Development and Characterization of Titanium Dioxide Gel with

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Development and Characterization of Titanium Dioxide Gel with Encapsulated Bacteriorhodopsin for Hydrogen Production Kaitlin E. Johnson, Sukriti Gakhar, Subhash H. Risbud, and Marjorie L. Longo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01471 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Development and Characterization of Titanium Dioxide Gel with Encapsulated Bacteriorhodopsin for Hydrogen Production Kaitlin E. Johnson‡, Sukriti Gakhar‡, Subhash H. Risbud†, and Marjorie L. Longo*,‡ ‡

Department of Chemical Engineering, University of California Davis, Davis, California 95616, United States †

Department of Materials Science and Engineering, University of California Davis, Davis, California 95616, United States

ABSTRACT: We study bacteriorhodopsin (BR) in its native purple membrane encapsulated within amorphous titanium dioxide, or titania, gels and in the presence of titania sol-particles to explore this system for hydrogen production. Förster resonance energy transfer between BR and titanium dioxide sol particles was used to conclude that there is nanometer-scale proximity of bacteriorhodopsin to the titanium dioxide. The detection of BR-titania sol aggregates by fluorescence anisotropy and particle sizing indicated the affinity amorphous titania has for BR without the use of additional cross-linkers. UV-Visible spectroscopy of BR-titania gels show that methanol addition did not denature BR at a 25 mM concentration presence as a sacrificial electron donor. Additionally, confinement of BR in the gels significantly limited protein denaturation at higher concentration of added methanol or ethanol. Subsequently, titania gels fabricated through the sol-gel process using a titanium ethoxide precursor, water and the addition of 25 mM methanol were used to encapsulate BR and a platinum reduction catalyst for the production of hydrogen gas under white light irradiation. The inclusion of 5 µM bacteriorhodopsin resulted in a hydrogen production rate of about 3.8 µmole hydrogen mL-1 hr-1,

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an increase of 52% compared to gels containing no protein. Electron transfer and proton pumping by BR in close proximity to the titania gel surface are feasible explanations for the enhanced production of hydrogen without the need to crosslink BR to the titania gel. This work sets the stage for further developments of amorphous, rather than crystalline, titania-encapsulated bacteriorhodopsin for solar-driven hydrogen production through water-splitting. INTRODUCTION Titanium dioxide, or titania, has been long considered to be a promising material for use in hydrogen production through photocatalytic water-splitting.1-3 When irradiated with ultraviolet (UV) light, titania produces an electron-hole pair capable of oxidizing water for the production of hydrogen protons and oxygen gas.1 The hydrogen protons can be reduced to H2 gas at an additional reduction catalyst. The large band gap of titania prevents this reaction from occurring when irradiated with lower energy wavelengths of light, making it an inefficient option for solar applications.1 Titania can be sensitized for use in visible light through the addition of metal dopants4 or charge-transfer dyes,5 however, these additions may still be limited by the surface area and morphology of the titania.6 Additionally, dopants may lead to the formation of additional phases that result in premature recombination of electron-hole pairs,7 limiting the effectiveness provided by the extended light absorbance of the titania. Instead of traditional charge-transfer dyes, the use of light-harvesting biomolecules may allow the opportunity to take advantage of the functionality of such biomolecules in addition to the increased sensitization to visible wavelengths. One such molecule is bacteriorhodopsin (BR), a light-harvesting integral membrane protein derived from Halobacterium salinarum that functions as a proton pump, moving hydrogen protons across its native purple membrane in response to photon absorption.8-10 This functionality of BR may help to increase the mass


