Bio-nano hybrid materials based on bacteriorhodopsin: Potential applications and future strategies Baharak Mahyad, Sajjad Janfaza, Elaheh Sadat Hosseini PII: DOI: Reference:
S0001-8686(15)00169-4 doi: 10.1016/j.cis.2015.09.006 CIS 1578
To appear in:
Advances in Colloid and Interface Science
Please cite this article as: Mahyad Baharak, Janfaza Sajjad, Hosseini Elaheh Sadat, Bionano hybrid materials based on bacteriorhodopsin: Potential applications and future strategies, Advances in Colloid and Interface Science (2015), doi: 10.1016/j.cis.2015.09.006
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Bio-nano hybrid materials based on bacteriorhodopsin:
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Potential applications and future strategies
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Baharak Mahyada, Sajjad Janfazab,a*, Elaheh Sadat Hosseinia
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares
b
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University, Jalal Ale Ahmad Highway, Tehran 14117, Iran
Young Researchers & Elite Club, Pharmaceutical Sciences Branch, Islamic Azad University,
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Tehran, Iran
*Corresponding author:
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Abstract:
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Email: [email protected]; Phone: +98-21-22954285
This review presents an overview of recent progress in the development of bionano hybrid materials based on the photoactive protein bacteriorhodopsin (bR). The interfacing of bR with various nanostructures including colloidal nanoparticles (such as quantum dots and Ag NPs) and nanoparticulate thin films (such as TiO2 NPs and ZnO NPs,) has developed novel functional materials. Applications of these materials are comprehensively reviewed in two parts: bioelectronics and solar energy conversion. Finally, some perspectives on possible future strategies in bR-based nanostructured devices are presented.
Keywords:
Bacteriorhodopsin,
bioelectronics, energy conversion.
nanomaterials,
bio-nano
hybrid
systems,
ACCEPTED MANUSCRIPT Contents 1. Introduction ....................................................................................................... 3
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2. Applications ....................................................................................................... 6
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2.1. Solar energy conversion .............................................................................. 6
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2.1.1 biomolecule-sensitized solar cells.......................................................... 6 2.1.2 Hydrogen production ............................................................................. 9
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2.2. Bioelectronics ........................................................................................... 12
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2.2.1. Carbon nanotubes ............................................................................... 12 2.2.2. Titanium dioxide nanowires ............................................................... 15
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2.2.3. Silver NPs .......................................................................................... 15
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2.2.4. Quantum dots ..................................................................................... 17
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3. Summary and perspectives ............................................................................... 17 Acknowledgments ............................................................................................... 24 References ........................................................................................................... 25
ACCEPTED MANUSCRIPT 1. Introduction Nanotechnology provides the ability to manipulate and characterize materials at
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the atomic and molecular levels and this has driven extensive research into
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possible applications of nanomaterials. This new emerging multidisciplinary field
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can be thought of an intersection of chemical, physical, biological, and engineering
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sciences on a scale of ∼1–100 nm [1, 2]. Over the last decade, combination of
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nanomaterials and biomolecules including nucleic acids, proteins and antibodies has been developed as a new interdisciplinary research field [3-6]. Among the bio-
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nano hybrids, integration of proteins with engineered nanomaterials has attracted
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materials [3-6].
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particular interest and has led to the design and development of new advanced
Bacteriorhodopsin (bR) is a unique photochromic protein that has been successfully incorporated with various nanomaterials, in order to develop novel nano-bio hybrid materials and nanostructured devices [7, 8]. This nano-scale molecular machine pumps protons across a purple membrane of Halobacterium salinarum powered by sunlight [9-11]. Bacteriorhodopsin contains seven transmembrane helices embedding a retinal chromophore in the middle that is linked to the side chain of lysine 216 (Lys216) via a protonated Schiff base [12]. The proton pumping of this protein is coupled with photochemical conversions, occurring during a photocycle of bR [13-18]. The
ACCEPTED MANUSCRIPT main photocycle of bR including several spectrally different intermediates is shown in Fig. 1.
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Each intermediate has a distinct absorbance maximum and different life times. The
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intermediates are commonly represented by a single letter code where the index
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represents the absorption maximum [13]. When exposed to light at 568 nm, the
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molecules in the initial state (bR568) convert to the short-living J625 and proceed to the K590 state [14-16]. During these initial steps, the isomerization from all-trans to
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13-cis retinal occurs. This photoreaction initiates a cyclic sequence of thermal
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conversions (L550, M412, N560, and O640), which finally lead back to the bR568 state
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[17, 18]. This rapid isomerization of retinal in combination with well-defined
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conformational changes in bR during the photocycle leads to the transport of a proton from the cytoplasmic to the extracellular side of the membrane [19]. The interesting point about the photocycle is that all the intermediate states are able to be photo-chemically switched back to the bR568 state by shining light at a wavelength that corresponds to the absorption peak of the respective intermediates [20, 21]. Its unique properties have made bR a promising material for a wide range of applications. The major advantages of the bR include [22-25]: (1) high quantum efficiency of converting light into a state charge, (2) wide range absorption of visible light, (3) high thermal and photochemical stability, (4) extremely large
ACCEPTED MANUSCRIPT optical nonlinearities, (5) robustness to degeneration by environmental perturbations, (6) low production cost, (7) environmental friendliness, and (8) the
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existence of genetic variants with enhanced spectral properties for specific device
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applications.
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Various mutants of bR have been reported to be used in bioelectronics as well as
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the native one [26-29]. Two classes of bR mutants have been suggested for solar cell application [30]. The first one includes the mutants, such as 3Glu, in which
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glutamate (Glu) is replaced by glutamine (Gln) [31, 32]. These replaced residues
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include Glu9, Glu74, Glu194 and Glu204 and are placed in the outer side of
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membrane. In comparison to native bR, these mutants are more capable of
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transferring electrons through redox electrolyte to the anode [28, 31, 32]. The second group of the mutant bR consists of cysteine (Cys), unlike the native. These mutants have Cys in 3, 36 and 247 positions of amino acid sequence and are able to attach to the gold surface by their sulfhydryl group through covalent bond [30]. Also, the mutant D96N with aspartic acid-96 to asparagine replacement [33, 34] has been well studied for various bioelectronics applications. Over the last two decades, some potential applications of both mutants and wildtype bR have been proposed ranging from medicine to electronics [35-40]. On the other hand, bR can be interfaced with various nanomaterials for fabrication of highly efficient devices and advanced functional materials. In more recent years,
ACCEPTED MANUSCRIPT nano-bio hybrid materials based on bacteriorhodopsin have attracted broad attention because of their excellent physical, chemical, and biological properties
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and its potential use in a variety of medical, optic, optoelectronic, and
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environmental applications.
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This review looks at bacteriorhodopsin-nanomaterials hybrids as promising
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materials for use in bioelectronics and energy conversion. We discuss here how bR can be conjugated to various nanomaterials such as carbon nanotubes (CNT), TiO2
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NPs, ZnO NPs, and quantum dots (QDs) in order to improve its functional
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2. Applications
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properties.
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2.1. Solar energy conversion
Renewable energies have attracted more and more attention due to the rise in energy consumption and environmental concerns. Nano-Bio hybrid materials based on semiconductor nanostructures and bacteriorhodopsin protein have been successfully used for solar energy conversion systems such as photovoltaic cells and water splitting photoelectrochemical cells. 2.1.1 biomolecule-sensitized solar cells Biomolecule-sensitized solar cells (BSSCs) have been developed as a promising “green” technology for low-cost photovoltaic power generation [41-43]. The mechanism of BSSCs is very similar to dye-sensitized solar cells (DSSCs). DSSCs
ACCEPTED MANUSCRIPT appeared as a new generation of photovoltaic device and have attracted much attention as potential alternatives to traditional photovoltaic devices [41]. The
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advantages of DSSCs over silicon-based solar cells lie in its simple production
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method, varieties of material sources, the low cost and possible fabrication of
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those of silicon-based solar cells [44, 45].
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flexible solar cells, even though their performances are still low compared with
In bR based BSSCs, the photoanode is made of a porous semiconductor
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nanoparticles (mainly TiO2 or ZnO NPs) sensitized with bacteriorhodopsin protein
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[22, 46].
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bR with a wide absorption range in the visible region, harvests the solar light,
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going up to the excited state, and injects photo-excited electrons into the conduction band (CB) of semiconductor. The CB of TiO2 is located at a lower energy level than the lowest occupied molecular orbital (LUMO) of the bR molecule, so injection of the photoelectrons from bR to TiO2 is energetically favorable [22]. The electrons migrate to the counter electrode through the external circuit. The oxidized bR accepts electrons from the electrolyte, regenerating the ground state (Figure 2) [22, 28]. The application of bR as a sensitizer in BSSCs was first reported by Thavasi et al.,[28] in which bR was involved in photo induced charge transfer into the titanium dioxide (TiO2) semiconductor upon immobilization. They demonstrated
ACCEPTED MANUSCRIPT that the electrostatic potential map of 3Glu mutant showed better likelihood to bind efficiently to TiO2, which led to higher photoelectric response of the bio-solar cell
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composed of the 3Glu mutant bR, than that composed by the wild type bR. In 3Glu
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mutant, three negative charged Glu residues (E9, E194 and E204) have been
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mutated to Gln (at the EC site of bR) which strongly changes the surface potential
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map of bR. This changes in potential map of bR most likely results in better attachment of bR to exposed oxygen atoms of anatase surface [28].
