본 연구에서는 단일벽 탄소나노튜브 (single-walled carbon nanotubes, SWCNTs)로 개질된 탄소종이 전극을 기반으로 , 포도당 산화효소 (GOx)-트레할로스 분해효소 (TREH)가 고정화된  anode와 빌리루빈 산화효소 (BOD)가 고정화된  cathode로 구성된 트레할로스 연료전지를 제작하고 그 성능을 평가하였다 . 특히 , ferrocene methanol (FcMeOH)과 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)를 각각  anode와 cathode에 효소와 함께 고정화하였다 . 주사전자현미경 -에너지분산형 분광분석을 통해 , SWCNTs, 효소 및 전자전달체가 전극 표면에 성공적으로 도입되었음을 확인하였다 . Cyclic voltammetry 분석에서는  0.2V 부근에서  FcMeOH의 산화전류 피크가 관찰되었으며 , SWCNTs가 도입된 전극 (CP-SWCNT)에서 더 강한 산화전류가 나타났다 . 또한 생화학적 분석에서도  CP-SWCNT 전극에 고정화된 GOx가 더 높은 효소 활성을 보였다 . 전기화학 임피던스 분광법 분석에서는  SWCNT 도입에 따라 전하전달 저항이 감 소하고 전자전달 특성이 향상됨을 확인하였다 . GOx-TREH 이효소 전극은 트레할로스 존재 시 높은 산화전류를 생성 하였고 , BOD 기반  cathode는 산소 포화 조건에서 높은 환원 전류를 보였다 . 최종적으로 제작된 트레할로스 연료전지 는 약 0.6 V의 개방회로 전압과 최대  11.7±0.9 μW/cm²의 전력밀도를 나타내어 , SWCNTs 기반 전극 개질 , 효소 및 전 자전달체 고정화 전략이 연료전지 성능 향상에 효과적임을 입증하였다 .

 In this study, a trehalose enzymatic fuel cell was fabricated and evaluated using carbon paper electrodes modified with single-walled carbon nanotubes (SWCNTs), consisting of a glucose oxidase (GOx)-trehalase (TREH)- immobilized anode and a bilirubin oxidase (BOD)-immobilized cathode. In particular, ferrocene methanol (FcMeOH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were co-immobilized with the enzymes on the anode and cathode, respectively. SEM-EDS analysis confirmed the successful introduction of SWCNTs, enzyme, and electron transfer mediator onto the electrode surfaces. Cyclic voltammetric analysis revealed a distinct oxidation peak of FcMeOH near 0.2 V, and the SWCNT-modified electrode (CP-SWCNT) exhibited a higher oxidation current than the bare CP. In addition, biochemical analysis demonstrated that GOx immobilized on the CP-SWCNT electrode showed higher enzymatic activity. EIS analysis indicated that the introduction of SWCNTs reduced the charge transfer resistance and improved the electron transfer properties of the electrode. The GOx-TREH bienzyme electrode generated a high oxidation current in the presence of trehalose. In addition, the BOD-based cathode exhibited a high reduction current under oxygen-saturated conditions. The fabricated trehalose enzymatic fuel cell exhibited an open- circuit voltage of approximately 0.6 V and a maximum power density of 11.7±0.9 μW/cm², demonstrating that the SWCNT-based electrode modification and enzyme/electron transfer mediator immobilization strategy were effective for improving enzymatic fuel cell performance.

Trehalose fuel cell, Single-walled carbon nanotubes, Enzyme-mediator co-immobilization, Cascade enzyme reaction, Ferrocene methanol

1. Xia, X., Xia, H., Wu, R., Bai, L., Yan, L., Magner, E., Cosnier, S., Lojou, E., Zhu, Z. and Liu, A., “Tackling the Challenges of Enzymatic (Bio)Fuel Cells,” Chem. Rev., 119, 9509-9558(2019).
2. Moehlenbrock, M. J., Toby, T. K., Pelster, L. N. and Minteer, S. D., “Metabolon Catalysts: an Efficient Model for Multi-enzyme Cas- cades at Electrode Surfaces,” ChemCatChem., 3, 561-570(2011).