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transport within a titania system, moving the hydrogen protons produced at the surface of the titania to the reduction catalyst for full hydrogen evolution in addition to injecting electrons excited from photon absorption by the protein’s co-retinal to the conduction band of titania.8 In fact, BR crosslinked or adsorbed on crystalline titania has been investigated for the production of photocurrent under irradiation by white light and has been suggested to improve the overall yield in such photoelectrochemical systems.8-9 The impact of BR on hydrogen production under white light has previously only been studied using crystalline titania nanopowder.9-10 Methanol at a concentration of 25 mM was used as an electron donor in the system9 and it is posited to be a source of electrons for excitement in the absence of a tradition electrolyte as the breakdown of methanol results in the production of electrons.11 Amorphous titania has been largely ignored in the past due to the slightly larger band gap energy and the increased potential for defects within the disordered structure which may lead to electron-hole pair recombination.4, 12 However, the high surface area afforded by the disorder of amorphous titania has been shown to be beneficial for photocatalytic applications as higher rates of adsorption on the surfaces are possible and the lack of a calcination or crystallization stage makes synthesis cheaper, easier, and faster than crystalline titania.2 Sol-gel-derived amorphous titania gels may allow for the development of high surface area titania for use with BR without the need for crosslinking chemicals or lengthy adsorption stages. During the sol-gel process, titania forms a porous network through polycondensation of a hydrolyzed titanate precursor.13-14 Biomolecules such as proteins can be added during this polycondensation or gelation stage for encapsulation within the final matrix structure.15 In the case of BR, the protein’s carboxylic groups can also chemically adsorb to the hydroxyls of the titania surface.9 Figure 1 illustrates the expected encapsulation environment of BR and platinum nanoparticle


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reduction catalyst within amorphous titania gels. Additionally, the sol-gel synthesis process allows for the production of highly tunable products that have been shown to improve the stability of confined biomolecules when exposed to damaging environmental factors.16

Figure 1. Proposed encapsulation environment of bacteriorhodopsin and platinum nanoparticle reduction catalyst within amorphous titania gel monoliths for the production of hydrogen under white light irradiation. In our previous work, amorphous titania gel monoliths were shown to achieve photocatalytic breakdown of a model organic dye, methylene blue at a degree and rate comparable to that of commercial crystalline titania nanopowder.17 Additionally, BR was encapsulated within titania and shown to retain the ability to make reversible conformational changes associated with the photocycle.17 This indicates BR is likely to retain the functionality necessary to perform proton pumping within the pores of the gel. An important question that remained was whether BR is in close proximity to the titania surface in order to enhance the mass transfer through proton pumping without the need for a cross-linker. Förster resonance energy transfer (FRET), a phenomenon in which the photonic energy of a fluorophore is transferred to an acceptor when the two molecules are less than 10 nm apart,18 has been used in the past to study the distance between BR and nanoparticles.19 In this work, BR and surrounding titania are probed with fluorescence spectroscopy and FRET is used to study their spatial


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proximity within the pores of the titania and to more fully understand the association between titania and BR in eliminating the necessity of a cross-linker. Additionally, we explore the impact of synthesis conditions on the stability of encapsulated BR and photocatalytic performance, focusing on the addition of methanol as a sacrificial electron donor. Subsequently, amorphous titania gel monoliths, with and without encapsulated BR, are investigated for the production of hydrogen through water-splitting under white light irradiation. MATERIALS AND METHODS Materials. Titanium ethoxide (~80%), absolute ethanol and methanol, propylene oxide (≥99.5%), methylene blue (≥82%), platinum nanoparticle (3 nm) dispersion (99.99%), and P25 titanium dioxide nanopowder (≥99.5%) were purchased from Sigma-Aldrich, Inc. Hydrochloric acid (12.1M) was purchased from Aqua Solutions, Inc. (Deer Park, TX). Purple membrane associated bacteriorhodopsin derived from Halobacterium salinarum was purchased from Bras del Port, S.A. (Santa Pola, Alicante, Spain). A Barnstead Nanopure System (Barnstead Thermolyne, Dubuque, IA) was used to purify water used in all experiments. Synthesis of Amorphous Titania Gel Matrix. During a typical synthesis of titania gels, 170 µL concentrated hydrochloric acid was added to up to 10 mL of diluted methylene blue solution. The solution was then stirred vigorously and 1 mL titanium ethoxide was dispensed slowly into the stirred solution. To produce gels with 0-5.2 M added ethanol, a solution of titanium ethoxide and up to 3 mL absolute ethanol was produced and slowly dispensed into the methylene blue solution. Once the titania precursor had been completely incorporated, up to 1 mL of propylene oxide was added to the solution to encourage gelation. The solution, or sol, was then filtered through a 0.2 µm filter and aliquoted in 3.5 mL acrylic cuvettes. The cuvettes were then sealed and stored in the dark, at room temperature to allow for gelation. Gels typically solidified within