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Recently, Molaeirad et al. have demonstrated that enhanced photovoltaic
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performance of BSSCs can be achieved by using the co-sensitization of bR and
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bacterioruberin, which are complementary in their spectral responses [42]. More
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recently, they have immobilized an Langmuir−Blodgett (LB) monolayer of bacteriorhodopsin on ZnO nanoporous film and improved the bio-solar cell performance [46]. ZnO nanoparticles with high isoelectric point (IEP) of about 9.5 is an appropriate substrate to immobilize bR, a low IEP protein, by electrostatic interactions with high binding stability [46]. On the other hand, loading the nanostructured photoelectrode with more than one layer of bR can significantly decrease the charge transfer rate in the TiO2/bR interface [22]. Therefore, higher efficiency of the photovoltaic cell can be ascribed to immobilization of LB monolayer of bacteriorhodopsin on the ZnO nanoparticulate film as well as those intrinsic features of ZnO thin film.
ACCEPTED MANUSCRIPT In 2015, Mohammadpour and Janfaza designed a highly efficient bR based BSSC with optimization of the materials at the nano-bio interface and also morphology
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design [22]. They showed that TiCl4 treatment of TiO2 nanoparticle surface and
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controlling the bR loading time could enhance the charge transfer rate in TiO2/bR
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interface. Thus, by employing these two strategies, as well as coating a layer of
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light-scattering paste consists of TiO2 nanofibers, the power conversion efficiency of 0.35%, open circuit voltage (Voc) of 533 mV, and short-circuit current of (Jsc) of
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1 mA cm-2 were obtained [22]. Table 1 summarizes photovoltaic characteristics
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of different bR-based BSSCs reported in the literature.
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However, efficiency of bR-based BSSCs is not high enough compared with
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DSSCs. In contrast to conventional DSSCs that use expensive and toxic ruthenium complexes, bR-based BSSCs employ a low cost, renewable, environment friendly, and non-carcinogenic without any disposal problems as light harvesters [42, 43]. The hope is that the novel bio-sensitizers, like wild-type bR and its mutants, will be acceptable alternative to common dyes. 2.1.2 Hydrogen production Bio-conjugated nanomaterials seem to be very promising for solar hydrogen generation systems [47-50]. Bacteriorhodopsin has been considered as an appropriate biomolecule for incorporation with nanosemiconductors to produce hydrogen via water-splitting. There are two basic approaches to achieve bR based
ACCEPTED MANUSCRIPT solar hydrogen generation: a particle system and an electrode system, as schematically presented in Fig. 3.
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Allam et al. reported the assembly and use of a bR/TiO2 nanotube array (TNA)
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hybrid electrode system for enhanced photoelectrochemical water splitting [47].
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bR was attached onto TiO2 surfaces either with or without linker. Physically
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adsorbed protein showed instability upon conducting the photoelectrochemical tests. But, when using 3-Mercaptopropionic acid as a linker, high stability of the
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fabricated hybrid electrode was revealed [47]. Under the illumination of AM 1.5, a
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photocurrent density of 0.65 mA/cm2 was recorded which was a 50% increase over
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that measured for pure TiO2 nanotubes (0.43 mA/cm2) fabricated and tested under
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the same conditions. They proposed that the enhanced photocurrent generation is because of the proton pumping effect of bR. In addition, in the presence of redox electrolyte (0.02M I-/I3-), the maximum photocurrent of 0.87 mA/cm2 was obtained. This 33 per cent increase in the photocurrent can be mainly attributed to the electron transfer effect of bR [47]. Recently, Naseri et al. have decorated TiO2 nanoparticulate (TNP) and TNA electrodes with bR which caused a several-times increase in photoresponse of the systems (~ 7 and 1.7 times for TiO2 nanoparticulate and nanotubular film, respectively) [48]. They attributed the higher photoactivity improvement for TNP system than TNA one to more surface sites in nanoparticulate film for attachment
ACCEPTED MANUSCRIPT of submicron PM fragments. The photocurrent density of 0.64 mA cm-2 in the bias of +0.5 V was obtained for bR/TNP electrode, under white light irradiation [48].
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Therefore, based on these above-mentioned reports, remarkably improved
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modifying the nanostructured electrodes with bR.
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performances of photoelectrochemical water splitting can be achieved when
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It is of interest to compare photocurrents of bR-based photoelectrochemical water splitting systems with those of bR-sensitized solar cells. Results show that BSSCs
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can generate higher photocurrents under the same illumination conditions.
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The application of bacteriorhodopsin immobilized on Pt/TiO2 photocatalyst for
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visible light-driven hydrogen generation has been studied by A. Rozhkova group
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[49]. Under green and white light illumination in the presence of methanol (at pH 7), the nano-bio hybrid system produced 207 and 5275 μmole of H2 (μmole protein)−1 h−1 , respectively. This 25.5-fold increase in the photocatalytic efficiency of the system under white light illumination can also be due to additional electrons coming from UV excitation of TiO2 nanoparticles. In addition, they stated that bR can act as a visible light-harvesting molecule as well as, a proton pump [49]. Recently, it has been demonstrated that reduced graphene oxide (rGO) can improve photocatalytic performance of the above mentioned bR based hybrid system [50]. Under white light irradiation, the presence of the rGO causes hydrogen production rates of approximately 11.24 mmol of H2 (μmol protein)-1 h-1
ACCEPTED MANUSCRIPT which is 2 times higher than that of the Pt/TiO2-bR nano-bio catalyst alone without rGO (5.38 mmol of H2 (μmol protein)-1 h-1)
[50]. It is proposed that rGO acts
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similarly to bR and helps to sensitize TiO2 to visible light. Moreover, rGO as a
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nanoscaffold promotes an interface between bR, TiO2 NPs, and Pt NPs [50]. 2.2. Bioelectronics
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The use of different nanoscale materials in various fields, including physics,
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chemistry, biology and electronics have grown explosively, over the last decade. In recent years, the incorporation of bR as a unique photo-active protein and
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nanomaterials such as metallic and ceramic nanostructures has been studied to
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enhance the performance of both protein and nanostructures. These combinations
applications.
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offer promising strategies to fabricate novel nanobiomaterials for bioelectronics
The first sub-section (2.2.1) focuses on carbon nanotube as a favorable nanostructure in bioelectronics field. The next sub-sections (2.2.2 and 2.2.3) focus on ceramic and metallic nanoparticles. In sub-section 2.2.4 we will illustrate the potential application of bR/QD hybrid materials in bioelectronics. 2.2.1. Carbon nanotubes The application of carbon nanotubes in nanobiotechnology has become the subject of intense investigation since its discovery in 1991 [51]. Such considerable interest
ACCEPTED MANUSCRIPT reflects the unique behavior of CNT, including their high electrical conductivity, excellent biocompatibility, chemical stability and mechanical strength [52-55].
long-range
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and
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CNT with the advantages of high surface area, fast heterogeneous electron transfer, used
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develop
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nanobioelectronics in the last decade [29, 56-58]. Biomolecules (e.g., proteins and
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DNA) can also be electrostatically adsorbed onto the surface of CNTs and can be attached to functional groups on modified CNTs [59, 60].
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Bradley et al. demonstrated the integration of bacteriorhodopsin and nanotube
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network transistor as a nanoelectronic device [61]. It was found that both
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components kept their functionality while interacting with each other. The device
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provided additional details about some characteristics of bacteriorhodopsin like asymmetry of the bR charge distribution. Also, they suggested that with connecting the living cells directly to these nanoelectronic devices, monitoring of certain cell functions in different environments could be possible [61]. Integration of single-walled carbon nanotubes (SWNTs) and bR for potential use as building blocks for molecular optoelectronic devices was reported by Bertoncini and Chauvet [57]. They used sonication to debundle SWNT in buffer solution (pH 7.5) without surfactant before the addition of PM. Their results indicated that bR adsorbed onto the SWNT undergoes conformational changes [57]. Since the bR conformation is important in controlling the photocycle kinetics [62], it is expected
ACCEPTED MANUSCRIPT that the structural changes in bR will affect its function. However, based on the previous report on integration of bR and CNT transistors [61], it is stated that both
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components retain their functionality. Hence, it can be concluded that these
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conformational changes of bR are probably not enough to impair the function of
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protein.
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While in the previously mentioned report [61], wild-type bR was adsorbed on SWNTs; Ingrosso et al. investigated integration of D96N mutant of bR and
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SWNTs [58]; They effectively combined the sensitivity of D96N mutant with the
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ballistic transport of the electrons in SWNTs to fabricate an electronic sensor
The presence of these analytes causes the changes in
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and other vapors.