3. Paul, M. J., Primavesi, L. F., Jhurreea, D. and Zhang, Y., “Tre- halose Metabolism and Signaling,” Annu. Rev. Plant Biol., 59, 417-441(2008).
4. Nawab, S., Sha, C., Shah, S. B., Keerio, H. A., Zhu, Z. and Yong, Y., “Trehalose-powered Membraneless Enzymatic Fuel Cell Based on Flexible Alginate Composite Hydrogel Bioelec- trodes,” Process. Biochem., 153, 18-25(2025).
5. Rasmussen, M., West, R., Burgess, J., Lee, I. and Scherson, D., “Bifunctional Trehalose Anode Incorporating Two Covalently Linked Enzyme Acting in Series,” Anal. Chem., 83, 7408-7411
(2011).
6. Lee, Y. S., Lim, K. and Minteer, S. D., “Cascaded Biocatalysis and Bioelectrocatalysis: Overview and Recent Advances,” Annu. Rev. Phys. Chem., 72, 467-488(2021).
7. Hwang, E. T. and Lee, S., “Multienzymatic Cascade Reactions via Enzyme Complex by Immobilization,” ACS Catal., 9, 4402- 4425(2019).
8. Zhang, Y., Selvarajan, V., Shi, K. and Kim, C.-J., “Fabrication and Characterization of Glucose-oxidase-trehalose Electrode Based on Nanomaterial-coated Carbon paper,” RSC Adv., 13, 33918(2023).
9. U.S., J., Rewatkar, P. and Goel, S., “Miniaturized Polymeric Enzymatic Biofuel Cell with Integrated Microfluidic Device and Enhanced Laser Ablated Bioelectrodes,” Int. J. Hydrog. Energy., 46, 3183-3192(2021).
10. Shan, D., Yao, W. and Xue, H., “Electrochemical Study of Fer- rocenemethanol-modified Layered Double Hydroxides Composite Matrix: Application to Glucose Amperometric Biosensor,” Biosens. Bioelectron., 23, 432-437(2007).
11. Raad, F. A., Picot, P., Barruet, E., Caer, S. L., Schaming, D., Sicard, L. and Thill, A., “Redox Behavior of Ferrocene-methanol Encapsu- lated in Hydrophobic Nanotubes,” Appl. Clay Sci., 276, 107909 (2025).
12. Ariga, K., Rezki, M., Suzuki-Nagata, K., Mikawa, T. and Tsujimura, S., “MOF-based Hybrid Electrodes for Multi-enzyme Cascade Reaction with Stabilized Mediator Immobilization,” Chem. Com- mun., 61, 12309-12312(2025).
13. Rezki, M. and Tsujimura, S., “Hybrid Bioelectronic Interfaces with Supramolecularly Immobilized Redox Mediators for Ultra- stable and High-performance Enzyme Electrodes,” ACS Appl. Mater. Interfaces, 17, 44101-44111(2025).
14. Takeda, K., Minegishi, Y., Lundin, S. T. B., Yoshida, A., Nishina, Y., Nishihara, H., and Itoh, T., “Quinone-Based Mediator Immo- bilized Mesoporous Electrodes for Bioelectrocatalysis of Glu- cose Dehydrogenase,” ACS Appl. Bio Mater., 9(3), 1365-1373(2026).
15. Das, P., Das, M., Chinnadayyala, S. R., Singha, I. M. and Goswami,
rd
P., “Recent Advances on Developing 3 Generation Enzyme Elec- trode for Biosensor Applications,” Biosens. Bioelectron., 79, 386- 397(2016).
16. Pichardo, S., Gutierrez-Praena, D., Puerto, M., Sanchez, E., Grilo, A., Camean, A. M., and Jos, A., “Oxidative Stress Responses to Car- boxylic Acid Functionalized Single Wall Carbon Nanotubes on the Human Intestinal Cell Line Caco-2,” Toxicol. In Vitro, 26, 672-677(2012).
