Development of a method of depositing monolayers of Photoreaction Center proteins on Gold Electrodes Progress Report (Chem 449) Sylvester Zhang Bizzotto Group University of British Columbia May 13, 2020 1 / 30
Motivations (a) Rhodobacter sphaeroides used as (b) Binding and measurement of photosynthesis model and as source for protein on electrode biohybrid solar cells (c) Determine the method of producing monolayer, and characterize the properties of a RC monolayer 2 / 30
Introduction Figure: Structure of RC.Based on PDB 2J8C. 1. Embedded Membrane protein from Rhodobacter sphaeroides 2. Well studied as photosynthesis model 3. 3 peptides (H, L and M) surround photosynthesis factor cofactors 1 1 Jones, M. R. The Petite Purple Photosynthetic Powerpack. Biochm. Soc. Trans . 2009 , 37 (2), 400–407. 3 / 30
Introduction (a) Fate of an electron in an (b) Absorbance spectra of the Wild-Type RC. RC, and their relative Labeled are the peaks corresponding to the Ha and energies. P moieties in the RC. ◮ “P” center absorbs light ◮ Electrons cascade through cofactors “B” and “H” very quickly - energies determined by orientations of B and H ◮ Stable charge separated P+Q- state lasts for 100-1000 µ s 4 / 30
Introduction Figure: Cartoon of the binding mode and electron path in a “DM” RC. The hypothesized path of an electron is from the protein’s P center, through the protein, and ultimately into the electrode. The oxidized protein is then reduced by hydroquinol, which is oxidized, as hydroquinone (HQ[o]) ◮ RC bound to electrode by a gold-thiol bond introduced by cysteines mutated on the ventral face of the RC ◮ Each RC is roughly 6x10x4nm ◮ P center excited, donates electron to electrode. Hydroquinone in solution then reduces oxidized P , and allows the process to restart. 5 / 30
Introduction Figure: Cartoon of how MCH creates a monolayer. ◮ Binding of RCs expected to be both specifically (gold-thiol bound) and non-specifically (van der waals-esque) bound ◮ 6-Mercaptohexanol (MCH) displaces non-specifically bound RCs due to much stronger gold-thiol bond between MCH and gold ◮ Only specifically bound RCs left on surface. 6 / 30
Methods Figure: Schematic for deposition of RCs and MCH on electrode ◮ Clean electrode deposited in eppendorf containing solution of RCs in buffer for 1 hour ◮ Electrode then placed in eppendorf containing solution of MCH in buffer for 24 hours, and then briefly deposited in clean buffer solution 7 / 30
Methods Figure: Lockin Technique 1. Measure electrochemical current - background signal (mA) from electrode - redox species (hydroquinone) in solution 2. Light excites photoreaction center periodically at 13 hz - generate small desired signal (nA/pA) from reaction center to electrode transfer 3. Measure periodic modulation in current - consider this photocurrent 2 2 Jun, D.; Beatty, J. T.; Bizzotto, D. Highly Sensitive Method to Isolate Photocurrent Signals from Large Background Redox Currents on Protein-Modified Electrodes. ChemElectroChem 2019 , 6 (11), 2870–2875. 8 / 30
Methods (a) Gold bead formed by melting (b) AFM image of gold(111) face and slowly cooling a gold wire. of single-crystal gold bead. 100uM MCH was deposited on the surface for 24 hours. ◮ Single crystal gold bead formed ◮ Gold bead immersed in eppendorf containing solution of RCs in buffer for 1 hour ◮ Electrode then placed in eppendorf containing solution of MCH in buffer for 24 hours, and then briefly deposited in clean buffer solution ◮ Gold bead imaged by AFM in air, under tapping mode. 9 / 30
Concentration dependence of Photocurrent Figure: Faradaic Current (a) and photocurrent (b) vs Potential (Ag/AgCl) for DM RCs at varying concentrations at 805 nm, vs Ag | AgCl. ◮ Faradaic currents - from oxidation of HQ at electrode - remain similar ◮ Photocurrents increase with concentration of deposited RCs 10 / 30
Concentration dependence of Photocurrent Figure: Photocurrent vs Concentration ( µ M ) of RCs deposited for 1 hour, at various potentials (Ag/AgCl). ◮ Steady rise in photocurrent with concentration of RCs observed at positive potentials relative to OCP ◮ Plateaus after 2.5uM RC deposition conditions - expect to reach some kind of saturation point. 11 / 30
Impact of MCH on Photocurrent Figure: Faradaic current vs potential (a) and Photocurrent (nA) vs potential (b) (V) vs Ag | AgCl, for 5uM RCs deposited for 24 hours, with 1 hour of MCH treatment (pink), and without any MCH treatment (red). ◮ MCH - lowers the faradaic current consistently ◮ Photocurrent increases with more RCs, but when there’s too much RCs, it 12 / 30 decreases again