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device for detecting analytes as ammonia, aqueous vapor, chloroform, diethylether
electrochemical response of carbon nanotube field effect transistor (FET) or impedance sensors. In addition, they showed that the involvement of hydrophobic interactions between the SWNT walls and bR lead to a conformational change in protein [58]. Recently, it has been shown that the PM patches can wrap the SWNT (see Fig. 4) by π–π stacking, hydrophobic and electrostatic forces and bonds involving sp3 carbon atoms, concurrently undergoing a conformational change from the α I- to the αII-helix form [29]. The interaction between bR and SWNT is highly pH dependent. Due to a higher degree of nanotube functionalization, acid and basic
ACCEPTED MANUSCRIPT buffers are suitable for the interaction of SWNT with bR, while the binding of bR to SWNT markedly decreases at pH 7.5 [29]. Moreover, modification of the
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SWNT walls with the D96N mutant enhances the mechanical properties of carbon
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nanotube and p-dopes the nano-objects by charge transfer phenomena [29]. 2.2.2. Titanium dioxide nanowires
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A promising platform for the bioelectronics application of bR-nano hybrid
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materials has been reported by Li et al. [63]. They fabricated the bR modified TiO2 nanowires FET and investigated the effect of bR on the FET performance. Results
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showed that the dipole bio-originated from bR can tune the FET performance.
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After bR modification of the TiO2, the mobility of FET increased from 5.14 x 10-4
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cm2 V-1 s-1 (for bare TiO2 nanowires FET) to 9.93 x 10-4 cm2 V-1 s-1, under negative gate voltage (Fig. 5). Therefore, bR increased the hole mobility in the TiO2 nanowires FET and led to increase in both transfer and output characteristics for bR modified TiO2 nanowires FET in comparison with bare TiO2 nanowires FET [63]. 2.2.3. Silver NPs Among noble-metal nanomaterials, silver nanoparticles (Ag NPs) are one of the most commonly used metal-nanoparticles, which have received considerable attention in biological research [64, 65]. Due to their attractive physicochemical properties like the surface plasmon resonance, silver nanoparticles can affect the
ACCEPTED MANUSCRIPT bR photocycle and speed up the proton pumping process [66, 67]. So, integration of bR with Ag NPs can potentially allow for the fabrication of efficient nano-bio
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hybrid systems [66, 68].
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The effect of 40nm silver nanoparticles on the photocycle of bR has been studied
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using visible spectroscopy by Adamov et al. [67]. They reported that for bR
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modified Ag NPs, the plasmon resonance peak blue shifts (from 410 to 402 nm) but the position of the bacteriorhodopsin band remains unchanged (568 nm). In
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addition, the intensity of the absorption band at 400–410 nm was increased, with
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affect the bR photocycle [67].
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increasing the bR concentration. Therefore, it can be expected that nanoparticles
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It has been shown that when exposed to blue light, the M intermediate of bR returns to ground state in hundreds of nanoseconds. This bypassed photocycle, known as the blue light effect, significantly reduces the conventional photocycle period which takes 15 ms [69, 70]. Recently El-Sayed's group has found that the photocurrent generation from bR can be considerably enhanced in the presence of silver nanoparticles under illumination with blue light [66]. The surface plasmon resonance band of Ag NPs overlaps with the M intermediate absorption range (in the blue wavelength region) [70]. The plasmonic field of AgNPs greatly enhances the flux of blue photons which increases the blue light effect. This leads to shorten the timescale of the photocyle,
ACCEPTED MANUSCRIPT resulting in the enhanced photocurrent. So, plasmonic nanoparticle with both maximum plasmonic field and bR-Ag spectral overlap results in maximum
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photocurrent improvement [66]. For instance, 40-nm AgNPs are more efficient
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with the M intermediate absorption in bR [70].
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than the 8-nm, because of the much larger field strength and better spectral overlap
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Surface-enhanced Raman spectroscopy (SERS) is a well- known analytical method which has been extensively employed in the study of a wide range of biomolecules
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[71, 72]. Nabiev et al., for the first time, adsorbed the purple membrane on silver
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electrodes to study enhanced Raman spectra of bR [73, 74]. More recently, the
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large local electromagnetic fields induced by resonant surface plasmons of Ag NPs
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have been used in SERS for studying bR as a model membrane protein [75]. 2.2.4. Quantum dots
In this part of the paper we will present the details about bR-QD hybrids, their incredible features and the effect of quantum dots on bR activities. At first, quantum dots are going to be introduced. Quantum Dots (QDs) are core-shell containing semiconducting materials with broad absorption and narrow size-tunable photoluminescent (PL) emission spectra [76]. Thanks to their broad absorption spectra, QDs could be utilized as light harvesting nanoantenna when they are coupled with natural light harvesting systems [77]. This feature turns quantum dots to a perfect donor in FRET system.
ACCEPTED MANUSCRIPT FRET or Förster Resonance Energy Transfer is a phenomenon in which energy is transferred from a donor molecule in its excited state to an acceptor molecule in its
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FRET depends mainly on four factors [77, 78]:
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ground state by non-radiative dipole-dipole interaction [78]. The efficiency of
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1. Sixth power of distance between donor and acceptor.
maximum absorption.
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3. Emission quantum yield of donor.
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2. Spectral overlap between donor's maximum emission and acceptor's
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4. Dipole-dipole orientation between donor and acceptor.
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bR is a photoactive protein and similar to other photoactive proteins, it has a
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limited interaction with UV region of spectrum [77]. In other words, bR is sort of blind for these parts of spectrum. QDs can overcome this limitation by broaden absorption spectra of bR through FRET. QDs attachment to bR is being done in three ways [77, 79-82]: 1. electrostatic self-assembly 2. Bio-affinity interaction 3. Covalent conjugation Regarding the first method, although the surface of the PM is negatively charged overall, but where bR faces both outer and inner side of the membrane, it has some positively charged residues. Subsequently, it has some electrostatic interactions
ACCEPTED MANUSCRIPT with negatively charged QDs [77, 79-81]. Bio-affinity interaction is created between streptavidin-coated QD and biotin-labeled bR or PM by natural affinity of
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streptavidin to biotin [77, 82]. For covalent conjugation, QDs should be
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functionalized with one or more functional groups such as carboxyl groups. PEG-
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COOH-coated QDs are also being used for QD-bR attachment through a covalent
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interaction between an amino group of protein and the carboxyl group of QD [77, 79]. Among all these three methods, the electrostatic self-assembly is the easiest,
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fastest and most economical method [77].
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Although, the PM patches are usually several hundreds of nanometers in size, their
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thickness is almost 5nm (Fig. 6) [83]. Retinal is located at the center of bR in 2.5
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nm depth from each side of the membrane. As QD is immobilized on the surface of PM, minimum distance between QD and retinal is 2.5 nm and maximum distance is less than average suitable distance between donor and acceptor for FRET [77]. In 2012, Bounchonville et al. studied correlation of FRET efficiency between QD and bR with three factors consisting of Förster radius, donor emission and acceptor absorption spectrum overlap, and distance between QD and bR. They demonstrated that accurate control of distance between QD and bR and reasonable overlap between QD emission and bR absorption spectrum result in complete control on FRET efficiency. They showed that electrostatic self-assembled QDstreptavidin on PM-biotin has the highest FRET efficiency [84].
ACCEPTED MANUSCRIPT QD quenching in QD-bR hybrids occurs as powerful UV lights are absorbed by QD and are transferred to bR by FRET. But there is still an unclear point that
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should be proven. How do we know QD quenching happens because of energy
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transfer to bR during FRET? How can we prove that other phenomenon is not
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engaged with this quenching? Griep et al. designed an experiment to demonstrate
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QD quenching as a result of energy transfer to bR through FRET. They fixed QDs on three different substrates: 1) Glass 2) Bleached bR (bR without retinal) 3) bR.
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Then, they examined fluorescent properties of QDs. Fluorescent spectrums of QD
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on glass and bleached bR substrate were the same, but it showed significant
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decrease in case it was fixed on bR (Fig. 7) [82]. In 2010, Nabiev's group also
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demonstrated the quenching of QD in bR-QD hybrids by FRET. They used white membranes (WMs), PMs without retinal, as a control material due to studying FRET between bR and QDs. Structural and morphological similarity of protein and lipid component in WMs and PMs, provides equal chance for WMs and PMs to nonradiative quenching of QD throw their binding to either membranes. Their study showed 4-5 times less quenching of QDs by WMs than that of by PMs [79] . These experiments declare that FRET is the main mechanism of QD quenching in bR-QD hybrids.