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30 to 90 mins depending on water content. The final transparent gels had a concentration of 17 µM methylene blue. The final titania concentration of the gels was determined through an initial aging of the gels at 85°C for about 5-7 hours followed by heating at 150°C for 90 minutes to dehydrate and densify the titania. The weight of the densified titania was recorded and used with the known volume of the initial gels before ageing to calculate the concentration. Titania concentration was found to increase with the use of ethanol due to slowed hydrolysis. For ethanol extraction experiments, prior to the addition of propylene oxide, the sol was subjected to rotary evaporation at 55°C at 340 mbar reduced pressure for ten minutes. The resulting sol was filtered with a 0.2 µm filter and placed in an ice bath before the addition of up to 1 mL propylene oxide and the solution was mixed with a vortex mixer and aliquoted in 3.5 mL cuvettes and sealed for gelation. The concentration of ethanol in the pores of the gels was determined by allowing the gels to age at room temperature in sealed vials until 150 µL of pore fluid was expelled from the monolith. The ethanol concentration of this pore liquid was measured using an analog refractometer (TekcoPlus Ltd., Hong Kong) calibrated for ethanol. For gels in which methanol was used to substitute for ethanol, methanol was added to the methylene blue solution to achieve a final molarity of 2.6 or 5.2 M methanol as appropriate to match the concentration of ethanol added in ethanol gels. To this solution, 170 µL concentrated hydrochloric acid was added before slowly dispensing the titanium ethoxide precursor directly into the solution. The procedure following this stage matched the methods described for the previously described gels. Bacteriorhodopsin Encapsulation. Titania gels for BR encapsulation were synthesized following protocols described in the synthesis section, replacing the methylene blue solution with DI water. BR suspended in ultra-pure DI water was added to the titania sol following the filtration step. The sol with a final BR concentration of 5 µM, was gently mixed and allowed to


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gel while sealed and covered on the benchtop. Gelation typically occurred within 30 to 90 mins and the transparent gels were stored in the dark at 4°C for up to a month. The absorbance of the BR gels was measured using a SpectraMax M2 UV/Vis spectrophotometer (Molecular Devices, LLC., Sunnyvale, CA). Förster Resonance Energy Transfer. To initially probe tryptophan residues in BR, titania gels with and without encapsulated BR in 3.5 mL acrylic cuvettes were excited at 285 nm using a JASCO FP-8500 fluorescence spectrometer (JASCO, Inc., Easton, MD). The emission intensity was observed at wavelengths from 300 to 750 nm with excitation and emission slit sizes of 10 nm and a response time of 0.5 seconds with a scan speed of 2000 nm/min. As a result of Förster resonance energy transfer (FRET) from BR to titania, emission from BR tryptophan was quenched at high titania concentrations associated with the titania gels. To study quenching of tryptophan emission in presence of titania, 3 mL of 1 µM BR solution was titrated with increments of 10 µL of diluted titania sol with 7.66 mM titania and the fluorescence emission was recorded after each addition of sol. Titania concentration varied form 0-250 µM at an interval of 25 µM. The excitation wavelength used was 285 nm and emission was recorded from 300 to 600 nm. Both excitation and emission slit widths were 2.5 nm with a response time of 0.5 seconds and a scan speed of 2000 nm/min. Correction for Inner Filter Effects. In solutions with optical densities higher than 0.05 at excitation and emission wavelengths the observed fluorescence is attenuated. As titania has an absorbance higher than 0.05 at 285 nm between 25-250 µM concentrations (Figure S1 in Supporting Information), the observed emission intensity from BR in presence of titania was corrected for inner filter effects using Equation 1,

= 10( )/,


(1)

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where Aex and Aem are the absorbance values of titania at the excitation and emission wavelengths respectively. The excitation wavelength was 285 nm and the emission wavelength used was 337 nm, the wavelength for maximum fluorescence emission from BR tryptophan residues. Fluorescence Anisotropy of Tryptophan Residues in Bacteriorhodopsin and Dynamic Light Scattering. The fluorescence anisotropy of tryptophan residues in BR and particle size by dynamic light scattering was measured with increasing titania concentration to determine if there is aggregation of purple membrane fragments in presence of titania. The rotational speed of the fluorescent probe which is inversely related to its size is can be represented with anisotropy value (r) of the emitted fluorescence. Anisotropy can be described by the relationship between the intensity of the parallel and perpendicular polarized light emission as shown in Equation 2.20 =

