.03, -1.00, and -0.78 V. The highest occupied molecular orbital (HOMO) of EY, mTHPP, and GO therefore can be calculated from the differences between the Eg and LUMO. The corresponding HOMO values are 1.07, 1.18, and 1.63 V, respectively. Because the reduction potential of Mbpy and Mphen was -0.77 and -0.72 V (Table S1), there is a match of energy bands among EY/mTHPP, Mbpy/Mphen and GO. The electron transfer direction among EY/mTHPP, Mbpy/Mphen and GO was shown in Figure S13. Due the LUMO value of GO is lower than that of EY and mTHPP, and higher than that of M, so the photo-excited electrons would transfer from LUMO of PSs to M through 18


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LUMO of GO. Considering GO has great electroconductivity and charge carrier mobility, so that the GO doped in the shell of fibers not only provided tentacle points for PS and M binding, but also act as a “one-way expressway” to shorten the distance and speed up the electron transfer between PS and M. To further investigate the advantageous photochemical properties of G-Fiber based integrated APS, the photocurrent response versus time profiles of these four different PS-M combinations, either integrated in G-Fiber or in solution formations, were measured under an applied potential of 50 mV s-1 and light irradiation (λ>420 nm). According to the photocurrent-time (I-T) profiles shown in Figure 8, all the systems present prompt photocurrent response with ON/OFF cycles of simulated solar light, and the peak anodic photocurrent values of each of the integrated PS and M was higher than its corresponding free system. This indicates more-efficient electron excitation by simulated solar light and electron transfer from the sacrificial agent TEOA to the electrode through the integrated PS and M system. Moreover, the EY-Mphen combination presented the highest peak anodic photocurrent values, which was consistent with its highest efficiency in NADH photo-regeneration and methanol synthesis. In order to find out the contribution of G-Fiber for the enhanced activities, the pure PU hollow nanofibers and those doped with different materials were pasted on the surface of a working electrode to evaluate their effect on the I-T profiles of PS (EY) and electron mediator (Mphen). For the pure PU hollow nanofibers and those doped with only PAH, EY, and Mphen were supplemented in the electrolyte solution 19



Page 20 of 44

containing TEOA, because these two compounds could not be assembled on the fibers without the aid of GO. Meanwhile, for the GO-doped and G-Fiber, the EY and Mphen were pre-assembled through the GO facilitated π- π interactions. For I-T testing, the fibers were pasted on the surface of a working electrode and the amount of EY and Mphen was adjusted to the same level for each measurement. Results presented in Figure 9 show that pure PU nanofibers have a poor electrical conductivity leading to less than 3 µA cm-2 of photocurrent. With doping of linear cationic polyelectrolyte PAH, the conductivity of PU and electron transfer rate between PS and electron mediator was effectively increased allowing the photocurrent of PAH-doped nanofibers to reach about 9 µA cm-2. Due to the abundance of delocalized electrons from the conjugated sp2-bonded carbon network of GO, GO-doped hollow nanofibers have a more efficient electron transfer efficiency (12 µA cm-2). For G-Fiber, the highest photocurrent (15 µA cm-2) was obtained. Conclusion In

summary,

a

G-Fiber

based

integrated

APS

incorporating

the

photo-regeneration of NADH with biocatalyzed synthesis of methanol from CO2 was successfully developed by co-axial electrospinning and self-assembly technologies. Nearly 80% of NADH could be regenerated from NAD+ by this integrated APS within 3 h of visible light irradiation. Compared with the solution-based system, approximately 10-times higher methanol yield was accomplished by using the integrated APS. Based on detailed photochemical and electrochemical property investigations, it was confirmed that the electron-transfer efficacy of integrated APS 20


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and the overall reaction efficiency were correlated. The GO doped in the shell of fibers not only provided tentacle points for PS and M binding, but also act as a “one-way expressway” to shorten the distance and speed up the electron transfer along the PS-M-NAD+ electron transport chain. The perfect band match with TEOA, higher absorbance in the visible light region of EY, and stronger connection of Mphen with GO enabled the EY-Mphen combination to be significantly more efficient in catalyzing NADH regeneration and methanol formation than other PS-M combinations. Besides increased activity, this highly integrated system also demonstrated excellent recycling capability and stability as 95% of its original activity was retained after 10-times of reuse. This work lays a foundation for future development on photo-enzymatic reaction systems and reveals a new paradigm in integrated APS.