ACCEPTED MANUSCRIPT Besides FRET, QD affects bR in some ways which are not directly related to FRET. For instance because of being charged particles with intrinsic dipole
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moments, QDs have a significant influence on photocycle of bacteriorhodopsin. In
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2014, Zaitsev et al. acknowledge that QD570 prolong M-intermediate lifetime by an
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unclear mechanism [85]. Metastable M-intermediate is considered to be the most
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important intermediate for optical device applications, so the integration of bR with QDs will lead to more efficient optical devices.
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QDs can improve the performance of proton pumping of bR especially in low
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QD/bR molar ratios [79]. Indeed, bR uses the extra absorbed energy, provided by
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QDs, to improve the proton pumping process. In order to prove this fact, Rakovich
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and her colleagues designed an interesting test [79]. They embedded bR in proteoliposomes membrane inside out. By using this trick, bR which ordinary pumps protons to outside environment of membrane, pumps protons to the inside of the proteoliposoms, so the pH of outside solution rises. They showed that compare to solitary bR, bR-QD hybrids have got more than 25% increment in proton pumping [79]. Because of their notable properties, bR-QD hybrids can be utilized in various fields especially in nano-biosensor applications. In 2011 Griep et al. constructed a nanobiosensor platform based on QD-bR [86]. They used bR as transducer in the bio-nano sensing device and reported that by FRET mechanism, electrical output
ACCEPTED MANUSCRIPT of bR has been improved up to 300% and sensing ability of device has been prospered. Maltose molecule has been chosen as analyte for sensing. Due to the
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fact that this system is not capable of sensing any molecule intrinsically, they used
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layer of MBPs (Maltose binding proteins) and immobilized it on a layer of QD-bR
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(Fig. 8). In order to quench QDs, They used dark quenchers (DQ). DQs and
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maltose are competitor of each other in binding to MBP. Two-layered sensing device containing QD and bR layers has a significant 0.7 mV signal. Adding a
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layer of Maltose Binding proteins to this system has reduced the intensity of signal
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but not that much. The dramatic reduction in photovoltage has occurred in the
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system when DQs has been added to previous layers. As DQ quenches QD, no
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energy transfer occurs between QD and bR; so, electrical output of sensing device decreased. By replacing the DQs by Maltose molecules photovoltage increased. This was the first nanobiosensor based on QD-bR hybrids for real time detection of Maltose [86].
Bouchonvill et al. after several hours of observing bR-QD by AFM, recognized in the presence of some detergents like Triton X100, QD accelerated bR monomerization process [87]. Monomeric and trimeric bR are different in some properties; for instance bR in its monomeric state has lower thermal stability than trimeric one [88]. So all interactions between QD and bR are not desired and for
ACCEPTED MANUSCRIPT constructing bR-QD hybrids and applying them in various fields it's better to consider every aspects.
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To conclude, QDs mostly improve optical absorption, photocycle, and proton
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pumping of bR.
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3. Summary and perspectives
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Biotechnology, nanotechnology and novel immobilization strategies broaden scientist’s horizons toward nano-biomaterials and nano-bio hybrid systems.
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Therefore, they are becoming more and more widely used.
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Nano-bio hybrid materials based on bacteriorhodopsin are widely studied in the
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field of bioelectronics and energy conversion. bR-based solar energy conversion
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devices, such as biomolecule-sensitized solar cells and photoelectrochemical cells are economical, green and relatively efficient but they are still in the evolution stage. In order to achieve high power conversion efficiency, further studies will also be required to investigate following issues: 1. Enhancement of charge transfer rate in semiconductor- biomolecule interface. 2. Co-sensitization of bR with QDs or biomolecules with complementary absorption spectra. 3.
Development of bR mutants with increased stability and high quantum
efficiency of the photoconversion.
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improvement of both bR and nanomaterials. For instance, quantum dots with two
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noticeable properties including broad absorption region and adjustable sharp
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emission spectrum by size can improve bR optical cycle, features and proton
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pumping.
On the other hand, the attachment of bR to the surface of nanostructures is another
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important step. The strong bonding provides an obstacle to the removal of bR from
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the surfaces of the nanomaterials. Also, for some applications, such as BSSCs,
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charge transfer rate in nanostructure/bR interface must be considered. Time, pH,
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and temperature are the important factors for the protein immobilization. However, the integration of bR with the above mentioned materials have been proposed for various applications in bioelectronics. There are plenty of materials to be conjugated with bR. As an example, according to the reports on light-controlled spin filtering in bR [89], magnetic nanoparticles such as nickel and iron oxide can be an area of interest for further research. Considering all of these aspects, it can be stated that these new hybrid bionanomaterials with excellent physical, chemical, and biological properties offer the potential to develop new bio-photonic devices. Acknowledgments
ACCEPTED MANUSCRIPT The authors are grateful to Dr. Naimeh Naseri for critical comments on the manuscript. We gratefully acknowledge financial support from Young Researchers
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& Elite Club.
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References
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[89] Einati H, Mishra D, Friedman N, Sheves M, Naaman R. Nano Lett. 2015;15:1052. [90] Ito S, Murakami TN, Comte P, Liska P, Grätzel C, Nazeeruddin MK, et al. Thin Solid Films. 2008;516:4613.
ACCEPTED MANUSCRIPT Table 1 Photoelectrochemical parameters of bR-based BSSCs and DSSC.
Nanostructured
scattering
TiCl4
Jsc −2
layer
treatment
(mA cm )
3Glu mutant
TiO2 NPs
no
no
0.09
wild-type bR
ZnO NPs
no
no
0.39
bR+bacterioruberin
TiO2 NPs
no
yes
0.45
wild-type bR
TiO2 NPs
yes
yes
1
N719
TiO2 NPs
yes
yes
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18.2
Efficiency
(mV)
(%)
350
-
[28]
500
0.1
[46]
570
0.16
[42]
533
0.35
[22]
789
10.1
[90]
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semiconductor
Voc
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Sensitizer
Reference
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Figure 2. (a) Schematic illustration of biomolecule-sensitized solar cell, utilizing bR as
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biophotosensitizer. (b) Simple energy level diagram of BSSC; the absorption of light by bR
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causes the excitation of electrons from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), resulting in injection of an electron from bR into
the electrolyte. Adapted from reference [22].
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conduction band of the TiO2 that causes to charge separation and transportation of holes through
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Figure 3. Schematic illustration of bR-based solar H2 generation systems: (a) electrode systems and (b) particle systems. There are two possible functions for bR: 1.proton pumping 2.visible
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light-harvesting.
Figure 4. TEM and AFM images of bare SWNTs (a and c) and SWNTs modified by PM patches
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(b and d). Adapted from reference [29].
Figure 5. Mobility of the bare and bR modified FET. After modification with bR, the mobility of the TiO2 nanowires FET is increased by a factor of 2. Adapted from reference [63]. Figure 6. Schematic of FRET between QDs (donor) and PM (acceptor). Figure 7. QD emission spectrum. The emission spectrum of QDs while attached to bleached bR, the glass (control) and bR. Adapted from reference [82]. Figure 8. a) QD-PM, the photovoltage is approximately 0.7 mV. b) QD-PM-MBP, the photovoltage decreases to 0.6 mV. c) There is a dramatic decrease in QD-PM-MBP/DQ photovoltage which barely reaches to 0.2 mV. d) QD-PM-MBP/QD + Maltose, compared to c plot the photovoltage has risen. Adapted from reference [86].
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Figure 1. The photocycle of bacteriorhodopsin.
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Figure 2. (a) Schematic illustration of biomolecule-sensitized solar cell, utilizing
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bR as biophotosensitizer. (b) Simple energy level diagram of BSSC; the absorption of light by bR causes the excitation of electrons from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), resulting in injection of an electron from bR into conduction band of the TiO 2 that causes to charge separation and transportation of holes through the electrolyte. Adapted from reference [22].
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Figure 3. Schematic illustration of bR-based solar H2 generation systems: (a) electrode systems and (b) particle systems. There are two possible functions for bR: 1.proton pumping 2.visible light-harvesting.
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Figure 4. TEM and AFM images of bare SWNTs (a and c) and SWNTs modified by PM patches (b and d). Adapted from reference [29].
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Figure 5. Mobility of the bare and bR modified FET. After modification with bR, the mobility of the TiO2 nanowires FET is increased by a factor of 2. Adapted from reference [63].
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Figure 6. Schematic of FRET between QDs (donor) and PM (acceptor).
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Figure 7. QD emission spectrum. The emission spectrum of QDs while attached to bleached bR, the glass (control) and bR. Adapted from reference [82].
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Figure 8. a) QD-PM, the photovoltage is approximately 0.7 mV. b) QD-PMMBP, the photovoltage decreases to 0.6 mV. c) There is a dramatic decrease in QD-PM-MBP/DQ photovoltage which barely reaches to 0.2 mV. d) QD-PMMBP/QD + Maltose, compared to c plot the photovoltage has risen. Adapted from reference [86].
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Graphical abstract
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Bacteriorhodopsin is promising for use in bioelectronics and energy
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conversion.
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QDs can improve the proton pumping performance of bR.
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There are two basic approaches to achieve bR based hydrogen generation.