∥ ∥

(2)

Diluted samples of titania sol were added to 1 µM BR in an acrylic cuvette and excited with a light polarized along the z-axis at 285 nm and both the parallel and perpendicular emission intensities at 337 nm were recorded as temperature was maintained at 22°C. A slit width of 2.5 nm for both excitation and emission was used for all measurements. Dynamic light scattering was completed with a 90Plus Particle Size Analyzer (Brookhaven Instruments Corp., Holtsville, NY). Photocatalytic Activity. Titania gels containing methylene blue were irradiated from above in the dark with 365 nm light from a Spectroline ENF-280C dual-wavelength UV lamp (Spectronics, Corp., Westbury, NY) at an intensity of 1.80±0.01 mW/cm2 for 3 hours. The intensity of the light irradiating samples was regularly monitored using a UV512AB Digital UV AB Light Meter (General Tools & Instruments, LLC., New York). Photocatalytic activity in the


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gels was previously compared with the behavior of P25 nanopowder under the same conditions.17 A SpectraMax M2 UV/Vis spectrophotometer (Molecular Devices, LLC., Sunnyvale, CA) was used to determine the absorbance of all samples before and after UV irradiation. Degradation of the methylene blue within samples was calculated by monitoring the peak height at 665 nm as this is directly proportional to the concentration of methylene blue through the Beer-Lambert Law. Photocatalytic performance was normalized by titania concentration. Hydrogen Production and Detection. Titania gels were synthesized using up to 5 µM BR using the previously described method and dispensed into 20 mL reaction vials, leaving 10 mL of headspace. Following the addition of the BR to the sol during the polycondensation stage, 0.25 wt% platinum nanoparticles were added to the solution and mixed to uniformity. The samples were allowed to gel covered in the dark and typically required an hour to completely gel. The gels were then stored covered at 4°C for up to a day to slow aging and prevent the gels from shrinking or cracking before use. Prior to irradiation, the vials were sealed with a rubber septum and purged with nitrogen gas (99.995%). Gels were then irradiated for five hours with 100 mW/cm2 white light from 330 to 700 nm using a 300 W Lambda LS-XL xenon arc lamp (Sutter Instrument Co., Novato, CA) with heat-absorbing glass (Edmund Industrial Optic, Inc., Barrington, NJ). A LED light meter (FLIR Systems, Inc., Wilsonville, OR) was used to monitor the intensity of the white light. To characterize the hydrogen concentration in the headspace, 500 µL of the headspace was extracted with a gas-tight syringe and injected into a Varian 3800 gas chromatograph (Varian, Inc., Palo Alto, CA) equipped with a 5 Å column molesieve PLOT column. The column was run isothermally at 50°C with argon gas (99.997%) at a pressure of 425 kPa as the carrier gas. Hydrogen was detected using a thermal conductivity detector (TCD) and a


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1% hydrogen in nitrogen standard (Sigma-Aldrich, Inc., St. Louis, MO) was used for calibration under the same conditions. RESULTS AND DISCUSSION Fluorescence Characterization of Confined Bacteriorhodopsin. Previously, we characterized the UV-visible absorbance spectra of the retinal chromophore of encapsulated BR in titania gels which confirmed the ability of the protein to make conformational changes associated with the proton pumping photocycle under confinement.17 Those titania gels were synthesized without the addition of ethanol because we found this formulation to result in high amounts of stable confined BR without significant levels of denaturation, in contrast to gels synthesized with 2.6 M and 5.2 M added ethanol which denatured the BR. Here, titania gels with encapsulated BR were characterized using fluorescence spectroscopy. Upon excitation of the eight tryptophan residues of BR21-22 at 285 nm, the expected emission at 337 nm is not present and appears to be fully quenched and absent as shown in Figure 2A. Moreover, when comparing the emission of gels with encapsulated BR to those without, an increase in the fluorescence emission corresponding to the titania emission spectra was found to occur as shown in Figure 2A. This indicates that the system is likely subject to the phenomenon of Förster resonance energy transfer (FRET) in which photonic energy is transferred from the tryptophan residues of BR donor species to the titania acceptor, increasing the apparent fluorescence from the titania.23 Evidence for the potential for this phenomenon can also be found in Figure 2A. The overlap between the fluorescence emission of BR tryptophan residues and the absorbance of the titania, may allow for the transfer of photonic energy from the protein to the titania. This highest increase in titania emission (see 0 M added ethanol BR gel in Figure 2A and Table S1 in Supporting Information) occurred in the gel with the lowest amount of denatured BR17. Successive inclusion of ethanol lowered the extent of