Experimental Section Materials Dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer, [Cp*RhCl2]2 was supplied by Alfa. Cofactor (oxidized or reduced nicotinamide adenine dinucleotide (NADH/NAD+)), and dehydrogenases including alcohol dehydrogenase (ADH, EC 1.1.1.1),

formaldehyde

dehydrogenase

(FaldDH,

EC.1.2.1.46),

and

formate

dehydrogenase (FateDH, EC.1.2.1.2) were purchased from Sigma-Aldrich Chemical Co. Other products including poly(allylamine hydrochloride) (PAH), triethanolamine (TEOA), Eosin Y (EY), 5,10,15,20-tetrakis(3-hydroxyphenyl)-21H,23H-porphine 21



Page 22 of 44

(mTHPP), 2,2'-bipyridine, 1,10-phenanthroline, and N, N-dimethylacetylamide (DMAc), were all supplied by Sigma-Aldrich Chemical Co. Graphene oxide (GO) was purchased from Nanjing Xianfeng nano Co. (Nanjing, China). Polyurethane A85E (PU) pellet was purchased from Xiamen Jinyouju Chemical Agent Co. (Xiamen, China). The

electron

mediators,

(pentamethyl-cyclopentadienyl-2,2’-bipyridine-aqua) (Mbpy=[Cp*Rh(bpy)H2O)]2+,

Cp*=C5Me5,

including rhodium-(III)

bpy=2,2’-bipyridine

(pentamethyl-cyclopentadienyl-1,4-phenanthroline-aqua)

rhodium-

and (III)

(Mphen=[Cp*Rh(phen)H2O)]2+, Cp*=C5Me5, phen=1,10-phenanthroline) Mbpy and Mphen were synthesized according to previous reports. 35, 41 Fabrication of G-Fiber General procedure for preparation of G-Fiber by co-axial electrospinning technology was performed as following. Firstly, a specific amount of GO was dissolved in DMAc solution with the final polymer concentration of 1 wt.%, and underwent ultrasonic treatment for 3 h, then PU pellets were added into the above-mentioned DMAc solution and stirred for 24 h. After PU pellets were dissolved completely, the GO and PU mixed DMAc solution was used as shell-phase solution. Secondly, core-phase solution was prepared by adding 20 mg FateDH, 20 mg FaldDH, 20 mg ADH, and 28 mg NAD+ to 200 µL of 0.1 M pH 7.0 phosphate buffer solution containing pre-dissolved 20 mg PAH. The solution was then well mixed with 800 µL glycerol. The other parameters of co-axial electrospinning were the same as reported 22


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previously.19, 30 Construction of Integrated APS by Self-Assembly of PS and M Generally, the prepared G-Fiber membrane were suspended in Mbpy or Mphen aqueous solution (5 mL, 10mg/mL) containing NaCl (0.5 M). After shaking for 3 h, the G-Fiber membrane coated with Mbpy or Mphen layer were taken out from the solution using tweezers and washed three times with fresh buffer solution to remove the residual Mbpy or Mphen. Then, the fiber membrane was immersed into EY or mTHPP solution (5 mL, 0. 1mg/mL) and shook for 60 min. After being washed twice with water, the G-Fiber assembled with one layer of electron mediator (Mbpy or Mphen) and one photosensitizer (EY or mTHPP) layer was obtained. Characterization Morphologies and hollow structure of the hybrid hollow nanofibers were detected by scanning electronic microscope (SEM, JSM-6700F, JEOL, Japan). The distribution of encapsulated enzymes and cofactors were detected by confocal laser scanning microscopy (CLSM). Firstly, FateDH, FaldDH, and ADH were pre-labeled by FITC. CLSM observation was performed with Leica TCS SP5 microscope (Leica Camera AG, Germany). Due to the autofluorescence of NADH, the distribution of NAD(H) could be detected directly without fluorescence labeling with the excitation and emission wavelength at 351 nm and 460 nm, respectively. An energy dispersive X-ray spectroscope (EDS) (Inca X-MAX, Oxford, UK) connected with SEM was applied to detect the assembly of photosensitizer (EY) and electron mediator (Mbpy or Mphen) on the surface of hollow nanofibers. 23