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Charge transfer rate in bR-TiO2 interface is effective on BSSCs performance.
S0001-8686(15)00169-4 doi: 10.1016/j.cis.2015.09.006 CIS 1578
To appear in:
Advances in Colloid and Interface Science
Please cite this article as: Mahyad Baharak, Janfaza Sajjad, Hosseini Elaheh Sadat, Bionano hybrid materials based on bacteriorhodopsin: Potential applications and future strategies, Advances in Colloid and Interface Science (2015), doi: 10.1016/j.cis.2015.09.006
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Bio-nano hybrid materials based on bacteriorhodopsin:
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Potential applications and future strategies
a
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Baharak Mahyada, Sajjad Janfazab,a*, Elaheh Sadat Hosseinia
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares
b
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University, Jalal Ale Ahmad Highway, Tehran 14117, Iran
Young Researchers & Elite Club, Pharmaceutical Sciences Branch, Islamic Azad University,
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Tehran, Iran
*Corresponding author:
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Abstract:
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Email: [email protected]; Phone: +98-21-22954285
This review presents an overview of recent progress in the development of bionano hybrid materials based on the photoactive protein bacteriorhodopsin (bR). The interfacing of bR with various nanostructures including colloidal nanoparticles (such as quantum dots and Ag NPs) and nanoparticulate thin films (such as TiO2 NPs and ZnO NPs,) has developed novel functional materials. Applications of these materials are comprehensively reviewed in two parts: bioelectronics and solar energy conversion. Finally, some perspectives on possible future strategies in bR-based nanostructured devices are presented.
Keywords:
Bacteriorhodopsin,
bioelectronics, energy conversion.
nanomaterials,
bio-nano
hybrid
systems,
ACCEPTED MANUSCRIPT Contents 1. Introduction ....................................................................................................... 3
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2. Applications ....................................................................................................... 6
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2.1. Solar energy conversion .............................................................................. 6
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2.1.1 biomolecule-sensitized solar cells.......................................................... 6 2.1.2 Hydrogen production ............................................................................. 9
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2.2. Bioelectronics ........................................................................................... 12
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2.2.1. Carbon nanotubes ............................................................................... 12 2.2.2. Titanium dioxide nanowires ............................................................... 15
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2.2.3. Silver NPs .......................................................................................... 15
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2.2.4. Quantum dots ..................................................................................... 17
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3. Summary and perspectives ............................................................................... 17 Acknowledgments ............................................................................................... 24 References ........................................................................................................... 25
ACCEPTED MANUSCRIPT 1. Introduction Nanotechnology provides the ability to manipulate and characterize materials at
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the atomic and molecular levels and this has driven extensive research into
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possible applications of nanomaterials. This new emerging multidisciplinary field
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can be thought of an intersection of chemical, physical, biological, and engineering
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sciences on a scale of ∼1–100 nm [1, 2]. Over the last decade, combination of
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nanomaterials and biomolecules including nucleic acids, proteins and antibodies has been developed as a new interdisciplinary research field [3-6]. Among the bio-
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nano hybrids, integration of proteins with engineered nanomaterials has attracted
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materials [3-6].
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particular interest and has led to the design and development of new advanced
Bacteriorhodopsin (bR) is a unique photochromic protein that has been successfully incorporated with various nanomaterials, in order to develop novel nano-bio hybrid materials and nanostructured devices [7, 8]. This nano-scale molecular machine pumps protons across a purple membrane of Halobacterium salinarum powered by sunlight [9-11]. Bacteriorhodopsin contains seven transmembrane helices embedding a retinal chromophore in the middle that is linked to the side chain of lysine 216 (Lys216) via a protonated Schiff base [12]. The proton pumping of this protein is coupled with photochemical conversions, occurring during a photocycle of bR [13-18]. The
ACCEPTED MANUSCRIPT main photocycle of bR including several spectrally different intermediates is shown in Fig. 1.
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Each intermediate has a distinct absorbance maximum and different life times. The
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intermediates are commonly represented by a single letter code where the index
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represents the absorption maximum [13]. When exposed to light at 568 nm, the
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molecules in the initial state (bR568) convert to the short-living J625 and proceed to the K590 state [14-16]. During these initial steps, the isomerization from all-trans to
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13-cis retinal occurs. This photoreaction initiates a cyclic sequence of thermal
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conversions (L550, M412, N560, and O640), which finally lead back to the bR568 state
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[17, 18]. This rapid isomerization of retinal in combination with well-defined
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conformational changes in bR during the photocycle leads to the transport of a proton from the cytoplasmic to the extracellular side of the membrane [19]. The interesting point about the photocycle is that all the intermediate states are able to be photo-chemically switched back to the bR568 state by shining light at a wavelength that corresponds to the absorption peak of the respective intermediates [20, 21]. Its unique properties have made bR a promising material for a wide range of applications. The major advantages of the bR include [22-25]: (1) high quantum efficiency of converting light into a state charge, (2) wide range absorption of visible light, (3) high thermal and photochemical stability, (4) extremely large
ACCEPTED MANUSCRIPT optical nonlinearities, (5) robustness to degeneration by environmental perturbations, (6) low production cost, (7) environmental friendliness, and (8) the
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existence of genetic variants with enhanced spectral properties for specific device
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applications.
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Various mutants of bR have been reported to be used in bioelectronics as well as
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the native one [26-29]. Two classes of bR mutants have been suggested for solar cell application [30]. The first one includes the mutants, such as 3Glu, in which
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glutamate (Glu) is replaced by glutamine (Gln) [31, 32]. These replaced residues
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include Glu9, Glu74, Glu194 and Glu204 and are placed in the outer side of
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membrane. In comparison to native bR, these mutants are more capable of
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transferring electrons through redox electrolyte to the anode [28, 31, 32]. The second group of the mutant bR consists of cysteine (Cys), unlike the native. These mutants have Cys in 3, 36 and 247 positions of amino acid sequence and are able to attach to the gold surface by their sulfhydryl group through covalent bond [30]. Also, the mutant D96N with aspartic acid-96 to asparagine replacement [33, 34] has been well studied for various bioelectronics applications. Over the last two decades, some potential applications of both mutants and wildtype bR have been proposed ranging from medicine to electronics [35-40]. On the other hand, bR can be interfaced with various nanomaterials for fabrication of highly efficient devices and advanced functional materials. In more recent years,
ACCEPTED MANUSCRIPT nano-bio hybrid materials based on bacteriorhodopsin have attracted broad attention because of their excellent physical, chemical, and biological properties
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and its potential use in a variety of medical, optic, optoelectronic, and
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environmental applications.
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This review looks at bacteriorhodopsin-nanomaterials hybrids as promising
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materials for use in bioelectronics and energy conversion. We discuss here how bR can be conjugated to various nanomaterials such as carbon nanotubes (CNT), TiO2
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NPs, ZnO NPs, and quantum dots (QDs) in order to improve its functional
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2. Applications
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properties.
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2.1. Solar energy conversion
Renewable energies have attracted more and more attention due to the rise in energy consumption and environmental concerns. Nano-Bio hybrid materials based on semiconductor nanostructures and bacteriorhodopsin protein have been successfully used for solar energy conversion systems such as photovoltaic cells and water splitting photoelectrochemical cells. 2.1.1 biomolecule-sensitized solar cells Biomolecule-sensitized solar cells (BSSCs) have been developed as a promising “green” technology for low-cost photovoltaic power generation [41-43]. The mechanism of BSSCs is very similar to dye-sensitized solar cells (DSSCs). DSSCs
ACCEPTED MANUSCRIPT appeared as a new generation of photovoltaic device and have attracted much attention as potential alternatives to traditional photovoltaic devices [41]. The
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advantages of DSSCs over silicon-based solar cells lie in its simple production
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method, varieties of material sources, the low cost and possible fabrication of
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those of silicon-based solar cells [44, 45].
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flexible solar cells, even though their performances are still low compared with
In bR based BSSCs, the photoanode is made of a porous semiconductor
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nanoparticles (mainly TiO2 or ZnO NPs) sensitized with bacteriorhodopsin protein
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[22, 46].
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bR with a wide absorption range in the visible region, harvests the solar light,
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going up to the excited state, and injects photo-excited electrons into the conduction band (CB) of semiconductor. The CB of TiO2 is located at a lower energy level than the lowest occupied molecular orbital (LUMO) of the bR molecule, so injection of the photoelectrons from bR to TiO2 is energetically favorable [22]. The electrons migrate to the counter electrode through the external circuit. The oxidized bR accepts electrons from the electrolyte, regenerating the ground state (Figure 2) [22, 28]. The application of bR as a sensitizer in BSSCs was first reported by Thavasi et al.,[28] in which bR was involved in photo induced charge transfer into the titanium dioxide (TiO2) semiconductor upon immobilization. They demonstrated
ACCEPTED MANUSCRIPT that the electrostatic potential map of 3Glu mutant showed better likelihood to bind efficiently to TiO2, which led to higher photoelectric response of the bio-solar cell
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composed of the 3Glu mutant bR, than that composed by the wild type bR. In 3Glu
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mutant, three negative charged Glu residues (E9, E194 and E204) have been
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mutated to Gln (at the EC site of bR) which strongly changes the surface potential
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map of bR. This changes in potential map of bR most likely results in better attachment of bR to exposed oxygen atoms of anatase surface [28].