Impact of MCH on Photocurrent response time Figure: Current (A) vs Time (s) for 5uM RCs deposited on a gold electrode without MCH treatment, believed to be a multilayer situation (left) and for a 5um RCs deposited on a gold electrode with MCH treatment, a monolayer situation (right) ◮ Light turned on at T=0 and left on, before being turned off ◮ Current rises slowly versus time for the multilayer ◮ Current does not really rise with time for the MCH-treated monolayer, and sharp peaks - show - maybe charging? 13 / 30
Diffusion control for Photocurrent response time in multilayers (a) Cartoon of the hypothesized mechanisms of mediator (b) Faradaic current vs potential (a) and replenishment in the monolayer (left) and the multilayer Photocurrent (nA) vs potential (b) (V) vs Ag | AgCl, for 5uM RCs deposited for 24 hours, (right) scenarios. In a multilayer, presumably mediator must diffuse through the multilayer to replenish RCs. with 1 hour of MCH treatment (pink), and without any MCH treatment (red). √ D ◮ Cotrell equation: i ∝ √ t √ 1 ◮ Linear with respect to time ( s ) - possibly diffusion controlled? 14 / 30
MCH is critical for forming monolayers Figure: Cartoon of how MCH creates a monolayer. ◮ Multilayers form initially from non-specific adsorption ◮ MCH competitively displaces non-specifically adsorbed RCs ◮ MCH is needed to obtain monolayers 15 / 30
Atomic Force Microscopy counting of RCs (a) Single crystal gold (111) (b) Height distribution of bumps surface with 2.5uM RCs deposited. 16 / 30
Atomic Force Microscopy counting of RCs Figure: Raw (a), and segmented (b) image of RCs on gold electrode ◮ Bumps are about 6nm tall - as expected for an RC lying on its side. ◮ Can count RCs ˜300/ µ m 2 due to the smoothness of underlying gold/MCH. ◮ Photocurrent per RC ≈ 10 electrons per second - 3.71 cm 2 area of electrode, and ≈ 210 nA current 17 / 30
Atomic Force Microscopy counting of RCs (a) AFM image of a RCs (b) Single crystal gold (c) AFM image of gold(111) deposited for 24 hours, on (111) surface with 2.5uM face of single-crystal gold gold (111) surface. Z scale RCs deposited. Z scale bead with 100uM MCH was ranges 30nm ranges 20nm deposited on the surface for 24 hours. Z scale ranges 3nm ◮ Multilayer very rough - no gold layer can be seen by AFM ◮ Compare to bare gold, and monolayer, where gold can be seen, and RCs can be counted. 18 / 30
Future Work (d) Marcus theory cartoon. (e) Marcus theory vs MHC theory. Reorganization energy is the Adapted from (3). intersection of the parabolas. ◮ Marcus theory - reorganization energy is intersection of two parabolas - energ of donor, and the acceptor ◮ Marcus-Hush-Chidsey - the acceptor energy states are the entire band of electronic states in the gold electrode. ◮ Key prediction: No “Marcus inverted region”. 3 3 Zeng, Y, Smith, R. B., Bai, P . and Bazant, M. Z., "Simple formula for Marcus-Hush-Chidsey kinetics", Journal of Electroanalytical Chemistry 735 (2014) , pp. 77-83. 19 / 30
Future Work Figure: Marcus-Hush-Chidsey kinetics fits for the multilayer (5uM RCs for 24 hours, and no MCH), and monolayer (5uM RCs for 1 hour, and 100uM MCH for 24 hours) ◮ Marcus-Hush-Chidsey re-organization energy: 0.4eV ◮ Compare to 0.5eV calculated for re-organization energy of P + Q − in implicit solvent and membrane lipids 4 ◮ Reorganization energy of 1eV - typically a very tightly packed material, lower values - easier to reorganize. 20 / 30
Future Work (a) Faradaic current (a) and Photocurrent (b) vs potential (b) Absorbance spectra of the Wild-Type RC. for 5.0uM RCs excited at various wavelengths of light Labeled are the peaks corresponding to the Ha and P moieties in the RC. ◮ Wavelength of exciting light has impact on photocurrent detected ◮ Unclear if its due to absorbance - it is not known what a monolayer absorbance spectra is - absorbance spectra in solution may not reflect absorbance of a monolayer adsorbed on gold ◮ Must determine the monolayer absorbance, and activity spectrum (action spectrum) 21 / 30
Acknowledgements Prof. Daniel Bizzotto Dept. Microbiology and Immunology Ms. Tianxiao Ma Prof. Thomas Beatty Mr. Adrian Grzedowski Dr. Daniel Jun Ms. Jessica Shi Ms. Amita Mahej 22 / 30
Control - Photocurrent Signal Origins (c) Excited electron donates to (d) Photoreaction center electrode flexes when excited, resulting in a 13 Hz change in background signal ◮ Electron transfer from protein to electrode ◮ Conformational flexing of reaction center changing effective concentration of mediators near electrode ◮ 13 hz signal from excited mediator to electrode ◮ Anything else that may occur at 13hz 23 / 30
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