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increase in the titania emission (see 2.6 and 5.2 M added ethanol BR gel in Figure 2A and Table S1 in Supporting Information). This suggests that properly folded membrane associated BR is in closer proximity to the titania surface than denatured BR.

Figure 2. (A) The spectral overlap between the titania sol absorbance spectra (in molar extinction coefficient, M-1cm-1) with the fluorescence emission from BR tryptophan residues and fluorescence emission from titania gels excited at 285 nm with and without BR and added ethanol. (B) The fluorescence emission and excitation spectra of BR in solution and titania gels and excitation spectra of BR-titania sol particles. The concentration of the BR in solution and gels was 5 µM. FRET may be present in systems in which spectral overlap between the fluorescence emission of a donor molecule and the absorbance spectra of an acceptor occurs.18,

24-25

The

spectral overlap of titania absorbance (in M-1cm-1) with the regressed BR emission spectrum (see Supporting Information and Figure S2) is shown in Figure 2A. Titania absorbs light in the UV range between 230-350 nm (250-350 nm shown) and BR tryptophan residues fluorescently emit between 270-450 nm. The spectral overlap between titania absorbance and BR emission therefore occurs in the wavelength range of 270-350 nm. The spectral overlap combined with the apparent tryptophan quenching and increase in titania gel fluorescence with encapsulated BR


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protein allows us to conclude FRET from BR tryptophan residues to titania likely occurs within the gels. The possibility of quenching mechanisms other than FRET, were also considered in the system. Quenching of fluorescence can also occur due to dynamic and static quenching mechanisms. These mechanisms are characterized using Stern-Volmer constant (KSV) shown in Equation 3.

= 1 + []

(3)

is the ratio of emission intensity of the fluorophore (tryptophan) in the absence and presence

of the quencher and [Q] is the quencher concentration. The KSV value represents accessibility of the fluorophore being quenched to the solvent if the quenching mechanism is dynamic. In order to explore the possibility of this mechanism, titania sol was synthesized using the typical method as previously described and diluted with DI water to a final concentration of 7.66 mM titania to prevent gelation. The diluted sol was added in 10 µL increments to a 1 µM suspension of BR and the fluorescence emission of BR tryptophan was recorded after each addition. The KSV value determined from the linear regression of the experimental data is 4592 M-1 at 293 K as shown in Figure 3. The biomolecular quenching constant, kq, obtained from this KSV (kq× ) value is 7.91 × 10#$ M-1 s-1 using lifetime of tryptophan fluorescence in BR, , as 0.58 ns.26 This biomolecular quenching constant is 3 orders of magnitude larger than the highest reported value of tryptophan quenching with molecular oxygen (~10#' M-1 s-1).20 Thus, the dynamic quenching mechanism was rejected for the observed quenching behavior of BR tryptophan fluorescence in the presence of titania.


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Figure 3. Fluorescence quenching of BR tryptophan with increasing concentration of titania at different temperatures. The linear fits are using Equation 3. Slope represents Ksv values. Static quenching occurs due to a fluorophore-quencher complex formation which is nonfluorescent and then decreases the total fluorescence intensity. Increasing temperature breaks the complex and results in a decrease in observed quenching. We observe no such temperature pattern as shown in Figure 3, therefore, tryptophan fluorescence quenching with titania cannot be concluded as static quenching. Both dynamic and static quenching need close approach of the fluorophore and quencher molecule (10 mM) required to observe quenching.20 Quenching of BR fluorescence takes place at comparatively lower concentrations of titania (

Development and Characterization of Titanium Dioxide Gel with

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