DOI: 10.1002/celc.201402035 Immobilisation of Flavin-Adenine-Dinucleotide-Dependent Glucose Dehydrogenase a Subunit in Free-Standing Graphitised Carbon Nanofiber Paper Using a Bifunctional Cross-Linker for an Enzymatic Biofuel Cell Deby Fapyane, [a] Yooseok Lee, [a] Chyi Yan Lim, [a] Jou-Hyeon Ahn, [b] Seon-Won Kim, [c] and In Seop Chang* [a] Free-standing graphitised carbon nanofiber paper (GCNFp) is fabricated using a dispersion–filtration method and modified by non-covalent functionalisation with 1-pyrenebutyric acid N-hydroxysuccinimide ester—a bifunctional linker reagent— through p–p stacking. This modified GCNFp is then used to immobilise enzymes, and together they form an electrode for enzymatic biofuel cell (EBFC) applications. This fabrication method is shown to be capable of providing a practical plat- form for enzyme–electrode electrical communication that is faster than comparable systems based on other carbon materi- als, as calculated from the heterogeneous electron-transfer rate constant. The GCNFp-based EBFC reaches a maximum power density at a glucose concentration of 100 mm, yielding 834.9  200, 262.9  15.6 and 147.2  4.70 mW cm 2 for flavin- adenine-dinucleotide-dependent glucose dehydrogenase (FADGDH)–menadione, glucose oxidase (GOX)–menadione, and GOX-only systems (as the anode), respectively, with lac- case as the cathode. In recent years, there has been much interest in the study of nanotechnology and its application in a diverse range of fields. Developments in nanotechnology might lead to the establish- ment of new fields of functionality, such as nano-catalysis. When nano-catalysts are supported by nanomaterials, the sur- face area, and therefore the efficiency, of the catalytic process increases due to smaller reaction spaces and faster rates. [1] Since the discovery of carbon nanotubes (CNTs) by Iijima, carbon nanomaterials have been highlighted as the most promising materials for use in nano-catalysis. [2] CNTs, as well as other carbon nanomaterials such as fullerene, graphene, and carbon nanofibers (CNFs), have been extensively studied due to the exceptional nature of their properties including nano- porosity, mechanical strength, and excellent conductivity for bio-catalysis applications. [2, 3] As one of the potential applications of bio-catalysis, the en- zymatic biofuel cell (EBFC) is an electrochemical system for electricity generation through enzymatic substrate oxidation or oxidant reduction. Generally, enzymes are used at the anode and cathode, and are immobilised on the surface of a conduct- ing electrode material such as carbon to allow fast and effi- cient electron transfer. Carbon nanomaterials are the preferred supports for EBFC enzyme–electrode configurations due to their stable chemical and physical properties. [4] Up to now, several approaches have been proposed to im- mobilise enzymes in nanomaterials, such as physical entrap- ment, [5] covalent bonding through carbodiimide chemistry, [6] and non-covalent bonding through bifunctional linkers. [7] Phys- ical entrapment of enzymes in the nanomaterial is limited by several issues related to enzyme stability, which correlate to the overall system stability. Covalent bonds formed through carbodiimide chemistry can be the solution to stability prob- lems as strong amide bonds are formed between amine resi- dues of the enzyme and carbonyl residues in the carbon nano- material. [6] This approach, however, requires the oxidation of carbon nanomaterials to form hydroxyl and carbonyl groups, thus a proportion of sp 2 -hybridised carbons are transformed to sp 3 , thereby reducing the conductivity of the material carbon- yls are not sp 3 . [8] One nanomaterial that has not yet been widely explored is CNF. CNF is a carbon nanomaterial possessing a unique struc- ture that makes it possible to activate its entire surface. [9] CNFs have a stacked cup-like structure that can be readily decorated with enzymes for biosensor applications [10] and other electro- chemical applications such as biofuel cells, and CNFs have sim- ilar electrical and mechanical properties to CNTs. CNFs are more structurally accessible for enzyme immobilisation due to a larger active-surface area than CNTs and therefore they might be more suitable electrode materials. [10, 11] Direct immo- bilisation on the active CNF surface might be sufficient for bio- sensor applications, as the electrode will only be used a limited number of times. However, direct immobilisation might not be [a] D. Fapyane, Y. Lee, C. Y. Lim, Prof. I. S. Chang School of Environmental Science and Engineering Gwangju Institute of Science and Technology (GIST) 261 Cheomdan-gwagiro (Oryong-dong) Buk-gu, Gwangju 500-712 (Republic of Korea) E-mail : ischang@gist.ac.kr [b] Prof. J.-H. Ahn Department of Chemical and Biological Engineering Gyeongsang National University Jinju 660-701 (Republic of Korea) [c] Prof. S.-W. Kim Division of Applied Life Science (BK21 Plus) Plant Molecular Biology and Biotechnology Research Center Gyeongsang National University Jinju 660-701 (Republic of Korea) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/celc.201402035.  2014 Wiley-VCH Verlag GmbH & Co. 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