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Recently, Molaeirad et al. have demonstrated that enhanced photovoltaic
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performance of BSSCs can be achieved by using the co-sensitization of bR and
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bacterioruberin, which are complementary in their spectral responses [42]. More
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recently, they have immobilized an Langmuir−Blodgett (LB) monolayer of bacteriorhodopsin on ZnO nanoporous film and improved the bio-solar cell performance [46]. ZnO nanoparticles with high isoelectric point (IEP) of about 9.5 is an appropriate substrate to immobilize bR, a low IEP protein, by electrostatic interactions with high binding stability [46]. On the other hand, loading the nanostructured photoelectrode with more than one layer of bR can significantly decrease the charge transfer rate in the TiO2/bR interface [22]. Therefore, higher efficiency of the photovoltaic cell can be ascribed to immobilization of LB monolayer of bacteriorhodopsin on the ZnO nanoparticulate film as well as those intrinsic features of ZnO thin film.
ACCEPTED MANUSCRIPT In 2015, Mohammadpour and Janfaza designed a highly efficient bR based BSSC with optimization of the materials at the nano-bio interface and also morphology
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design [22]. They showed that TiCl4 treatment of TiO2 nanoparticle surface and
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controlling the bR loading time could enhance the charge transfer rate in TiO2/bR
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interface. Thus, by employing these two strategies, as well as coating a layer of
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light-scattering paste consists of TiO2 nanofibers, the power conversion efficiency of 0.35%, open circuit voltage (Voc) of 533 mV, and short-circuit current of (Jsc) of
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1 mA cm-2 were obtained [22]. Table 1 summarizes photovoltaic characteristics
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of different bR-based BSSCs reported in the literature.
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However, efficiency of bR-based BSSCs is not high enough compared with
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DSSCs. In contrast to conventional DSSCs that use expensive and toxic ruthenium complexes, bR-based BSSCs employ a low cost, renewable, environment friendly, and non-carcinogenic without any disposal problems as light harvesters [42, 43]. The hope is that the novel bio-sensitizers, like wild-type bR and its mutants, will be acceptable alternative to common dyes. 2.1.2 Hydrogen production Bio-conjugated nanomaterials seem to be very promising for solar hydrogen generation systems [47-50]. Bacteriorhodopsin has been considered as an appropriate biomolecule for incorporation with nanosemiconductors to produce hydrogen via water-splitting. There are two basic approaches to achieve bR based
ACCEPTED MANUSCRIPT solar hydrogen generation: a particle system and an electrode system, as schematically presented in Fig. 3.
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Allam et al. reported the assembly and use of a bR/TiO2 nanotube array (TNA)
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hybrid electrode system for enhanced photoelectrochemical water splitting [47].
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bR was attached onto TiO2 surfaces either with or without linker. Physically
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adsorbed protein showed instability upon conducting the photoelectrochemical tests. But, when using 3-Mercaptopropionic acid as a linker, high stability of the
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fabricated hybrid electrode was revealed [47]. Under the illumination of AM 1.5, a
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photocurrent density of 0.65 mA/cm2 was recorded which was a 50% increase over
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that measured for pure TiO2 nanotubes (0.43 mA/cm2) fabricated and tested under
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the same conditions. They proposed that the enhanced photocurrent generation is because of the proton pumping effect of bR. In addition, in the presence of redox electrolyte (0.02M I-/I3-), the maximum photocurrent of 0.87 mA/cm2 was obtained. This 33 per cent increase in the photocurrent can be mainly attributed to the electron transfer effect of bR [47]. Recently, Naseri et al. have decorated TiO2 nanoparticulate (TNP) and TNA electrodes with bR which caused a several-times increase in photoresponse of the systems (~ 7 and 1.7 times for TiO2 nanoparticulate and nanotubular film, respectively) [48]. They attributed the higher photoactivity improvement for TNP system than TNA one to more surface sites in nanoparticulate film for attachment
ACCEPTED MANUSCRIPT of submicron PM fragments. The photocurrent density of 0.64 mA cm-2 in the bias of +0.5 V was obtained for bR/TNP electrode, under white light irradiation [48].
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Therefore, based on these above-mentioned reports, remarkably improved
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modifying the nanostructured electrodes with bR.
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performances of photoelectrochemical water splitting can be achieved when
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It is of interest to compare photocurrents of bR-based photoelectrochemical water splitting systems with those of bR-sensitized solar cells. Results show that BSSCs
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can generate higher photocurrents under the same illumination conditions.
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The application of bacteriorhodopsin immobilized on Pt/TiO2 photocatalyst for
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visible light-driven hydrogen generation has been studied by A. Rozhkova group
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[49]. Under green and white light illumination in the presence of methanol (at pH 7), the nano-bio hybrid system produced 207 and 5275 μmole of H2 (μmole protein)−1 h−1 , respectively. This 25.5-fold increase in the photocatalytic efficiency of the system under white light illumination can also be due to additional electrons coming from UV excitation of TiO2 nanoparticles. In addition, they stated that bR can act as a visible light-harvesting molecule as well as, a proton pump [49]. Recently, it has been demonstrated that reduced graphene oxide (rGO) can improve photocatalytic performance of the above mentioned bR based hybrid system [50]. Under white light irradiation, the presence of the rGO causes hydrogen production rates of approximately 11.24 mmol of H2 (μmol protein)-1 h-1
ACCEPTED MANUSCRIPT which is 2 times higher than that of the Pt/TiO2-bR nano-bio catalyst alone without rGO (5.38 mmol of H2 (μmol protein)-1 h-1)
[50]. It is proposed that rGO acts
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similarly to bR and helps to sensitize TiO2 to visible light. Moreover, rGO as a
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nanoscaffold promotes an interface between bR, TiO2 NPs, and Pt NPs [50]. 2.2. Bioelectronics
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The use of different nanoscale materials in various fields, including physics,
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chemistry, biology and electronics have grown explosively, over the last decade. In recent years, the incorporation of bR as a unique photo-active protein and
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nanomaterials such as metallic and ceramic nanostructures has been studied to
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enhance the performance of both protein and nanostructures. These combinations
applications.
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offer promising strategies to fabricate novel nanobiomaterials for bioelectronics
The first sub-section (2.2.1) focuses on carbon nanotube as a favorable nanostructure in bioelectronics field. The next sub-sections (2.2.2 and 2.2.3) focus on ceramic and metallic nanoparticles. In sub-section 2.2.4 we will illustrate the potential application of bR/QD hybrid materials in bioelectronics. 2.2.1. Carbon nanotubes The application of carbon nanotubes in nanobiotechnology has become the subject of intense investigation since its discovery in 1991 [51]. Such considerable interest
ACCEPTED MANUSCRIPT reflects the unique behavior of CNT, including their high electrical conductivity, excellent biocompatibility, chemical stability and mechanical strength [52-55].
long-range
electron
transfer,
has
been
widely
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and
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CNT with the advantages of high surface area, fast heterogeneous electron transfer, used
to
develop
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nanobioelectronics in the last decade [29, 56-58]. Biomolecules (e.g., proteins and
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DNA) can also be electrostatically adsorbed onto the surface of CNTs and can be attached to functional groups on modified CNTs [59, 60].
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Bradley et al. demonstrated the integration of bacteriorhodopsin and nanotube
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network transistor as a nanoelectronic device [61]. It was found that both
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components kept their functionality while interacting with each other. The device
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provided additional details about some characteristics of bacteriorhodopsin like asymmetry of the bR charge distribution. Also, they suggested that with connecting the living cells directly to these nanoelectronic devices, monitoring of certain cell functions in different environments could be possible [61]. Integration of single-walled carbon nanotubes (SWNTs) and bR for potential use as building blocks for molecular optoelectronic devices was reported by Bertoncini and Chauvet [57]. They used sonication to debundle SWNT in buffer solution (pH 7.5) without surfactant before the addition of PM. Their results indicated that bR adsorbed onto the SWNT undergoes conformational changes [57]. Since the bR conformation is important in controlling the photocycle kinetics [62], it is expected
ACCEPTED MANUSCRIPT that the structural changes in bR will affect its function. However, based on the previous report on integration of bR and CNT transistors [61], it is stated that both
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components retain their functionality. Hence, it can be concluded that these
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conformational changes of bR are probably not enough to impair the function of
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protein.
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While in the previously mentioned report [61], wild-type bR was adsorbed on SWNTs; Ingrosso et al. investigated integration of D96N mutant of bR and
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SWNTs [58]; They effectively combined the sensitivity of D96N mutant with the
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ballistic transport of the electrons in SWNTs to fabricate an electronic sensor
The presence of these analytes causes the changes in
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and other vapors.
