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Direct extraction of photosynthetic electrons from the whole photosynthetic cells such as plant cells or algal cells can be highly efficient and sustainable compared to other approaches based on isolated photosynthetic apparatus such as photosystems I, II, and thylakoid membranes. However, insertion of nanoelectrodes (NEs) into individual cells are time-consuming and unsuitable for scale-up processes. We propose simple and efficient insertion of massively-populated NEs into cell films in which algal cells are densely packed in a monolayer. After stacking the cell film over an NE array, gentle pressing of the stack allows a large number of NEs to be inserted into the cells in the cell film. The NE array was fabricated by metal-assisted chemical etching (MAC-etching) followed by additional steps of wet oxidation and oxide etching. The cell film was prepared by mixing highly concentrated algal cells with alginate hydrogel. Photosynthetic currents of up to 106 nA/cm2 was achieved without aid of mediators, and the photosynthetic function was maintained for 6 days after NE array insertion into algal cells.

Harvesting photosynthetic electrons (PEs) from plant or algal cells can be a highly efficient and environmentally friendly way of generating renewable energy. Recent work on nanoelectrode insertion into algal cells has demonstrated the possibility to directly extract PEs from living algal cells with high efficiencies. However, the instability of the inserted cells limits the practicality of this technology. Here, the impact of nanoelectrode insertion on intracellular extraction of PEs is characterized with the goal of stabilizing algal cells after nanoelectrode insertion. Using nanoelectrodes

Introduction Microneedle (MN) has been developed to diagnose and detect the biomarkers which are related to various diseases such as diabetes mellitus (DB) and caner. The biomarkers could be extracted from interstitial fluid (ISF) in the human body by using MN with a minimally-invasive and pain-free manner. In particular, glucose sensing has widely been investigated due to increase of DB patients who suffered inconvenience of frequent monitoring of glucose levels. MN-based sensors targeting ISF glucose monitoring was introduced and showed the possibility to replace the existing blood-based glucose sensing technologies [1-3]. We hypothesized that the coating of a MN surface with swellable material such as hydrogel could increase detection sensitivity and time for stabilization. Thus, we developed a swellable hydrogel coated MN sensor which could extract the glucose from ISF with swelling for a short time. Methacrylated hyaluronic acid (MeHA), photo-crosslinkable hydrogel, was utilized to absorb ISF containing biomarkers for electrochemical reaction with the metallized surface of a MN. Cyclic voltammetry (CV) with a model redox species, ferricyanide, were performed to characterize the MeHA-coated MN sensors for the detection of glucose. Experimental Methods To fabricate the MeHA coated MN sensor, 3×3 SU-8 MN array was transfer molded on the 1 cm × 2 cm of the glass wafer. The female PDMS mold which was patterned MN shapes were filled with SU-8 in a vacuum condition and the mold was gently transferred on the glass wafer. After UV curing, the MN electrode was prepared by the Ti-gold sputtering. MeHA was synthesized by modifying from the hydroxyl groups to the methyl group in basic condition. Dimethylformamide, methacrylic anhydride, and sodium chloride were mixed in the 2% of hyaluronic acid solution while a solution was controlled in basic condition. The precursor was washed three times by ethanol and was freeze-dried to obtain the MeHA sponges. Then, the MeHA sponges were dissolved into the deionized water which was contained 0.05% of the photoinitiator for preparing the MeHA solution. The MeHA solution was poured on the surface of gold-sputtered SU-8 MN arrays and the swellable MN sensor was fabricated after drying. Before measuring the glucose, potassium ferricyanide (K3[Fe(CN)6]) which was the standard redox solution to conduct CV was used as an alternative of the biomarker. The MeHA coated MN electrode was used as a working electrode and a Pt mesh electrode was used as a counter electrode. The MN sensor was immersed into the potassium ferricyanide solution and the CV was conducted while the concentration chose 10 mM, 10 μM, and 1 μM to confirm sensitivity and detection limit of the three-electrode system with MeHA coated MN sensor. Results and Discussion The 3×3 array of SU-8 MNs fabricated on a glass wafer had the height of 640 μm and the aspect ratio of 1.6. The MeHA was successfully synthesized and the MeHA film had a swelling ratio of 450% after 10 sec swelling in the phosphate buffered saline. By using the three-electrode system including the MeHA uniform coated MN electrode, in vitro sensing tests were performed with various concentrations of potassium ferricyanide. As the results of the CV analysis, when the concentration of potassium ferricyanide was 10 mM, the anodic and cathodic peak currents of the measured I-V curve were 135 and 144 μA, respectively. Although the peaks were observed from the CV plots, it was hard to measure the exact value of peak currents when the concentration of potassium ferricyanide, 10 μM, and 1 μM. Nevertheless, the current peaks of 10 μM was slightly higher than the peaks of the 1 μM sample. Since the peak current was increased in proportion to the level of potassium ferricyanide, it was concluded that the MeHA coated MN sensor system could be used to measure the biomarker. These results also support that the biomarkers could be extracted from the ISF by the swelling behavior of the MeHA coated on the surface of MNs. Summary The MeHA coated MN sensor was fabricated with simple molding techniques and various photocurable materials such as SU-8 2010 and MeHA. The ferricyanide could be extracted by using the swelling behavior of the MeHA. As the results of in vitro test, the various concentration of potassium ferricyanide solution was measured with the three-electrode system including MeHA coated MN sensor and the detection limit was observed at least 1 μM. Reference 1. Sharma, Sanjiv, et al. "A pilot study in humans of microneedle sensor arrays for continuous glucose monitoring." (2018). 2. Caliò, A., et al. "Polymeric microneedles based enzymatic electrodes for electrochemical biosensing of glucose and lactic acid." Sensors and Actuators B: Chemical 236 (2016): 343-349. 3. Ribet, Federico, Göran Stemme, and Niclas Roxhed. "Real-time intradermal continuous glucose monitoring using a minimally invasive microneedle-based system." Biomedical microdevices 20.4 (2018): 101.