17. Wang, X., Kim, J. H., Choi, Y. B., Kim, H.-H and Kim, C.-J., “Fabrication of Optimally Configured Layers of SWCNTs, Gold Nanoparticles, and Glucose Oxidase on ITO Electrodes for High- power Enzymatic Biofuel Cells,” Korean J. Chem. Eng., 36, 1172- 1183(2019).
18. Sheldon, R. and Pelt, S. V., “Enzyme Immobilization in Bioca- talysis: Why, What and How,” Chem. Soc. Rev., 42, 6223-6235(2013).
19. Wang, L., Xu, Rong, Chen, Y. and Jiang, R., “Activity and Sta- bility Comparison of Immobilized NADH Oxidase on Multi-walled Carbon Nanotubes, Carbon Nanospheres, and Single-walled Car- bon Nanotubes,” J. Mol. Catal.B Enzym., 69, 120-126(2011).
20. Asuri, P., Bale, S. S., Pangule, R. C., Shah, D. A., Kane, R. S. and Dordick, J. S., “Structure, Function, and Stability of Enzymes Covalently Attached to Single-Walled Carbon Nanotubes,” Langmuir, 23, 12318-12321(2007).
21. Loew, N., Ofuji, T., Shitanda, I., Hoshi, Y., Kitazumi, Y., Kano, K., and Itagaki, M., “Cyclic Voltammetry and Electrochemical Impedance Simulation of the Mediator-type Enzyme electrode Reaction Using Finite Element Method,” Electrochim. Acta., 367, 137483(2021).
22. Gao, P., Kasama, T., Shin, J., Huang, Y., and Miyake, R., “A Medi- ated Enzymatic Electrochemical Sensor Using Paper-based Laser- induced Graphene,” Biosensors., 12, 995(2022).
23. Lasia, A., “Impedance of Porous Electrodes,” Curr. Opin. in Electro- chem., 54, 101764(2025).
24. Mei, B., Lau, J., Lin, T., Tolbert, S. H., Dunn, B. S., and Pilon, L., “Physical Interpretations of Electrochemical Impedance Spectros- copy of Redox Active Electrodes for Electrical Energy Stor-age,” J. Phys. Chem. C. 122, 24499-24511(2018).
25. Torrigino, F., Nagel, M., Hartmann, M., and Herkendell, K. “Electro- chemical Impedance Spectroscopy As a Characterization Method for Enzymatic Fuel Cell Bioanodes,” ChemElectroChem., 11, e202400329(2024).
26. Han, J, Hwang, S., and Kim, J. H., “Electrochemical Impedance Spectroscopy Analysis of Plasma-treated, Spray-coated Single-walled Carbon-nanotube Film Electrodes for Chemical and Electrochemi- cal Devices,” Microchemical J., 188, 108446(2023).
27. Thangavel, B., Venkatachalam, G. and Shin, J. H., “Emerging Trends of Bilirubin Oxidases at the Bioelectrochemical Interface: Paving the Way for Self-Powered Electrochemical Devices and Biosen- sors,” ACS Appl. Bio. Mater., 7, 1381-1399(2024).
28. Thomas, J. H., Drake, J. M., Paddock, J. R., Conklin, S., Johnson, J. and Seliskar, C. J., “Characterization of ABTS at a Polymer- modified Electrode,” Electroanal., 16, 547-555(2004).
29. Rasmussen, M., Ritzmann, R. E., Lee, I., Pollack, A. J., and Scher- son, D., “An Implantable Biofuel Cell for a Live Insect,” J. Am. Chem. Soc., 134, 1458-1460(2012).
30. Shoji, K., Akiyama, Y., Suzuki, M., Hoshino, T., Nakamura, N., Ohno, H., and Morishima, K., “Insect Biofuel Cells using Treha- lose Included in Insect Hemolymph Leading to an Insect-mount- able Biofuel Cell,” Biomed. Microdevices, 14, 1063-1068(2012).