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device for detecting analytes as ammonia, aqueous vapor, chloroform, diethylether
electrochemical response of carbon nanotube field effect transistor (FET) or impedance sensors. In addition, they showed that the involvement of hydrophobic interactions between the SWNT walls and bR lead to a conformational change in protein [58]. Recently, it has been shown that the PM patches can wrap the SWNT (see Fig. 4) by π–π stacking, hydrophobic and electrostatic forces and bonds involving sp3 carbon atoms, concurrently undergoing a conformational change from the α I- to the αII-helix form [29]. The interaction between bR and SWNT is highly pH dependent. Due to a higher degree of nanotube functionalization, acid and basic
ACCEPTED MANUSCRIPT buffers are suitable for the interaction of SWNT with bR, while the binding of bR to SWNT markedly decreases at pH 7.5 [29]. Moreover, modification of the
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SWNT walls with the D96N mutant enhances the mechanical properties of carbon
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nanotube and p-dopes the nano-objects by charge transfer phenomena [29]. 2.2.2. Titanium dioxide nanowires
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A promising platform for the bioelectronics application of bR-nano hybrid
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materials has been reported by Li et al. [63]. They fabricated the bR modified TiO2 nanowires FET and investigated the effect of bR on the FET performance. Results
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showed that the dipole bio-originated from bR can tune the FET performance.
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After bR modification of the TiO2, the mobility of FET increased from 5.14 x 10-4
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cm2 V-1 s-1 (for bare TiO2 nanowires FET) to 9.93 x 10-4 cm2 V-1 s-1, under negative gate voltage (Fig. 5). Therefore, bR increased the hole mobility in the TiO2 nanowires FET and led to increase in both transfer and output characteristics for bR modified TiO2 nanowires FET in comparison with bare TiO2 nanowires FET [63]. 2.2.3. Silver NPs Among noble-metal nanomaterials, silver nanoparticles (Ag NPs) are one of the most commonly used metal-nanoparticles, which have received considerable attention in biological research [64, 65]. Due to their attractive physicochemical properties like the surface plasmon resonance, silver nanoparticles can affect the
ACCEPTED MANUSCRIPT bR photocycle and speed up the proton pumping process [66, 67]. So, integration of bR with Ag NPs can potentially allow for the fabrication of efficient nano-bio
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hybrid systems [66, 68].
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The effect of 40nm silver nanoparticles on the photocycle of bR has been studied
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using visible spectroscopy by Adamov et al. [67]. They reported that for bR
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modified Ag NPs, the plasmon resonance peak blue shifts (from 410 to 402 nm) but the position of the bacteriorhodopsin band remains unchanged (568 nm). In
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addition, the intensity of the absorption band at 400–410 nm was increased, with
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affect the bR photocycle [67].
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increasing the bR concentration. Therefore, it can be expected that nanoparticles
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It has been shown that when exposed to blue light, the M intermediate of bR returns to ground state in hundreds of nanoseconds. This bypassed photocycle, known as the blue light effect, significantly reduces the conventional photocycle period which takes 15 ms [69, 70]. Recently El-Sayed's group has found that the photocurrent generation from bR can be considerably enhanced in the presence of silver nanoparticles under illumination with blue light [66]. The surface plasmon resonance band of Ag NPs overlaps with the M intermediate absorption range (in the blue wavelength region) [70]. The plasmonic field of AgNPs greatly enhances the flux of blue photons which increases the blue light effect. This leads to shorten the timescale of the photocyle,
ACCEPTED MANUSCRIPT resulting in the enhanced photocurrent. So, plasmonic nanoparticle with both maximum plasmonic field and bR-Ag spectral overlap results in maximum
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photocurrent improvement [66]. For instance, 40-nm AgNPs are more efficient
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with the M intermediate absorption in bR [70].
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than the 8-nm, because of the much larger field strength and better spectral overlap
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Surface-enhanced Raman spectroscopy (SERS) is a well- known analytical method which has been extensively employed in the study of a wide range of biomolecules
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[71, 72]. Nabiev et al., for the first time, adsorbed the purple membrane on silver
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electrodes to study enhanced Raman spectra of bR [73, 74]. More recently, the
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large local electromagnetic fields induced by resonant surface plasmons of Ag NPs
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have been used in SERS for studying bR as a model membrane protein [75]. 2.2.4. Quantum dots
In this part of the paper we will present the details about bR-QD hybrids, their incredible features and the effect of quantum dots on bR activities. At first, quantum dots are going to be introduced. Quantum Dots (QDs) are core-shell containing semiconducting materials with broad absorption and narrow size-tunable photoluminescent (PL) emission spectra [76]. Thanks to their broad absorption spectra, QDs could be utilized as light harvesting nanoantenna when they are coupled with natural light harvesting systems [77]. This feature turns quantum dots to a perfect donor in FRET system.
ACCEPTED MANUSCRIPT FRET or Förster Resonance Energy Transfer is a phenomenon in which energy is transferred from a donor molecule in its excited state to an acceptor molecule in its
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FRET depends mainly on four factors [77, 78]:
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ground state by non-radiative dipole-dipole interaction [78]. The efficiency of
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1. Sixth power of distance between donor and acceptor.
maximum absorption.
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3. Emission quantum yield of donor.
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2. Spectral overlap between donor's maximum emission and acceptor's
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4. Dipole-dipole orientation between donor and acceptor.
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bR is a photoactive protein and similar to other photoactive proteins, it has a
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limited interaction with UV region of spectrum [77]. In other words, bR is sort of blind for these parts of spectrum. QDs can overcome this limitation by broaden absorption spectra of bR through FRET. QDs attachment to bR is being done in three ways [77, 79-82]: 1. electrostatic self-assembly 2. Bio-affinity interaction 3. Covalent conjugation Regarding the first method, although the surface of the PM is negatively charged overall, but where bR faces both outer and inner side of the membrane, it has some positively charged residues. Subsequently, it has some electrostatic interactions
ACCEPTED MANUSCRIPT with negatively charged QDs [77, 79-81]. Bio-affinity interaction is created between streptavidin-coated QD and biotin-labeled bR or PM by natural affinity of
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streptavidin to biotin [77, 82]. For covalent conjugation, QDs should be
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functionalized with one or more functional groups such as carboxyl groups. PEG-
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COOH-coated QDs are also being used for QD-bR attachment through a covalent
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interaction between an amino group of protein and the carboxyl group of QD [77, 79]. Among all these three methods, the electrostatic self-assembly is the easiest,
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fastest and most economical method [77].
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Although, the PM patches are usually several hundreds of nanometers in size, their
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thickness is almost 5nm (Fig. 6) [83]. Retinal is located at the center of bR in 2.5
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nm depth from each side of the membrane. As QD is immobilized on the surface of PM, minimum distance between QD and retinal is 2.5 nm and maximum distance is less than average suitable distance between donor and acceptor for FRET [77]. In 2012, Bounchonville et al. studied correlation of FRET efficiency between QD and bR with three factors consisting of Förster radius, donor emission and acceptor absorption spectrum overlap, and distance between QD and bR. They demonstrated that accurate control of distance between QD and bR and reasonable overlap between QD emission and bR absorption spectrum result in complete control on FRET efficiency. They showed that electrostatic self-assembled QDstreptavidin on PM-biotin has the highest FRET efficiency [84].
ACCEPTED MANUSCRIPT QD quenching in QD-bR hybrids occurs as powerful UV lights are absorbed by QD and are transferred to bR by FRET. But there is still an unclear point that
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should be proven. How do we know QD quenching happens because of energy
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transfer to bR during FRET? How can we prove that other phenomenon is not
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engaged with this quenching? Griep et al. designed an experiment to demonstrate
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QD quenching as a result of energy transfer to bR through FRET. They fixed QDs on three different substrates: 1) Glass 2) Bleached bR (bR without retinal) 3) bR.
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Then, they examined fluorescent properties of QDs. Fluorescent spectrums of QD
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on glass and bleached bR substrate were the same, but it showed significant
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decrease in case it was fixed on bR (Fig. 7) [82]. In 2010, Nabiev's group also
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demonstrated the quenching of QD in bR-QD hybrids by FRET. They used white membranes (WMs), PMs without retinal, as a control material due to studying FRET between bR and QDs. Structural and morphological similarity of protein and lipid component in WMs and PMs, provides equal chance for WMs and PMs to nonradiative quenching of QD throw their binding to either membranes. Their study showed 4-5 times less quenching of QDs by WMs than that of by PMs [79] . These experiments declare that FRET is the main mechanism of QD quenching in bR-QD hybrids.
ACCEPTED MANUSCRIPT Besides FRET, QD affects bR in some ways which are not directly related to FRET. For instance because of being charged particles with intrinsic dipole
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moments, QDs have a significant influence on photocycle of bacteriorhodopsin. In
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2014, Zaitsev et al. acknowledge that QD570 prolong M-intermediate lifetime by an
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unclear mechanism [85]. Metastable M-intermediate is considered to be the most
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important intermediate for optical device applications, so the integration of bR with QDs will lead to more efficient optical devices.
