Influence of Surface Adsorption on the Interfacial Electron Transfer of Flavin Adenine Dinucleotide and Glucose Oxidase at Carbon Nanotube and Nitrogen-Doped Carbon Nanotube Electrodes Jacob M. Goran, † Sandra M. Mantilla, ‡ and Keith J. Stevenson* ,† † Department of Chemistry and Biochemistry, Center for Electrochemistry, Center for Nano- and Molecular Science and Technology, The University of Texas at Austin, 1 University Station, A5300, Austin, Texas 78712, United States ‡ Department of Biomedical Engineering, Florida International University, 10555 West Flagler Street, EAS 2600, Miami, Florida 33174, United States * S Supporting Information ABSTRACT: The adsorption of flavin adenine dinucleotide (FAD) and glucose oxidase (GOx) onto carbon nanotube (CNT) and nitrogen-doped CNT (N-CNT) electrodes was investigated and found to obey Langmuir adsorption isotherm characteristics. The amount adsorbed and adsorption max- imum are dependent on exposure time, the concentration of adsorbate, and the ionic strength of the solution. The formal potentials measured for FAD and GOx are identical, indicating that the observed electroactivity is from FAD, the redox reaction center of GOx. When glucose is added to GOx adsorbed onto CNT/N-CNT electrodes, direct electron transfer (DET) from enzyme-active FAD is not observed. However, efficient mediated electron transfer (MET) occurs if an appropriate electron mediator is placed in solution, or the natural electron mediator oxygen is used, indicating that GOx is adsorbed and active on CNT/N-CNT electrodes. The observed surface- confined redox reaction at both CNT and N-CNT electrodes is from FAD that either specifically adsorbs from solution or adsorbs from the holoprotein subsequently inactivating the enzyme. The splitting of cathodic and anodic peak potentials as a function of scan rate provides a way to measure the heterogeneous electron-transfer rate constant (k s ) using Laviron’s method. However, the measured k s was found to be under ohmic control, not under the kinetic control of an electron-transfer reaction, suggesting that k s for FAD on CNTs is faster than the measured value of 7.6 s -1 . C arbon nanotubes (CNTs) have been explored for a vast array of biosensing applications, either by themselves 1-4 or modified with enzymes such as glucose oxidase (GOx). 5-8 The ideal enzymatic biosensor would be a so-called “third- generation” biosensor, where the electrode would have direct electrical access to the redox-active center of a functioning enzyme. Given that the protein shell is designed to protect the redox-active center and impart selectivity, only a few enzymes naturally exhibit this behavior on traditional electrode surfaces; 9-11 however, novel methods have been employed to facilitate electron transfer between electrode surfaces and other enzymes. 12-17 CNTs have been noted as an ideal material for direct electron transfer (DET) due to their small size and excellent electronic conductivity. 18 The literature contains many reports of DET between CNT electrodes and the enzyme GOx. 18-32 Flavin adenine dinucleotide (FAD), the redox-active center of GOx, is tightly bound inside a deep pocket of GOx, but is not covalently bound. 33 A hypothesis of the DET process between CNTs and GOx is that the small diameter CNTs are able to penetrate into the protein or glycoprotein shell deep enough for electron tunneling to occur. 18 The electrochemical behavior of FAD has been studied on electrode materials such as mercury, 34-36 glassy carbon (GC), 37,38 modified GC, 39 gold, 40 titanium, 41 TiO 2 , 42 and graphite, 43,44 but has not been adequately investigated on CNTs. This study examines the spontaneous adsorption of FAD and GOx on CNTs and nitrogen-doped CNTs (N- CNTs), which offers a unique way to modify the surface and extend our understanding of the surface-confined redox reaction. Our study is unique in that oxidative acids such as sulfuric and nitric 18,22,23,29,30 are not used to create oxygen functionalities and assist in the dispersion of the hydrophobic CNTs into aqueous solutions. In addition, we do not employ surfactants like cetyltrimethylammonium bromide (CTAB, a cationic surfactant), 19 Triton X-100, 32 or 3-aminopropyltrie- thoxysilane (APTES) 20 to assist suspension, or binders such as Nafion 19,24,25,29 to ensure adhesion of the FAD or GOx to the electrode. Furthermore, GOx or FAD was not covalently attached to the CNTs with carbodiimide coupling, 22,32 dispersed in an immobilizing film, 27 or constructed from layer-by-layer assembly with cationic films such as polyethyle- Received: September 26, 2012 Accepted: January 5, 2013 Published: January 5, 2013 Article pubs.acs.org/ac © 2013 American Chemical Society 1571 dx.doi.org/10.1021/ac3028036 | Anal. Chem. 2013, 85, 1571-1581