Thylakoids are the photosynthetic complexes that absorb light energy to produce photosynthetic electrons (PEs) in the chloroplast of plant cells. PE production starts from water splitting at photosystem II (PSII) in the thylakoid membrane and they are excited by absorbing photon energy. Over the last decade, many researchers have attempted to extract PEs from thylakoid by enhancing attachment of thylakoid membranes to an electrode or using artificial mediators. However, these have some limitations including increased ohmic resistance from the length of tethering molecules and energy loss due to artificial mediators. In this study, we propose a photosynthetic electrochemical cell based on RuO2 nanosheet modified electrode for the photosynthetic energy harvesting from thylakoids. RuO2 is known as one of the most conductive metal oxides. Metal oxides have partially positive charges that can improve attachment of negatively charged thylakoids to the electrode. Also, partially positively and negatively charged characteristics of RuO2 surfaces induce ensemble docking between electron carriers and electrode surface, which can attract both negatively charged electron carriers and positively charged electron carriers. Furthermore, RuO2 nanosheets show a unique characteristic of proton adsorption, which can sustain positive electrical potential during PEs extraction. For photosynthetic energy harvesting, the photosynthetic electrochemical cell was constructed with anode based on thylakoid and air-cathode based on Pt-C, which separated by a proton exchange membrane. The light was controlled by a 100 W halogen lamp to operate the photosystem so that excited PEs can be extracted to the electrode. The surface potential of RuO2 nanosheets modified electrodes was 250 mV vs. Ag/AgCl, which shifted positively more than 100 mV than that of the bare Au electrode. From the inspection of RuO2 lattice by x-ray absorption spectroscopy, positively shifted surface potential is the result of proton adsorption. A positively biased electrode surface can induce a large amount of PEs extraction from the electron carriers than the Au electrode. When the light is illuminated on the thylakoids deposited electrodes, 8.9 ± 6.3 mV and 88.7 ± 13.8 mV of the surface potentials were measured from the Au and the RuO2 modified electrode, respectively. These lowered electrode potentials are due to the redox potential of the electron carriers on the electron transport chain of the thylakoid membrane. The open-circuit voltages (OCV, the subtracted potential between the cathode and the anode) were measured 575.1 ± 6.4 mV and 424.0 ± 17.2 mV from the Au and the RuO2 electrode, respectively. Although OCV of the RuO2 electrode is lower than the Au electrode, the power density of the RuO2 electrode shows 4 times higher than the Au electrode, and this is due to the enhanced PEs extraction by both the surface charge and the proton adsorption characteristics of RuO2 nanosheets.

