Optimization of a redox protein-carbon nanotube conjugate biosensor by siteselective binding

Abstract

We report on a redox protein highly ordered carbon nanotube (CNT) array conjugate system that exhibits an exceptionally high level of bioelectrocatalytic activity. The performance of the conjugated system is dependent upon site-selective placement of the protein on the nanotube. Enzymes immobilized on the nanotube tip have generated electrical biosignals more than 60 times greater than enzymes bound to the nanotube side walls, and have shown electron transfer rates on the order of 1500 sQ1. The substrate concentration dependence of the bioelectrocatalytic signal was measured by CV, and a detection limit in the single micromolar range was achieved. We conclude that the covalent attachment of redox enzymes to CNT tips enhances electron transfer efficiency by orders of magnitude when compared to side-wall adsorption, and that the resulting system enables real-time monitoring ofbiomolecular activities. The nanoelectronic platform that has enabled this novel course of study is a hexagonally ordered array of aligned multi-walled carbon nanotubes (MWNTs). Unique features of this array critical to this study include identical exposed nanotube length across the entire array, highly regular center-to-center CNT spacing, and electrical insulation of individual nanotubes within an aluminum oxide nanopore array. This configuration of nanotubes allows us to differentiate between the two distinctly different regions of the CNTs: the side walls which are highly hydrophobic and amenable to protein adsorption, and the tips that are easily oxidized and can be covalently modified with enzymes by carboxyl-amine coupling. We have exploited these properties to independently study the bioelectrocatalytic activity of enzymes bound to the two regions. A CNT array electrode was first treated with the surfactant arabic gum (GA) to prevent protein adsorption to the side wall and then the enzyme glucose oxidase (GOx) was covalently bound to the nanotube tips. On another sample, the nanotube tips were capped with ethanolamine to remove any free carboxylic acid groups, and then GOx was adsorbed to the protein sidewall. Gold nanoparticle labeling experiments were used to visually confirm the successful selective immobilization of GOx at the CNT tips, and an ELISA was performed to quantify the extent of enzyme coverage on the tips and on the side walls. We determined there to be more than 60 times more protein adsorbed to the side walls than GOx covalently linked to the CNT tips. Cyclic voltammetry (CV) measurements revealed the current density of each sample to be nearly the same (-l20±A*cm-2). This means that each enzyme bound to the CNT tip is contributing, on average, more than 60 times the electrical signal as GOx adsorbed to the nanotube side wall. A saturation assay was then performed to determine the unimolecular electron transfer rate (kET) of the tip-bound GOx-CNT conjugate system. Based on the peak current density (330pA*cm2) and the surface enzyme coverage determined using the ELISA (1 x 10-12 mol. cm2) we have calculated a kET Of 1500s-1 using a method previously established'. This rate is higher than the established rate for GOx in vivo2. There are several factors that may be contributing to this unusually high rate. Firstly, the nature of the covalent linkage at the CNT tip is such that the protein conformation is minimally stressed, and the enzyme should therefore retain its full bioelectrocatalytic function. In addition, because the CNT is electrically insulated from its neighbors, any voltage applied during CV measurements will result in a maximal electric field at the nanotube tip, and the CNT will act essentially as a lightning rod for efficient electron conduction. Such a high rate of electron transfer distinguishes this enzyme-CNT conjugate as an ideal biosensing system. To quantify the potential sensitivity and detection limit of a biosensor relying on this conjugated system, further CV measurements were performed on a CNT array sample in low glucose concentrations. Levels down to a single micromole were detected, resulting in a signal on the order of tens of nanoamps. Already, this compares favorably to other enzyme-based biosensor systems that generally do not venture below the millimolar range. However, the limit could even be pushed further, likely to the lower nanomolar levels, by using an ammeter capable of measuring currents in the picoampere range. These GOx-CNT electrode conjugate biosensors are very robust, demonstrating only a 10% loss of activity after two weeks storage in 50mM sodium phosphate buffer (pH 7.0) at 4°C and 50% loss after one month, measured once daily. [1] Xiao, Y. et al. Science 2003, 299, 1877-1881. [2] Bourdillon, C. et al. J Am. Chem. Soc. 1993, 115, 12264-12269.