Enhanced Harvesting of Photosynthetic Electrons from Isolated Thylakoid Membranes By Geometry-Optimized Microelectrodes
Authors: Ryu DongHyun , Kim Yongjae , Hong Hyeonaug , Ryu WonHyoung 
Published: February 2020

In photosynthesis of plants, photosystem II, embedded in thylakoid membranes, splits water into oxygen, proton and electron. The photosynthetic electrons (PEs) excited by light, are transferred along the series of proteins in thylakoid membranes and finally this energy is used to synthesize NADPH. Many researches have investigated efficient ways to harvest these PEs with high energy from thylakoid membranes. In order to extract PEs from thylakoid membranes, electrically conductive pathways between thylakoid membranes and a collector electrode surface need to be established. In previous researches, several chemical approaches have been attempted to tether thylakoid membranes to electrodes. Multi-walled carbon nanotubes were used to link thylakoid membranes near the surface of a conductive electrode. In addition, glassy carbon electrodes modified with benzene-4-carboxyphenyl were adapted to covalently bond thylakoid membranes to an electrode surface. Moreover, physical surface modifications have been attempted such as gold-nanoparticle-modified electrode for increasing surface area. Although there have been rapid advances like above, further enhancement of PE harvesting is desired for practical development of the thylakoid-based energy harvesting. In this study, we propose that design of electrode geometry to maximize the contact between the electrode and isolated thylakoid can increase direct exchange of PEs between thylakoid membranes and electrode surfaces. Firstly, we isolated chloroplasts from intact spinach by blending, mesh filtering and centrifuging. Because thylakoid membranes exist inside of the chloroplast membranes, osmotic shock was applied to break chloroplast membranes. Then, the size distribution of thylakoid membranes was analyzed with scanning electron microscopy images after dehydration treatments. Thylakoid membranes had disk-like shapes and it was found out that their sizes were distributed between 500 nm and 2.5 μm. About half of them had diameters between 1 μm and 1.4 μm. By considering three-dimensional morphologies of thylakoid membranes, an array of micro-pillar electrodes were fabricated such that thylakoid could fit tightly between the array of electrodes and maximize the direct contact as large as possible. Based on the size distribution of thylakoid membranes, micro-pillar electrodes were fabricated by a combination of lithographic patterning of metal and metal-assisted chemical (MAC) etching. Silicon wafers were used as substrates for the fabrication of micro-pillar array electrode. We used a photomask with hexagonal circle patterns. Each circular pattern had 1 μm diameter, and the center to center distance was 2 μm. Negative photoresist was used to deposit gold layer on the outside of the circles by a lift-off process. Wet etching was conducted with solution consisting of hydrofluoric acid (HF) and hydrogen-peroxide (H2O2). Because the gold layer on the silicon works as catalyst, silicon under the gold layer was etched away. Etching depth could be easily controlled by etching time. Surface area of the micro-pillar electrode was proportional to the etching depth. Photosynthetic currents were measured without any electron mediator to extract PEs only from direct electron transfer. Photosynthetic currents were measured in a three-electrode setup comprised of Ag/AgCl as a reference electrode and Pt mesh as a counter electrode at a 0.4V bias. Thylakoid membrane solutions were drop-cast on the electrodes and dried for a day for immobilization. Micro-pillar electrodes with low aspect-ratio showed larger photosynthetic currents compared to flat electrode. The enhancement in photosynthetic currents was proportional to the depth of micro-pillar electrodes. In particular, micro-pillar electrode with 1.7 μm depth showed 140% enhanced photosynthetic currents. This result corresponded to the increment of surface area which was about 150%. These results suggest that increased direct contacts between thylakoid membranes and micro-pillar electrodes can enhance direct electron transfer. Moreover, further increase was observed by adding an electron mediator, potassium ferricyanide, which can transfer electrons from thylakoid membranes to the electrode surface. Furthermore, cyclic voltammetry results showed two anodic peaks which originated from cytochrome b6f complex and plastocyanin of thylakoid membranes.