Long-range electrical contacting of redox enzymes by SWCNT connectors.

The combination of biological molecules and novel nanomaterial components is of great importance in the process of developing new nanoscale devices for future biological, medical, and electronic applications. The electrical contacting of redox enzymes with electrodes is a subject of extensive research over the last decade, with important implications for developing biosensing enzyme electrodes, biofuel cells and bioelectronic systems. Tethering of redox-relay units to enzymes associated with electrodes, the immobilization of enzymes in redox-polymers, and the reconstitution of apo enzymes on relay-cofactor units associated with electrodes were reported as means to establish electrical communication between redox proteins and electrodes. Recently, the reconstitution of the apo–flavoenzyme glucose oxidase, GOx, with a single Au nanoparticle functionalized with the flavin adenine dinucleotide (FAD) cofactor was reported.The assembly of the Au-nanoparticle/GOx biocatalyst on an electrode led to an effective electrically contacted enzyme electrode. Single-walled carbon nanotubes (SWCNTs) exhibit unique structural, mechanical, and electronic properties, and recent studies have demonstrated the use of SWCNTs in nanodevices and sensors. Several research activities have addressed the generation of biomaterial–SWCNT hybrid systems, protein-linked CNT and nucleic acid-functionalized SWCNT. The oriented assembly of short SWCNT normal to electrode surfaces was accomplished by the covalent attachment of the CNT to the electrode surfaces. This structural alignment of the SWCNTs allows the secondary association of redox-active components to the CNT, and the examination of charge transport through the SWCNT. Herein we wish to report on the structural alignment of the enzyme glucose oxidase, GOx, on electrodes by using SWCNTs as electrical connectors between the enzyme redox centers and the electrode. We demonstrate that the surfaceassembled GOx is electrically contacted to the electrode by means of the SWCNTs, which acts as conductive nanoneedles that electrically wire the enzyme redox-active site to the transducer surface. The effect of the length of the SWCNTon controlling the electrical-communication properties between the enzyme redox center and the electrode is discussed. SWCNTs (Carbolex, Sigma) were first purified by heating the as-received nanotubes in refluxing 3m nitric acid for 24 h and then washing the resulting nanotubes with water by using a 0.6 mm polycarbonate membrane filter (Millipore). The purified long SWCNTs were chemically shortened by oxidation in a mixture of concentrated sulfuric and nitric acids (3:1, 98% and 70%, respectively) that was subjected to sonication for 8 h in an ice/water bath. This procedure yields shortened SWCNTs with a broad length distribution and that have terminal carboxyl functionalities. The shortened SWCNTs were purified by dialysis and filtering. The resulting SWCNTs suspension was further stabilized by sonication for 3 minutes in 1wt% sodium dodecylsulfate (SDS) as surfactant. After some macroscopic particles had settled down, the SWCNTs supernatant dispersion was loaded onto a controlled pore glass (CPG 3000 >, MPG) chromatographic column to perform length fractionation of the SWCNTs and finally dialyzed against a 1wt% Triton X-100 solution. The eluted SWCNTs fractions (ca. 50 fractions) were analyzed by atomic force microscopy (AFM), to determine the length distribution of SWCNTs in each fraction. The average SWCNTs length decreased as the fraction number increased, and the AFM histograms derived from each fraction showed a relative narrow length distribution. Previous studies have shown that the chemical shortening of SWCNTs by strong acids leads to the formation of carboxylic (and phenolic) groups at the nanotube ends (and sidewall defect sites), thus allowing the covalent immobilization of the SWCNTs on surfaces. A 2thioethanol/cystamine mixed monolayer (3:1 ratio) was assembled on an Au electrode and the length fractionalized SWCNTs were coupled to the surface in the presence of the coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as depicted in Figure 1. The incorporation of 2-thioethanol in the mixed monolayer was anticipated to prevent nonspecific adsorption of the surfactant-protected SWCNTs onto the electrode surface (and presumably to prevent lying of the SWCNT pipes on the surface). Figure 2 shows the AFM images of the 50 nm SWCNTs-modified surfaces upon coupling the CNTs to the modified surface for different time-intervals. Longer coupling times lead to higher surface coverage of the SWCNTs. As illustrated in Figure 2A, isolated needlelike protrusions are clearly seen on the surface after 30 minutes of surface modification, the density of which increases gradually, until a densely packed, needlelike pattern of standing SWCNTs is obtained after five hours of coupling. We could not find by AFM measurements SWCNTs that lie on the surface. There could be as many as eight carboxy groups at each end of the 1.3 nm diameter SWCNT of a (16,0) zigzag structure. Therefore, eight amide bonds could be created between each nanotube and the gold surface, thus leading to a preferred standing conformation of the SWCNTs onto the surface. We found, that the height of the surface-standing nanotubes (determined by AFM) relates directly to the length of the corresponding SWCNTs fraction, although the height measured by AFM is always lower than the length of the free SWCNTs adsorbed on mica surfaces and measured by AFM, [*] F. Patolsky, Y. Weizmann, Prof. I. Willner Institute of Chemistry The Hebrew University of Jerusalem Jerusalem 91904 (Israel) Fax: (+972)2-6527715 E-mail: willnea@vms.huji. ac.il