Preparation of NADPH-Tcpp Immobilized Electrode and Photoinduced Oxidation
Authors: Ando Hironao , Asakura Noriyuki 
Published: December 2020

The development of new strategies for converting light source to chemical energy or useful compounds have been required. In order to accomplish light-driven reaction, photosensitizer and enzymes are utilized as a component of reaction system. Moreover, the light-driven reaction prepared on solid phase is considered to be useful device on the system.
This study shows that photoinduced oxidation system on ITO electrode with porphyrin is prepared and some oxidase reaction by light irradiation occurs. A photoexcited state of porphyrins are able to accept electrons from oxidase via mediator such as NADPH, while an electron of the LUMO is donate to ITO electrode on which the porphyrin is immobilized. Therefore, photoinduced oxidation occur on ITO electrode.
NADPH-porphyrin immobilized ITO electrode was prepared and photoanodic reaction, photoinduced electron transfer to ITO electrode, is carried out. Firstly, redox cycle of NADPH immobilized electrode was investigated. Basically, immobilized NADPH redox reaction is hard to observe without excess over potentials, so that NADPH is immobilized on Au electrode by covalently binding due to decrease in an interfacial resistance between electrode surface and NADPH. Enzymatic reaction using NADPH immobilized electrode was carried out and investigated that NADPH monolayer mediate electron from enzymes.
In addition, NADPH-porphyrin immobilized ITO electrode was prepared and photoinduced oxidation with malate dehydrogenase was carried out.
NADPH monolayer on Au electrode was prepared as follows. Au electrode surface was modified with a self-assembled monolayer (SAM) of α-Lipoic acid, and 3-amino boronic acid was covalently linked to the carboxyl group of the α-Lipoic acid SAM. NADPH was covalently bound to the boronic acid, which was immobilized on α-Lipoic acid SAM.
In the case of the photoinduced oxidation experiment, ITO electrode on which NADPH-TCPP diads was absorbed (NADPH-TCPP-ITO). TCPP (tetrakis(4-carboxyphenyl)porphyrin) was used as a photosensitizer. ITO electrode was silanized with APTES (3-Aminopropyltriethoxysilane). The amino-functionalized layer on the ITO electrode was modified with TCPP and then 1,2-diamino ethane was bound to carboxy group of TCPP. Finally, NADPH was covalently bound to the amine group of diamino ethane-TCPP monolayer on the ITO electrode.
Experiment of photoinduced oxidation was carried out with NADPH-TCPP-ITO electrode. NADPH-TCPP-ITO electrode was immersed in 0.1M Tris buffer (pH9.0) containing 1 U of malate dehydrogenase (MDH) and 50 mM malic acid, and light irradiation under applied potential at 0 V. Current of this experiment was shown in Figure 2. Red line and black line show the current in the presence of MDH and in the absence of MDH, respectively. During light on 30 s~60 s, increase in current in the presence of MDH was observed, and the current was decrease immediately at the light off These result shows that photoinduced oxidation occurs, which NADPH donate electrons to HOMO of photoexcited state of TCPP and NADPH regenerate by the oxidation of malate with MDH. Otherwise, black line (in the absence of MDH) no current increase was observed during light on. Therefore, photoanodic reaction using MDH is able to proceed on NADPH-TCPP-ITO electrode.Figure 1 shows the cyclic voltammogram of NADPH immobilized Au electrode in 10 mM phosphate buffer (pH7.0). containing glucose dehydrogenase (GDH) and 10 mM glucose. CV was performed at 10 mV/s. Remarkably increase in anodic current was observed in the range of potential sweep. The increase in current indicate the catalytic current of GDH, which glucose was oxidized to gluconic acid Therefore, GDH in solution is able to access to the NADPH monolayer and occurs enzymatic reaction.

Figure 1