Photosynthesis converts solar energy to electricity in a highly efficient manner. Since only water is needed as fuel for energy conversion, this highly efficient energy conversion process has been rigorously investigated. In particular, photosynthetic apparatus, such as photosystem II (PSII), photosystem I (PSI), or thylakoids, have been isolated from various plants to construct bio-hybrid anodes. Although PSII or PSI decorated anodes have shown potentials, there still remain challenges, such as poor stability of PSII-based systems or need for electron donors other than water molecules of PSI-based systems. Thylakoid membranes are relatively stable after isolation and they contain all the necessary photosynthetic apparatus including the PSII and PSI. To increase electrical connections between thylakoids and anodes, nanomaterials such as carbon nanotubes, nanowires, nanoparticles, or graphene have been employed. However, since they rely on the secondary electrical connections between thylakoids and anodes; it is desired to achieve larger direct contacts between them. Here, we aimed to develop micro-pillar (MP) array anodes to maximize direct contact with thylakoids. The thylakoid morphology was analyzed and the MP array was designed to maximize direct contact with thylakoids. The performance of MP anodes and a photosynthetic fuel cell based on MP electrodes was demonstrated and analyzed.

Because of extremely high internal quantum efficiency of photosynthesis, photosynthetic bio-solar energy conversion has drawn much attention recently in the field of renewable energy. Thylakoid membranes (TMs) are the place where photosynthesis occurs. Since TMs can be easily isolated from plants or algae and remain stable after isolation, they are widely used in photosynthetic energy transfer systems. However, despite its high conversion efficiency from photons to photosynthetic electron (PE), their overall efficiency is limited due to limited absorption of incident photons and low extraction efficiency of PEs. Therefore, several approaches were developed to increase PE extraction by improving electrical connections between TMs and electrodes such as amine or carboxyl modified carbon nanotube electrode, titanium oxide (TiO2) modified electrode or reduced graphene oxide (rGO) modified electrode. Furthermore, PE extraction can also be increased by stimulating photosynthesis to produce more PEs. Recently, it was demonstrated that photosynthesis stimulation could be achieved by inducing localized plasmon field very close to the light energy harvesting complexes within isolated photosystem I or II. In this study, we aimed to enhance PE extraction by stimulating photosynthesis using plasmon induced resonance energy transfer (PIRET). Gold nanorod (AuNR) was used to induce plasmonic field close to the TMs for photosynthesis stimulation and the geometry of AuNR was carefully selected to match its plasmon resonance wavelength with the absorption wavelengths of photosystem I or II within TMs. Therefore, AuNRs of 17 nm width and 45 nm length were chosen to have their plasmon resonance peak at 675 nm. On the other hand, since plasmonic energy can be transferred only in a few nanometer ranges, AuNR should be located very close to TM. Therefore, a tethering molecule which can link AuNR and TM was added to attach the AuNRs to TMs. The tethering molecule has poly ethylene glycol (PEG) chain and two functional groups of thiol group (-SH) and succinimide ester (NHS ester) group at each terminal end. To make AuNR-PEG-TM conjugation, the tethering molecule solution was added to an AuNR in distilled water (DIW) suspension to form AuNR-PEG-NHS. Then, a TM solution with phosphate buffer saline (PBS) was added to the mixture so that AuNR-PEG-TM conjugation was finally formed. Each intermediate product was characterized using SEM, TEM, optical, fluorescence spectroscopy, and XPS analysis. The PE currents from AuNR-PEG-TM was measured and compared to PE currents from pure TM. With the AuNR-PEG-TM conjugation, the PE current was increased by 400% compared to the PE currents of pure TM.