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QDs can improve the performance of proton pumping of bR especially in low
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QD/bR molar ratios [79]. Indeed, bR uses the extra absorbed energy, provided by
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QDs, to improve the proton pumping process. In order to prove this fact, Rakovich
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and her colleagues designed an interesting test [79]. They embedded bR in proteoliposomes membrane inside out. By using this trick, bR which ordinary pumps protons to outside environment of membrane, pumps protons to the inside of the proteoliposoms, so the pH of outside solution rises. They showed that compare to solitary bR, bR-QD hybrids have got more than 25% increment in proton pumping [79]. Because of their notable properties, bR-QD hybrids can be utilized in various fields especially in nano-biosensor applications. In 2011 Griep et al. constructed a nanobiosensor platform based on QD-bR [86]. They used bR as transducer in the bio-nano sensing device and reported that by FRET mechanism, electrical output
ACCEPTED MANUSCRIPT of bR has been improved up to 300% and sensing ability of device has been prospered. Maltose molecule has been chosen as analyte for sensing. Due to the
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fact that this system is not capable of sensing any molecule intrinsically, they used
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layer of MBPs (Maltose binding proteins) and immobilized it on a layer of QD-bR
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(Fig. 8). In order to quench QDs, They used dark quenchers (DQ). DQs and
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maltose are competitor of each other in binding to MBP. Two-layered sensing device containing QD and bR layers has a significant 0.7 mV signal. Adding a
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layer of Maltose Binding proteins to this system has reduced the intensity of signal
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but not that much. The dramatic reduction in photovoltage has occurred in the
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system when DQs has been added to previous layers. As DQ quenches QD, no
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energy transfer occurs between QD and bR; so, electrical output of sensing device decreased. By replacing the DQs by Maltose molecules photovoltage increased. This was the first nanobiosensor based on QD-bR hybrids for real time detection of Maltose [86].
Bouchonvill et al. after several hours of observing bR-QD by AFM, recognized in the presence of some detergents like Triton X100, QD accelerated bR monomerization process [87]. Monomeric and trimeric bR are different in some properties; for instance bR in its monomeric state has lower thermal stability than trimeric one [88]. So all interactions between QD and bR are not desired and for
ACCEPTED MANUSCRIPT constructing bR-QD hybrids and applying them in various fields it's better to consider every aspects.
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To conclude, QDs mostly improve optical absorption, photocycle, and proton
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pumping of bR.
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3. Summary and perspectives
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Biotechnology, nanotechnology and novel immobilization strategies broaden scientist’s horizons toward nano-biomaterials and nano-bio hybrid systems.
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Therefore, they are becoming more and more widely used.
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Nano-bio hybrid materials based on bacteriorhodopsin are widely studied in the
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field of bioelectronics and energy conversion. bR-based solar energy conversion
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devices, such as biomolecule-sensitized solar cells and photoelectrochemical cells are economical, green and relatively efficient but they are still in the evolution stage. In order to achieve high power conversion efficiency, further studies will also be required to investigate following issues: 1. Enhancement of charge transfer rate in semiconductor- biomolecule interface. 2. Co-sensitization of bR with QDs or biomolecules with complementary absorption spectra. 3.
Development of bR mutants with increased stability and high quantum
efficiency of the photoconversion.
ACCEPTED MANUSCRIPT The integration of bR with engineered nanomaterials such as CNT, TNA, TiO2 NPs, ZnO NPs, and quantum dots can lead to the functional properties
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improvement of both bR and nanomaterials. For instance, quantum dots with two
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noticeable properties including broad absorption region and adjustable sharp
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emission spectrum by size can improve bR optical cycle, features and proton
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pumping.
On the other hand, the attachment of bR to the surface of nanostructures is another
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important step. The strong bonding provides an obstacle to the removal of bR from
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the surfaces of the nanomaterials. Also, for some applications, such as BSSCs,
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charge transfer rate in nanostructure/bR interface must be considered. Time, pH,
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and temperature are the important factors for the protein immobilization. However, the integration of bR with the above mentioned materials have been proposed for various applications in bioelectronics. There are plenty of materials to be conjugated with bR. As an example, according to the reports on light-controlled spin filtering in bR [89], magnetic nanoparticles such as nickel and iron oxide can be an area of interest for further research. Considering all of these aspects, it can be stated that these new hybrid bionanomaterials with excellent physical, chemical, and biological properties offer the potential to develop new bio-photonic devices. Acknowledgments
ACCEPTED MANUSCRIPT The authors are grateful to Dr. Naimeh Naseri for critical comments on the manuscript. We gratefully acknowledge financial support from Young Researchers
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& Elite Club.
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References
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ACCEPTED MANUSCRIPT Table 1 Photoelectrochemical parameters of bR-based BSSCs and DSSC.
Nanostructured
scattering
TiCl4
Jsc −2
layer
treatment
(mA cm )
3Glu mutant
TiO2 NPs
no
no
0.09
wild-type bR
ZnO NPs
no
no
0.39
bR+bacterioruberin
TiO2 NPs
no
yes
0.45
wild-type bR
TiO2 NPs
yes
yes
1
N719
TiO2 NPs
yes
yes
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18.2
Efficiency
(mV)
(%)
350
-
[28]
500
0.1
[46]
570
0.16
[42]
533
0.35
[22]
789
10.1
[90]
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semiconductor
Voc
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Sensitizer
Reference
ACCEPTED MANUSCRIPT Figure captions: Figure 1. The photocycle of bacteriorhodopsin.
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Figure 2. (a) Schematic illustration of biomolecule-sensitized solar cell, utilizing bR as
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biophotosensitizer. (b) Simple energy level diagram of BSSC; the absorption of light by bR
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causes the excitation of electrons from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), resulting in injection of an electron from bR into
the electrolyte. Adapted from reference [22].
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conduction band of the TiO2 that causes to charge separation and transportation of holes through
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Figure 3. Schematic illustration of bR-based solar H2 generation systems: (a) electrode systems and (b) particle systems. There are two possible functions for bR: 1.proton pumping 2.visible
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Figure 4. TEM and AFM images of bare SWNTs (a and c) and SWNTs modified by PM patches
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(b and d). Adapted from reference [29].
Figure 5. Mobility of the bare and bR modified FET. After modification with bR, the mobility of the TiO2 nanowires FET is increased by a factor of 2. Adapted from reference [63]. Figure 6. Schematic of FRET between QDs (donor) and PM (acceptor). Figure 7. QD emission spectrum. The emission spectrum of QDs while attached to bleached bR, the glass (control) and bR. Adapted from reference [82]. Figure 8. a) QD-PM, the photovoltage is approximately 0.7 mV. b) QD-PM-MBP, the photovoltage decreases to 0.6 mV. c) There is a dramatic decrease in QD-PM-MBP/DQ photovoltage which barely reaches to 0.2 mV. d) QD-PM-MBP/QD + Maltose, compared to c plot the photovoltage has risen. Adapted from reference [86].
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Figure 1. The photocycle of bacteriorhodopsin.
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Figure 2. (a) Schematic illustration of biomolecule-sensitized solar cell, utilizing
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bR as biophotosensitizer. (b) Simple energy level diagram of BSSC; the absorption of light by bR causes the excitation of electrons from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), resulting in injection of an electron from bR into conduction band of the TiO 2 that causes to charge separation and transportation of holes through the electrolyte. Adapted from reference [22].
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Figure 3. Schematic illustration of bR-based solar H2 generation systems: (a) electrode systems and (b) particle systems. There are two possible functions for bR: 1.proton pumping 2.visible light-harvesting.
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Figure 4. TEM and AFM images of bare SWNTs (a and c) and SWNTs modified by PM patches (b and d). Adapted from reference [29].
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Figure 5. Mobility of the bare and bR modified FET. After modification with bR, the mobility of the TiO2 nanowires FET is increased by a factor of 2. Adapted from reference [63].
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Figure 6. Schematic of FRET between QDs (donor) and PM (acceptor).
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Figure 7. QD emission spectrum. The emission spectrum of QDs while attached to bleached bR, the glass (control) and bR. Adapted from reference [82].
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Figure 8. a) QD-PM, the photovoltage is approximately 0.7 mV. b) QD-PMMBP, the photovoltage decreases to 0.6 mV. c) There is a dramatic decrease in QD-PM-MBP/DQ photovoltage which barely reaches to 0.2 mV. d) QD-PMMBP/QD + Maltose, compared to c plot the photovoltage has risen. Adapted from reference [86].
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Graphical abstract
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Bacteriorhodopsin is promising for use in bioelectronics and energy
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conversion.
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QDs can improve the proton pumping performance of bR.
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There are two basic approaches to achieve bR based hydrogen generation.
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Charge transfer rate in bR-TiO2 interface is effective on BSSCs performance.