To fabricate the working electrode to detect the photocurrent-time of the hollow nanofiber membrane, the membrane was connected to the glassy carbon through a conducting resin and were connected to a Cu wire using silver paste. Then, a specific area of approximately 1 cm2 was exposed to light irradiation and the other area was entirely covered by insulating epoxy resin. Enzyme Activity Analysis Activity units of FateDH, FaldDH, and ADH were defined as the amount of enzyme needed to reduce 1 µmol formic acid, formaldehyde, or methanol, respectively, in 1 min at pH 7.0 and 25oC. The specific details of this procedure’s dehydrogenase activity detections were the same as our previously reported work.19, 27 Photoregeneration of NADH The photocatalyzed reduction of NAD+ to NADH was performed in a quartz reactor containing 10 mL of buffer solution (100 mM PBS, pH 7.0) with pre-dissolved 1 mM NAD+, and free or integrated APS with definite concentration of Mbpy or Mphen (0.25 mM), EY or mTHPP (0.05 mM), and TEOA (15% w/v) under irradiation from a 400 W Xenon lamp covered with a 420 nm cut-off filter as a light source (CEL-HXF 300, CEAULIGHT, Beijing, China). Before placing the reactor under irradiation, the reactor was incubated in a dark environment for 1 h. The concentration of reduced NADH was monitored by measuring the absorbance at 340 nm (εNADH = 6.22 mM-1 cm-1). Methanol Formation from CO2 Catalyzed by Integrated APS The conversion of CO2 to methanol was also performed in a quartz cuvette reactor 24


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equipped with a 450 W Xenon lamp covered with a 420 nm cut-off-filter as the light source. In summary, 10 mL buffer solution (100 mM PBS, pH 7.0) was bubbled with CO2 gas for 0.5 h and then a specific amount of dehydrogenases (including FateDH, FaldDH, and ADH), NAD+, PS (EY or mTHPP) and M (Mbpy or Mphen) was added obtaining the final concentrations of 0.1mg/mL, 0.2 mM, 0.05 mM, and 0.25 mM, respectively. The reaction system was also kept at a constant pressure of 0.3 MPa. At certain time intervals, 20 µL of sample was taken from the reaction mixture for methanol concentration measurement. The concentration of methanol was detected by a gas chromatography described in one of our previous reports.19, 30 Stability of the Integrated APS Reusability of the G-Fiber based integrated APS was examined by measuring the methanol yield during repeated usages defining the methanol yield at first cycle as 100%. For each cycle, the reactions lasted for 20 h, and the reaction was stopped by taking out the hollow nanofiber from the reaction solution using tweezers. After washing the hollow nanofiber membranes with buffer solution thrice, the membranes were put into a new reaction solution for the next reuse cycle. For the detection of stability of the integrated APS under light irradiation, the integrated APS, with EY as photosensitizer and Mphen as M, was placed in buffer solution with direct irradiation by a 450 W Xenon lamp. At different time intervals, a certain amount of the membrane was taken out to investigate the residual methanol formation activity as indication of light stability. For the detection of the thermal stability of the integrated APS, the hollow 25



