Full Paper Direct Bioelectrocatalysis of PQQ-Dependent Glucose Dehydrogenase Dmitri Ivnitski, a * Plamen Atanassov , a Christopher Apblett b a Department of Chemical & Nuclear Engineering, University of New Mexico, Albuquerque, NM 87131, USA *e-mail: ivnitski@unm.edu b Sandia National Laboratories, Albuquerque, NM, 87185, USA Received: April 23, 2007 Accepted: May 16, 2007 Abstract The direct bioelectrocatalysis was demonstrated for pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-dependent GDH) covalently attached to single-walled carbon nanotubes (SWNTs). The homogeneous ink-like SWNT suspension was used for both creating the SWNT network on the microelectrode carbon surface and for enzyme immobilization. Functionalization of the SWNT surface by forming active ester groups was found to considerably enhance SWNT solubility in water with a range from 0.1 to 1.0 mg/mL. The PQQ-dependent GDH immobilized on the surface of the SWNTs exhibited fast heterogeneous electron transfer with a rate constant of 3.6 s 1 . Moreover, the immobilized PQQ-dependent GDH retained its enzymatic activity for glucose oxidation. A fusion of PQQ-dependent GDH with SWNTs has a great potential for the development of low-cost and reagentless glucose sensors and biofuel cells. Keywords: PQQ-dependent glucose dehydrogenase, Carbon nanotubes, Direct electron transfer, Glucose DOI: 10.1002/elan.200703899 1. Introduction Direct bioelectrocatalysis of redox enzymes has attracted increasing attention for development of the next gener- ation of electronic micro- and nanoscale biomaterials and nanodevices for industrial, pharmaceutical, clinical, envi- ronmental, space exploration, and defense applications [1– 8]. A fusion of nanotechnology with biology has excellent potential for the development of disposable biochips with near perfect selectivity for a given target analyte. For development of biofuel cells, efficient elec- trical enzyme-electrode communication also has a poten- tial for high power-output [7, 8]. Moreover, direct electron transfer (DET) between active site of enzyme and elec- trode surface is beneficial for studies of structures and mechanisms of different enzymatic reactions in biological systems. Extensive research of DET between the enzyme and the electrode has been carried out over the past 20 years [1, 9 – 14].TheexperimentalevidenceofDEThasbeenreportedin the literature for both low molecular weight electron- transfer proteins and enzymes with large, more complicated structures such as laccase, peroxidase, and glucose oxidase [1, 9–11, 18, 24]. The DET has been observed with multicofactor redox enzymes, such as flavohemeprotein gluconate dehydrogenase [12] and quinohemoproteins [13]. For alcohol dehydrogenase and fructose dehydrogenase the DET between the electrode and active site of the enzyme has been reported [14–17]. Thus, these experimental studies have resulted in substantial advances in our under- standing and utilization of DET phenomenon. At the same time, the efficient DET remains a scientific challenge because the active sites of enzymes are typically located deeply in the apoenzyme structure [1, 10 – 13, 25 – 29]. According to Marcus) theory [30], the kinetics of electron transfer between two redox species is determined by the driving force (e.g., the potential difference), the reorganizational energy and the distance between the two redox centers. Unfavorable orientation of enzyme mole- cules on electrode surface is one of the reasons that may block the electron exchange between the electrode and the electroactive redox center of enzyme via a tunneling mechanism. Significant breakthrough in this area of re- search has been achieved by using carbon nanotubes (CNTs) as promoters of direct bioelectrocatalysis [18 – 24, 31]. The CNTs, which have nanometer size, high chemical stability and a range of electrical conductivity, have become excellent conducting nanowires for fast DET between the active site of an enzyme and the electrode surface. The CNTs may provide an orientation of the enzyme and its redox-active cofactor with respect to the electrode through specific CNT surface modification and treatment. We have previously shown that multi walled carbon nanotubes are excellent promoters for the DET between the active site of glucose oxidase and the carbon electrode [24]. The electron transfer rate constant was between 2.3 s 1 and 2.5 s 1 , which indicated that the heterogeneous DET process was faster than one observed with nonmodified electrodes. As hy- 1562 Electroanalysis 19, 2007, No.15, 1562–1568 # 2007 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim