Letter pubs.acs.org/journal/ascecg
Next Generation Designed Protein as a Photosensitizer for Biophotovoltaics Prepared by Expanding the Genetic Code K. Deepankumar,† A. George,‡ G. Krishna Priya,‡ M. Ilamaran,‡ N. R. Kamini,‡ T. S. Senthil,§ S. Easwaramoorthi,⊥ and N. Ayyadurai*,‡ †
Division of Materials Technology, School of Materials Science & Engineering, Nanyang Technological University, Singapore Biotechnology Laboratory, CSIR-Central Leather Research Institute (CSIR-CLRI), Adyar, Chennai 600020, India § Department of Physics, Erode Sengunthar Engineering College, Erode, Tamil Nadu 638057, India ⊥ Chemical Laboratory, CSIR-Central Leather Research Institute (CSIR-CLRI), Adyar, Chennai 600020, India ‡
S Supporting Information *
ABSTRACT: We explored the possibility of generating nonpoisonous, renewable, low cost and a completely biodegradable photosensitizer for dye-sensitized solar cells (DSSC) as an alternative to synthetic molecules that involve expensive, time-consuming tedious synthesis and purification procedures. Several natural dyes from plants and microbes had successfully been demonstrated as photosensitizers to develop biosensitized solar cells (BSSCs). The objective of this work is to develop a next generation cleaner sensitizer for BSSC using a green fluorescent protein (GFP) and its designer variant (GFPdopa) through an expanding genetic code approach. The designer protein showed higher adsorption with TiO2 surface through oriented immobilization. The nanostructured layer formed by GFPdopa with TiO2 resulted in 0.94% level of photon conversion efficiency with open circuit voltage of 0.60 V, short circuit current of 1.75 mA/cm2 and fill factor of 0.88. It is one of the better energy conversion efficiencies obtained for BSSC when compared to with earlier reported sensitizers generated through protein and chemical complex synergism. From the results obtained, it is suggested that designer fluorescent itself can generate similar photoconversion efficiency and also could serve as an environmental friendly photosensitizer. The research and efficiency level of BSSC is in the early stages, and our proof of principle opens a new avenue to synthesize biologically designer sensitizers for BSSC. It also could be widely applied to other proteins to develop efficient sensitizers for BSSC with a green approach. KEYWORDS: Biosensitized solar cells, TiO2, Green fluorescent protein, L-3,4-Dihydroxyphenylalanine, Noncanonical amino acid
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INTRODUCTION At present, generation of energy from renewable sources with economic viability is one of the most important scientific and technological challenges. Among them, the third generation dye-sensitized solar cells (DSSC) have attracted considerable attention owing to their higher efficiency, available choice of sensitizer, and electrolyte and electrode combinations.1,2 The conventional DSSC’s consist of a wide band gap semiconductor sensitized by molecules that absorbs a broad spectrum of light in visible and near-infrared region, which is responsible for higher energy conversion efficiency of the device. Many synthetic inorganic compounds based on Ruthenium and other metal complexes and metal free organic dyes have mainly been used as a sensitizer due to their remarkable device efficiency and good photo and thermal stability.3 However, the high cost, a relatively low abundance of ruthenium, and its carcinogenic nature suggest and force to find an alternative greener sensitizer for solar cells. Recently, biosensitized solarcell (BSSC), an analogous term for DSSC’s, has been coined when the traditional sensitizers are replaced by the compounds © 2016 American Chemical Society
derived from the natural resources with little or no synthetic modification.4 Several dyes extracted from the natural sources such as plants and microbes were employed as a photosensitizer in BSSC.4 Though these dyes were abundantly available, renewable, nontoxic, and completely biodegradable, not much work has been reported in the literature. The bacteriorhodopsin, microbial pigments, and plant chlorophyll were identified as promising sensitizers,5,6 and their performance were further enhanced by cosensitization with inorganic dyes, coating with different scattering layer, altering electrodes, etc.5,7,8 Although biophotosensitizers have been successfully demonstrated as a material for bioelectronics, the requirement of a large amount of organic solvent for extraction, laborious multistep purification hampers the utilization of natural dyes in BSSC. On the other hand, the advent of recombinant DNA technology will be the smarter way for large scale synthesis Received: August 17, 2016 Revised: November 3, 2016 Published: November 15, 2016 72
