Published: September 25, 2011 r2011 American Chemical Society 8123 dx.doi.org/10.1021/ac2016272 | Anal. Chem. 2011, 83, 8123–8129 ARTICLE pubs.acs.org/ac Amperometric Detection of L-Lactate Using Nitrogen-Doped Carbon Nanotubes Modified with Lactate Oxidase Jacob M. Goran, Jennifer L. Lyon, 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 b S Supporting Information E ver since the pioneering work of Clark and Lyon in 1962, electrochemical biosensing schemes have incorporated the inherent selectivity of enzymes. 1 Initial problems with measuring variations in oxygen (O 2 ), which is used as a reagent in the enzymatic reaction, caused a change in detection methods toward the enzymatic byproduct hydrogen peroxide (H 2 O 2 ). However, large overpotentials are necessary for the direct detec- tion of H 2 O 2 by either oxidation or reduction at the electrode surface. 2 In order to lower the operating potential, additional peroxidases 35 and/or redox mediators 6 have been employed. Electrochemical enzyme biosensors that measure a change in O 2 or H 2 O 2 are classified as first-generation biosensors. Redox mediators shuttle electrons between the enzyme’s active center and the electrode, providing the basis for second-generation biosensors. Although these first- and second-generation biosen- sors are still the most widely used and studied, there is a need to develop third-generation biosensors where the enzyme’s active center has a direct electrical connection to the transducer. Third- generation biosensors would operate close to the redox potential of the enzyme, eliminating the need for diffusional redox mediators, peroxidases, or additional redox shuttles for signal transduction. Although certain immobilized enzymes have exhibited direct electron-transfer characteristics, 4,5,7,8 most schemes suffer from slow electron-transfer kinetics, and ineffi- cient utilization of active enzymes, due to the inaccessibility of the redox-active site. 8,9 Steps have been taken to overcome these barriers, such as novel approaches to “wire” the enzyme’s active center to promote facile electron-transfer processes via redox hydrogel, 10 conducting polymer, 11 or reconstituted enzyme 12 methods. Carbon nanotubes (CNTs) are a relatively recent addition to biosensing schemes and are of particular interest as carbon materials are more biocompatible than most prototypical noble metal electrodes and have a higher resistance to surface fouling or denaturing processes. 13 Additionally, the rigidity of CNTs can facilitate direct electronic conduction between the enzyme’s active site and the electrode. 1420 CNT-based sensors have displayed intrinsic electrocatalytic behavior toward biogenic molecules such as H 2 O 2 . 13,21 The electrocatalytic behavior of CNTs is often attributed to their edge plane character, which facilitates fast electron transfer and facile oxidation and reduction kinetics. 22 The inclusion of heteroatoms, such as nitrogen, creates turbostratic disorder, further increasing the edge plane character of CNTs. 23 Thus, heteroatom-doped CNTs, and nitrogen-doped CNTs (N-CNTs) in particular, have received considerable attention for biosensor applications. 15,24 Our group has developed synthetic procedures for N-CNTs and assessed their unique properties. 23,2528 We have also demonstrated that the oxygen reduction reaction (ORR) at N-CNTs proceeds through a peroxide pathway, 28,29 where a fast catalytic dispro- portionation of peroxide provides an alternative to the traditional bienzymatic detection schemes. 30 Herein, we report the use of Received: June 24, 2011 Accepted: September 24, 2011 ABSTRACT: Nitrogen-doped carbon nanotubes (N-CNTs) provide a simple, robust, and unique platform for biosensing. Their catalytic activity toward the oxygen reduction reaction (ORR) and subsequent hydrogen peroxide (H 2 O 2 ) dispropor- tionation creates a sensitive electrochemical response to en- zymatically generated H 2 O 2 on the N-CNT surface, eliminating the need for additional peroxidases or electron-transfer media- tors. Glassy carbon electrodes were modified with 7.4 atom % N-CNTs, lactate oxidase (LOx), and a tetrabutylammonium bromide (TBABr)-modified Nafion binder. The resulting amperometric L-lactate biosensors displayed a sensitivity of 0.040 ( 0.002 AM 1 cm 2 , a low operating potential of 0.23 V (vs Hg/Hg 2 SO 4 ), a repeatability of 1.6% relative standard deviation (RSD) for 200 μM samples of lactate, a fabrication reproducibility of 5.0% (RSD), a limit of detection of 4.1 ( 1.6 μM, and a linear range of 14325 μM. Additionally, over a 90 day period, the repeatability for 200 μM samples of lactate remained below 3.4% (RSD). Direct electron transfer was observed between the LOx redox-active center and the N-CNTs with the electroactive surface coverage determined to be 0.27 nmol cm 2 .