Thylakoids embedded in the chloroplast of plant absorb solar energy and produce photosynthetic electrons (PEs) from water splitting during photosynthesis. PEs excited by absorbing light energy are transferred via electrical carriers in the thylakoid membrane and finally they are consumed to form NADPH. Recent studies have investigated to harvest these excited PEs from thylakoids more efficiently through various approaches such as physical modification of electrodes, tethering of multi-walled carbon nanotubes to thylakoids, or use of mediators. Although these researches contributed to improve the efficiency of PE harvesting, still further studies are needed to achieve practical uses of the photosynthesis-based energy. In this study, we propose that 2-dimensional (2D) ruthenium oxide (RuO2) nanosheet-deposited electrodes can increase the amount of extracted PEs from thylakoids. With large surface area, 2D RuO2 nanosheets have high electrical conductivity and capacity such that they do not only enhance PE extraction but they also increase electron storage. RuO2 nanosheets were synthesized by following processes. First, layered NaRuO2 was synthesized by a solid state reaction, and then Na2S2O8 solution was added to remove excess Na ions. 1 M HCl solution was treated for protonating Na0.2RuO2 before RuO2 exfoliated to RuO2 nanosheets by additional protonating with tetrabutylammonuim. To purify thylakoids, spinach leaves were blended, filtered and centrifugated to extract chloroplasts which contain thylakoids within their membrane. Then, the chloroplasts were osmotically shocked to destroy chloroplast membranes, and density gradient centrifugation was performed to obtain pure thylakoids. For electrode modification with RuO2, RuO2 nanosheets were deposited on Au electrodes by two different methods: drop casting and electrophoresis. 10 and 50 μL of RuO2 solution with 2 mg/mL concentration was dropped on 1 cm2 Au electrode such that 0.02 and 0.1 mg/cm2 of RuO2 nanosheets were deposited on the electrodes. For electrophoretic deposition, Au electrodes were immersed in an acetone solution with 0.01 mg/mL concentration of RuO2. Because the zeta potential of RuO2 nanosheets is negative, the Au electrode was biased with 20 V in order to deposit RuO2 nanosheets on electrode. The thickness of RuO2 nanosheet film was controlled with deposition time. RuO2 was deposited 70 ~ 90 nm during 30 sec and 1~ 1.6 μm during 300 sec. For PEs extraction, 30 μL of thylakoid solution with 3 mg/mL chlorophyll concentration was dropped and dried on each electrode including bare Au electrodes as a control. Bias potential was maintained at 400 mV verses Ag/AgCl and Pt mesh was used as a counter electrode. Photosynthetic currents evidently increased for Au electrodes modified by RuO2 nanosheets. The PE currents from electrodes coated with RuO2 nanosheets were more than 4 times greater than that of bare Au electrodes. To be specific, in case of 0.02 mg/cm2 drop-cast RuO2 electrodes and electrophoretically-deposited RuO2 samples, PE currents were up to 500 nA/cm2, while PE currents from 0.1 mg/cm2 drop-cast RuO2 electrodes were 350 nA/cm2. Also, electrophoretically deposited RuO2 films were stable, while the drop cast RuO2 films were detached after several hours of solution immersion for electrochemical measurement.

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.

Plant cells produce photosynthetic electrons (PEs) by splitting water during photosynthesis. In the beginning of photosynthesis, antenna complex embedded in thylakoid membrane absorbs photon energy and converts them into excited electrons with high quantum efficiency. These excited electrons, known as PEs, are transferred through electron acceptors in order to grow cell or store surplus energy as a form of carbonhydrates. However, since the energy state of the PEs is lowered once they are stored as organic matter, many researchers have tried to extract PEs from photosynthetic electron transfer chain before their conversion into organic matter. Such previous works include collection of PEs by electrochemical oxidizing through mediator as an electron interceptor and use of electrical linker materials such as CNTs, electrically-conducting polymer matrix, or conducting linker to working electrodes between photosynthetic components and the surface of electrodes. Although these works demonstrated the feasibility of harvesting PEs in large scale, there are still issues including efficiency decrease from mediator use and limited stability or requirement of additional electron donors of isolated photosynthetic extracts. In our previous study, we demonstrated direct extraction of PEs from living algal cell, chlamydomonas reinhardtii, by inserting nanoelectrode (NE) into cell membrane. However, in spite of highly efficient extraction of PEs, the maximum harvesting period of PEs was shorter than 1 hour. In this study, we developed a cantilever type of NE system in order to find the optimal shape and size of NE for extraction of PEs for a longer period of time. Fabrication of cantilever NE system started by milling the end of commercial AFM cantilever tip by focused ion beam (FIB). Then, Au was deposited on the cantilever by sputtering as a working electrode. Si3N4 was coated on top of the Au-coated NE to minimize noise signals. Then, additional FIB milling was performed to expose the Au layer at the tip of the NE for localized PE signal measurement. With this cantilever NE, we firstly investigated a relationship between NE size and the viability of inserted cells. It was confirmed that algal cells remained stable for up to 7 days when NE diameters smaller than 500 nm were used. Through continuous observation of inserted algal cells, their viability was ascertained by cell proliferation. After high stability of NE inserted cells was confirmed, direct extraction of PEs was attempted without any mediator. Light-triggered currents from the cells inserted by NEs were observed and the photosynthetic origin of the currents was confirmed by application of DCMU and the subsequent disappearance of the light-responsive currents. Since exposure to highly intense and continuous illumination can damage the pigments of the photosystems, the effect of illumination condition on the PE extraction was investigated. Up to the light intensity of 132 μmol photons m-2s-1, no photobleaching was observed from the NE-inserted cells and the stronger light intensity produced the higher photosynthetic currents. When NE-inserted cells were incubated in a dark except during illumination for photosynthetic current harvesting, about 150 fA of photosynthetic currents were measured over 7 days with 9 μmol photons m-2s-1 of light intensity at 400 mV against Ag/AgCl electrode. As another comparison, when NE-inserted cells were exposed to continuous illumination with 16.5 μmol photons m-2s-1 for 60 hours, more than twice amount of PEs was measured compared to dark incubated cells. Finally, NE-inserted cells were exposed to continuous illumination and th e photosynthetic currents were monitored simultaneously. It was shown that PEs were extracted steadily during several hours of illumination. These results indicated that PEs could be extracted for an extended period of time under various illumination conditions. In this work, the maximum photosynthetic currents measured were 2.5 pA after continuous illumination on a single NE-inserted cell.

Plant cells highly efficiently convert solar energy into photosynthetic electrons during photosynthesis. The photosynthetic electrons (PEs) are transported via redox reactions between molecules in the thylakoid membrane of plant cells. Most of the PEs are utilized for cell growth and small fractions are stored in a form of carbohydrates within plant cells. There have been various approaches to harvest the PEs from the photosynthetic electron transfer chain during photosynthesis. These works have focused on indirect extraction of PEs from isolated photosynthetic components such as photosystem I (PSI) or fragments of thylakoid membrane that contains most of molecules involved in photosynthetic electron transfer. However, despite many advances made from those approaches, there are still challenges such as limited stability of isolated photosynthetic molecules and lowered efficiency due to use of mediators. We previously reported the feasibility of “direct” extraction of PEs from living single algal cells using AFM-compatible nanoelectrodes (NEs). Although the result demonstrated continuous extraction of PEs from living cells at high efficiency, fabrication of the customized NEs was expensive and had poor yield. More importantly, gradual leakage from algal cells occurred after NE insertion into plant cells, resulting in limited PE extraction for only up to an hour. In this study, we aimed to study NE geometry that enabled longer direct extraction of PEs from living algal cells that our previous work without using any mediator. For this, we developed horizontally-tilted cantilever NE system by which NE insertion into living cells could be optically monitored in situ and light-triggered PE extraction could be performed. A commercial AFM tip was custom-shaped into a cantilever with a nanowire (NW) at one end using focused ion beam (FIB). The NW was coated with Au and insulated with a Si3N4 passivation layer by CVD processes. For highly-localized electrochemical functions, another FIB milling was performed to expose the embedded Au only at the tip of the NW. First, we studied how the NW size affected the long-term stability of algal cells after NW insertion. Algal cells were stable for up to about 17 hours when they were inserted by NWs thinner than 500 nm, and they even proliferated after NW insertion. The electrochemical performance of the NW electrode was confirmed with the characteristics of an ultramicro electrode by running cyclic voltammetry analysis using potassium ferricyanide (K3[Fe(CN)6], 0.01M) as a redox system and potassium nitrate (KNO3, 0.1M) as an supporting electrolyte. Light-triggered electrical currents were measured after NW insertion into algal cells with light intensity of 99 μmol·m-2s-1 and potential of 0.4V (versus Ag/AgCl) applied to the NW. Potential-dependent analysis for photocurrents was performed to identify the donor of PEs. Disappearance of the light-triggered currents upon application of photosystem inhibitors also confirmed the currents originated from photosynthesis. Finally, long-term monitoring of cell stability and extraction of PEs was demonstrated for 17 hours after NW insertion into algal cells.