nanofiber membranes were firstly placed at 60oC. Then, at specific time intervals, a certain amount of the membrane was taken out to investigate the methanol formation ability. Association content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Chemical structures, absorbance spectra and standard curves of EY, mTHPP, Mbpy, and Mphen; the SEM images of the surface and cross-sectional of freshly prepared PAH-doped PU nanofibers; schematic illustration of the π-π interactions driven assembly of M and PSs onto the G-Fiber, as well as the electron transfer mechanism in the integrated APS for photo-enzymatic synthesis of methanol from CO2; the light stability of integrated EY-Mphen system; and the cathodic peak current and reduction potential at the cathodic peak current of free and integrated photosensitizer and M in the absence and presence of NAD+. Acknowledgements The authors thank the support from the National Basic Research Program of China (973 Program, 2013CB733604), and the National Natural Science Foundation of China (Grant Nos. 21676276 and 91534126).

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(40) Wang, S.; Lian, J. S.; Zheng, W. T.; Jiang, Q., Photocatalytic property of Fe doped anatase and rutile TiO2 nanocrystal particles prepared by sol-gel technique. Appl. Surf. Sci. 2012, 263, 260-265. (41) Lo, H. C.; Leiva, C.; Buriez, O.; Kerr, J. B.; Olmstead, M. M.; Fish, R. H., Bioorganometallic chemistry. 13. regioselective reduction of NAD+ models, 1-benzylnicotinamde triflate and β-nicotinamide ribose-5 ‘-methyl phosphate, with in situ generated [Cp* Rh (Bpy) H]+: structure-activity relationships, kinetics, and mechanistic aspects in the formation of the 1, 4-NADH derivatives. Inorg. Chem. 2001, 40, 6705-6716.

32


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Scheme 1. Diagrammatic illustration of preparation of G-Fiber based integrated APS.

33



Figure 1. Characterization of G-Fiber based integrated APS: a) surface SEM images, b) cross-sectional SEM images, c) CLSM images of encapsulated FITC-labeled enzymes, d) CLSM images of encapsulated NAD(H), e) EDS element mappings of Rh (red), and f) EDS element mappings of Br (green).

34


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Figure 2. Surface zeta-potential tests of HNFs after consequential assembly of M (Mbpy or Mphen) and PSs (EY or mTHPP).

35



Figure 3. Time profiles of photochemical regeneration of NADH from NAD+ with a) free and b) integrated systems of different PS and electron mediator combinations: (△, ▲) EY-Mbpy, (□,■)EY-Mphen, (▽,▼) mTHPP-Mbpy, (○,●) mTHPP-Mphen. The initial NAD+ concentration was set to 1 mM in each of these four different systems.

36


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Figure 4. Time profiles of photo-enzymatic synthesis of methanol from CO2 with different PS and electron mediator combinations: a) EY-Mbpy, b) EY-Mphen, c) mTHPP-Mbpy, and d) mTHPP-Mphen in different formations. (■) Both biocatalytic and photo-catalytic NADH regeneration parts were in the solution,

(▲) biocatalyst

part was in solution while photo-catalytic part was being integrated, ( ▼ ) photo-catalytic part was in solution while biocatalytic part was being integrated, and (●) both parts were being integrated. e) The summary table of the yields of methanol synthesized from CO2 after 20 h reaction. In all cases, concentrations of electron mediator, PS, and TEOA were adjusted to the same level, at 0.25 mM, 50 µM, and 15%, respectively. Concentrations of FateDH, FaldDH, and ADH in integrated formation and free formation were 0.1 mg/mL and NAD+ concentration in the corresponding formations was 0.2 mM.

37



Figure 5. Performance of the G-Fiber based integrated APS for methanol synthesis from CO2. a) Reusability of the integrated artificial photosynthesis; b) thermal stability of the free and integrated artificial photosynthesis using EY and Mphen as their PS and M at 60oC.