DOI: 10.1021/acssuschemeng.6b01975 ACS Sustainable Chem. Eng. 2017, 5, 72−77
Letter
ACS Sustainable Chemistry & Engineering
NCAA into the recombinant protein. For the SDS-PAGE analysis, cell lysis was carried out by using Bug Buster protein extraction kit (Novagen, CA, USA) followed by sonication. Briefly, the collected cell pellets corresponding to 1 mL of culture was resuspended in 100 μL of lysis buffer, incubated at room temperature for 10 min and centrifuged at 9000g, 4 °C for 20 min. The supernatant was saved as a soluble protein fraction and analyzed by SDS-PAGE. Protein Purification, Quantification, and Fluorescence Analysis. The soluble proteins were purified using Ni-NTA HisBind Resin (Novagen). Accordingly, the protein samples were equilibrated with 50 mM sodium phosphate buffer (SPB) containing 10 mM imidazole. The column was washed with 5 column volumes of SPBcontaining 10 mM imidazole and elution was done using SPBcontaining 150 mM imidazole. Further, the fractions enriched with recombinant proteins were collected and dialyzed against SPB without imidazole at pH 7.4. The purified samples were subjected to gel permeation chromatography (GPC) in an AKTA FPLC system containing Superdex 75 HR column at 4 °C. The concentration of the purified protein samples was determined using a UV/vis spectrophotometer at 280 nm using extinction coefficient calculated from the protein sequences. Fluorescence analysis was performed at 2 μM protein concentration by exciting at 470 nm and the emission maximum was recorded using JASCO FP-777 spectrofluorimeter (Jasco, Tokyo, Japan) equipped with digital software. Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Analysis. Samples were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) R250 solution (Biorad, CA, USA). The proteins were eluted for MALDI-TOF as described in Shevchenko et al., 2006 with a few modifications. MALDI-TOF was performed with the sinapinic acid matrix on a Microflex LT instrument (Bruker Daltonik GmbH, Leipzig, Germany). The MALDI-TOF was operated in the linear mode, to give optimal resolution with a 25 kV extraction voltage and 13 kV reflectron potential. Fabrication of BSSC. Commercial titanium dioxide powder (P25, Degussa) was used as a TiO2 source. The paste was prepared by mixing 2.0 g of titanium dioxide powder with a mixture consisting of 5.0 g of α-terpineol and 0.5 g of cellulose in 20 mL of ethanol, which was sonicated for 24 h at 1200 W cm−2. By using the prepared paste TiO2 thin films were formulated by coating the paste on a fluorinedoped tin oxide (FTO) conducting glass plate (Hartford FTO, ∼30 Ω cm−2, 80% transmittance in the visible region) using the doctor blade technique. The prepared films were annealed at 450 °C for 30 min. In cell assembly section, the prepared TiO2 thin film electrodes were immersed in different concentration of GFP and GFPdopa (2 to 5 mg/mL) at room temperature for 5−7 h. A platinum coated FTO electrode was then placed over the dye-adsorbed TiO2 thin film electrode. The two electrodes were clamped firmly together using a binder clip. A redox electrolyte consists of 0.5 mol KI, 0.05 mol I2, and 0.5 mol 4-tert-butylpyridine was used as a solvent and a drop of the electrolyte solution was injected into the space between the clamped electrodes. The electrolytes were entered into the cell through capillary action. This resulted in the formation of sandwich-type cell. The photocurrent−voltage (I−V) curves were measured using white light from a xenon lamp (max. 150 W) using a sun 2000 solar simulator (Abet Technologies, CT, USA). Light intensity was adjusted using a standard Si solar cell to ∼AM-1.5. Incident light intensity and active cell area were 100 mW cm−2 (one sun illumination) and 0.25 cm2 (0.5 × 0.5 cm), respectively. Quartz Crystal Microbalance (QCM). The quartz crystals coated with TiO2 (5-MHz piezoelectric) were purchased from Q-Sense, Sweden. The crystals were immersed in 1% Hellmanex II and purified water (Milli-Q, Millipore) for 30 min and exposed to ozone for 10 min. The cleaned surfaces were then washed thoroughly with purified water, dried with nitrogen gas and used immediately. Real-time QCM measurements were performed using a Q-Sense E4 QCM (Q-Sense, Sweden) instrument with four-channel IPC pump (Ismatec SA, Switzerland). Two cleaned crystals were mounted in the microfluidic chambers of the QCM. On attaining stable resonant frequencies, 0.1 mg mL−1 of each protein was injected into the two different channels at a constant flow rate of 50 μL min−1. GFP and GFPdopa were
