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Lactate has long been known as the indicator for distinguishing aerobic and anaerobic metabolism, and thus is a useful marker for metabolic disorders and the state of exercise [1]. Especially, the continuous monitoring of sweat lactate is a useful non-invasive method for real-time tracking the progress of exercise, e.g. of athletes, or of physical therapy patients. In biosensors for continuous monitoring, the biological recognition element, e.g. lactate oxidase (LOx) has to be immobilized on the electrode. Our group previously reported of a glucose sensor in which glucose dehydrogenase (GDH) was immobilized covalently on screen-printed MgO-templated mesoporous carbon (MgOC) modified by graft polymerization, and showed improved stability [2]. Likewise, in this study, radicals were introduced on the surface of the MgOC by electron beam irradiation. Next, glycidyl methacrylate (GMA) was polymerized on the radicalized MgOC, which resulted in epoxy groups on the surface. Ink was prepared by dispersing this poly(GMA)-MgOC (GMgOC) and a polyvinylidene difluoride (PVdF) derivative in N-methyl-2-pyrrolidone (NMP). The ink then was used for the final layer of the working electrode (WE) of a screen-printed 3-electrode strip. Next, 1,2-naphthoquinone was deposited on the WE as mediator, which is insoluble in water and thus is unlikely to dissolve into sweat during the measurement. Finally, LOx derived from Enterococcus faecium was immobilized on the WE by binding covalently to the epoxy groups. This combination of LOx immobilized on screen-printed GMgOC and naphthoquinone promises the construction of stable lactate biosensors suitable for continuous measurements. Furthermore, in this study, two other points were considered for the design and construction of the sensor device. Firstly, the sensor should be constantly supplied with a minimum amount of sweat, without the supply being interrupted. Secondly, the materials and texture of the parts of the sensor in direct contact with the skin should not be irritating. An integrated flow system can solve both points; it ensures a constant supply of sweat, and limits the material and texture in direct contact with the skin. For the construction of the flow system, first, a mold was formed on a silicon wafer using an epoxy-based negative photoresist. Next a degassed mixture of polydimethylsiloxane (PDMS) monomer and catalyst was poured over the mold and cured at 80°C for 1 hour. In first trials, the sensor and flow system were pressed together tightly onto artificial sweat glands. Leakage was prevented by constructing a connector for the sensor out of conducting adhesive tape. Potassium phosphate buffer (100 mM, pH 7.0) containing various amounts of sodium lactate were pumped through the system at 80 µL/min using a syringe pump. Figure 1a shows the response of this system to varying concentrations of lactate. The spikes at the change of concentration are due to a change in the flow when switching the syringe. The delay between the switch of the syringe and the change in response current is due to the lag time until the lactate reached the sensor. The plot of the response current vs the lactate concentration (Fig. 1b) shows a concentration dependent response up to 20 mM lactate. These results indicate that this system is suitable for measuring sweat lactate continuously. Acknowledgement: This work was partially supported by JST-ASTEP Grant Number JPMJTS1513, JSPS Grant Number 17H02162 and Private University Research Branding Project (2017-2019) from Ministry of Education, Culture, Sports, Science and Technology, and Tokyo University of Science Grant for President's Research Promotion． References [1] M.E. Payne, et al.; Sci. Rep., 9, 13720 (2019). [2] I. Shitanda, et al.; Bull. Chem. Soc. Jpn., 93, 32-36 (2020). Figure 1

Lactate concentration in sweat is closely related to exercise intensity. Thus, real-time monitoring of lactate concentration is extremely important for improving athlete training and health management. There are many reports for monitoring the change in the lactic acid concentration in sweat over a long period of time using a wearable lactate biosensor 1,2). For practical application of a wearable lactate biosensor for long-term monitoring, it is important to suppress elution of the enzyme from the electrode by simple enzyme immobilization scheme. Recently, we have reported that long-term stability of a glucose oxidase (GOD)-immobilized electrode was dramatically improved by introducing a poly(glycidyl methacrylate) on a mesoporous carbon, namely MgO-templated carbon (MgOC), which binds to a GOD by coavalent bonding scheme. In this study, we applied the method mentioned above to a printed lactate biosensor. A lactate oxidase (LOx) was immobilized on the electrode by covalent bonding with a poly(glycidyl methacrylate)-modified carbon surface. Firstly, radicals were introduced on the MgOC surface by electron beam irradiation, and glycidyl methacrylate (GMA) having an epoxy group was polymerized according to the previously reported scheme3). We referred the poly(GMA)-immobilized MgOC was “GMgOC” in this study. GMgOC and polyvinylidene fluoride (PVdF) were dispersed in N-methyl pyrrolidone (NMP) to prepare a graft polymerized carbon ink for printing. A printed electrode tips were fabricated by screen printing. Then, an enzyme-immobilized electrode was prepared by modifying 1,2-naphthoquinone as a mediator and lactate oxidase as an enzyme. The stability of the enzyme-immobilized electrode was evaluated by 10 cycles of cyclic voltammetry. The measurement was performed in a 1 mol dm-3 phosphate buffer (pH 7.0) containing 25 mmol dm-3 lactic acid with a three-electrode system. A platinum wire as a counter electrode, and a sat. KCl/Ag/AgCl electrode as a reference electrode were used. The maximum current density was observed at about 0.13 V vs. Ag/AgCl. Figure 1 shows the maximum current of the cyclic voltammogram plotted against the number of cycles during multiple sweeps. In case of the GMgOC, the current value retained 95.7% after 10 cycles, but it was 68.4% in the case of unmodified MgOC. By performing graft polymerization on the MgOC surface to fabricate the electrode, no significant change in the current value was observed. These results indicated that LOx was stably immobilized on the GMgOC surface without degeneration. Acknowledgements This work was partially supported by JST-ASTEP Grant Number JPMJTS1513, JSPS Grant Number 17H02162 and Private University Research Branding Project (2017-2019) from Ministry of Education, Culture, Sports, Science and Technology, and Tokyo University of Science Grant for President's Research Promotion． References 1) W. Jia, A. J. Bandodkar, G. Valdés-Ramírez, J.R. Windmiller, Z. Yang,J. Ramírez ,G. Chan, J. Wang, Analytical Chemistry, 85 (2013), 6553. 2) R.A.Escalona-Villalpando,E.Ortiz-Ortega,J.P.Bocanegra-Ugalde,Shelley,D.Minteer,J.Ledesma-García, L.G. Arriaga, Journal of Power Sources, 412 (2019),496-504. 3) I. Shitanda ,T. Kato ,R. Suzuki ,T. Aikawa ,Y. Hoshi ,M. Itagaki , and S. Tsujimura , Bulletin of the Chemical Society of Japan, 93 (2019), 32-36． Figure 1

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