38


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Figure 6. Cyclic voltammograms of (a) EY-Mbpy system, (b) EY-Mphen system, (c) mTHPP-Mbpy system, and (d) mTHPP-Mphen system. The concentration of PSs (EY or mTHPP), M (Mbpy or Mphen), and NAD+ was 0.05mM, 0.25mM, 0.25mM, and 1mM, respectively. The potential was scanned at 100 mV s-1 using glassy carbon (working), silver-silver chloride (reference), and platinum (counter) electrodes in sodium phosphate buffer (100 mM, pH 7.0).

39



Figure 7. (a) Solid UV-visible diffuse reflectance spectra, (b) linear sweep voltammograms, and (c) schematic energy-level and electron movement diagram of G-fiber based APS with integrated EY-Mbpy, EY-Mphen, mTHPP-Mbpy, and mTHPP-Mphen. For linear sweep voltammograms analysis, the scan rate was 100 mVs-1 silver-silver chloride (reference), and platinum (counter) electrodes in sodium phosphate buffer (100 mM, pH 7.0). To fabricate the working electrode to detect the linear sweep voltammograms the integrated APS, the fibers membrane was connected to the glassy carbon through a conducting resin and were connected to a Cu wire using silver paste. Then, a specific area of approximately 1 cm2 was exposed to light irradiation and the other area was entirely covered by insulating epoxy resin.

40


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Figure 8. Photocurrent-time (I-T) profiles of (a) EY-Mbpy system, (b) EY-Mphen system, (c) mTHPP-Mbpy system, and (d) mTHPP-Mphen system under simulated solar light illumination. The concentrations of PSs (EY or mTHPP), M (Mbpy or Mphen), and TEOA are 0.05mM, 0.25mM, and 15wt%, respectively. The scan rate was 50 mV s-1 and the input power was 100 mW cm-2 using glassy carbon (working), silver–silver chloride (reference), and platinum (counter) electrodes in sodium phosphate buffer (100 mM, pH 7.0).

41



Figure 9. Photocurrent-time profile of pure PU hollow nanofibers, PAH-doped hollow nanofibers, GO-doped PU hollow nanofibers, and G-Fiber electrodes under simulated solar light illumination. The pure PU hollow nanofibers and those doped with PAH, EY, and Mphen were supplemented in the electrolyte solution containing TEOA. The GO-doped and GO-PAH-doped PU hollow nanofibers, the EY, and Mphen were pre-assembled through the GO facilitated π- π interactions. The scan rate was 50 mV s-1 and the input power was 50 mW cm-2 using glassy carbon (working), silver-silver chloride (reference), and platinum (counter) electrodes in sodium phosphate buffer (100 mM, pH 7.0).

20 Photocurrent (µA/cm2)

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Page 42 of 44

PU hollow nanofiber PAH-doped PU hollow nanofiber GO-doped PU hollow nanofiber GO-PAH-doped PU hollow nanofiber

15 10 5 0

0

10

20

30 40 Time (s)

50

60

42


70

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Table 1 Enzyme Loading and Activities of Multi-Enzyme System Encapsulated Inside Hollow Chamber of G-Fiber Loading

Activity recovery Specific activity Gross activity

Enzymes(a) (mg/g-fiber)

(U/mg-enzyme)(b)

(%)

(U/g-fiber)

FateDH

7.78±0.51

71.85±3.25

3.19±0.39

24.82±0.20

FaldDH

7.78±0.95

83.82±1.56

3.00±0.11

23.34±0.10

ADH

7.78±0.46

72.64±30.01

31.07±0.69

241.72±0.32

(a) The specific activities of FateDH, FaldDH, and ADH in their free formation were determined to be 4.44±0.12, 3.58±0.07, and 42.77±0.23 U/mg-enzyme, respectively. (b) The specific activity of each of the immobilized dehydrogenases were determined separately.

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Synopsis A highly integrated artificial photosynthesis based on graphene oxide (GO) and polyelectrolyte PAH co-doped hollow nanofibers for efficient conversion of methanol from CO2.

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A Graphene Oxide and Polyelectrolyte Composed One-Way

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