of biophotosensitizers through cloning and overexpression of the target pigment-producing gene in microbes. Once the pigment coding gene has been cloned, its functionality and stability would be improved through a rational and mutagenesis approach. This would allow fine-tuning of protein by replacing the existing amino acids with other canonical amino acid building blocks to make the favorable interaction within the protein. These building blocks contain a limited number of chemically active groups (20 natural amino acids) and suggest that proteins require additional chemical functional side chains to evolve the proteins with new or enhanced properties.9,10 Herein, we aimed to employ the expanding the genetic code approach to introduce noncanonical amino acid (NCAA) in the green fluorescent protein (GFP) as an initiative for next generation biophotosensitizer for BSSC. Through this approach, the desired chemical side chains will be introduced into the fluorescent protein in order to modify the structural interaction and to assemble the sensitizer on TiO2 surface through oriented immobilization. In earlier reports, the biophotosensitizer was arbitrarily interacting with the semiconductor through carboxylic group or other peripheral acidic anchoring group, which may be responsible for the observed lower cell efficiency.4 Hence, the oriented immobilization of the biophotosensitizer with the semiconductor may allow the fast and energetically optimized charge transfer with the titanium dioxide. In this work, we have selected catechol containing tyrosine surrogate 3,4-dihydroxy-L-phenylalanine (DOPA) for genetic incorporation into GFP. In general, DOPA has formed through post-translational modification of rich tyrosine containing marine mussel adhesive proteins to provide attractive design paradigms for engineering synthetic polymers material.11,12 The self-polymerization nature (polydopamine) exhibited a stable interface with TiO2 by interacting through the strong bidentate ligand of catecholate (O, O) to prepare solar cells. We expect that the genetic incorporation of DOPA to manipulate GFP would be ideal sensitizer for protein based next generation biophotosensitizer for BSSC.
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EXPERIMENTAL SECTION
Materials. PCR reagents, T4 DNA ligase, and restriction endonucleases were purchased from Promega (WI, USA). IsopropylD-thiogalactopyranoside (IPTG) and sinapic acid were purchased from Sigma Chemicals (MO, USA). The host bacterium Escherichia coli XL1-blue (Stratagene, CA, USA) was used for plasmid DNA preparation. E. coli cells with plasmids were grown aerobically in Luria−Bertani (LB) broth (Difco Laboratories, MI, USA) or on LB agar plate, supplemented with appropriate antibiotics for the selection of transformants. The E. coli tyrosine auxotroph JW2581 were used as an expression host for production of recombinant GFP and congener protein variants preparation. The plasmid pQE-80L and pET30b vector were procured from Qiagen (Valencia, USA) and Novagen (CA, USA) respectively. Construction of Plasmids and NCAA Incorporation of LDOPA into GFP. The DNA manipulations were performed as described earlier.13 Briefly, the plasmid pQE-60 bearing GFP residues were amplified by gene specific primers and then cloned into pQE-80L using BamHI and HindIII restriction enzymes resulted with pQE80GFP. The construct pQE80-GFP was transformed into E. coli JW2581 tyrosine auxotroph for the NCAA incorporation. All these constructs were sequenced and verified for their target protein sequence. The tyrosine auxotrophic cells containing pQE80-GFP construct were grown in M9 minimal medium supplemented with 0.4% (w/v) of glucose, 0.1 mM CaCl2, 1.0 mM MgSO4, thiamine (35 μg/mL), 20 amino acids (40 mg/L), and ampicillin (10 μg/mL). The tyrosine auxotrophic cells were used for selective pressure incorporation of 73
DOI: 10.1021/acssuschemeng.6b01975 ACS Sustainable Chem. Eng. 2017, 5, 72−77
Letter
ACS Sustainable Chemistry & Engineering
Figure 1. Schematic representation of proposed next generation BSSC developed by fluorescent protein and its congener variant followed by the energy level generation diagram on BSSC.
Figure 2. (a) Absorption spectrum of GFP and GFPdopa on TiO2 thin film. (b) Optical UV-absorption spectrum of GFP and GFPdopa desorbed from photoanodes. (c) Protein adsorption efficiency with TiO2 confirmed through fluorescence analysis of GFP and GFPdopa. prepared in degassed 1× PBS buffer pH 8.0 at a concentration of 0.1 mg mL−1. Necessary experiments were repeated at least three times, with an s.d. of the resulting frequency change of
