Elsevier

Electrochimica Acta

Volume 89, 1 February 2013, Pages 206-211
Electrochimica Acta

Increased redox-active peptide loading on carbon nanotube electrodes

https://doi.org/10.1016/j.electacta.2012.10.108Get rights and content

Abstract

Carbon nanotube (CNT) electrodes for electrochemistry were fabricated from single- and double-walled carbon nanotubes. The electrodes were subsequently covalently loaded with a ferrocene modified α-aminoisobutyric acid peptide, and the electron transfer (ET) capabilities were probed with cyclic voltammetry. The CNT electrodes comprised of double walled CNTs (DWCNTs) demonstrated significantly higher peak current compared to their single walled counterparts (SWCNTs). This is attributed to a higher loading of the ferrocene modified peptide to the outer wall of the nanotube, through the presence of a larger number of defects sites within the sp2 carbon lattice for the DWCNTs. This higher loading was achieved without compromising the ET rate, indicating that DWCNTs may offer a useful alternative to SWCNTs in future electrochemical sensors and biosensors.

Highlights

► Double and single walled carbon nanotubes (DWCNT and SWCNT) were assembled on gold surfaces. ► CNT arrays were decorated with a ferrocene modified α-aminoisobutyric acid protein. ► A higher loading of redox protein was achieved in the DWCNT case. ► DWCNT electrode exhibited faster electron transfer kinetics. ► DWCNTs may be superior to SWCNTs in sensors and biosensing devices.

Introduction

Carbon nanotubes (CNTs) are a highly desirable material for incorporation into electrochemical [1], [2] and biological sensing devices [3], [4] owing to their fast heterogeneous electron transfer (ET), high surface area and electrochemical stability [5], [6]. Furthermore the conductivity of CNTs has been shown to be remarkably sensitive to changes in surface adsorbates, making them ideal for highly sensitive nanoscale sensors [7]. To date many different carbon nanotube based electrochemical sensors and biosensors exist in the literature, consisting of either randomly dispersed [8], [9] or well-ordered nanotube arrays [6], [10], [11]. However, in all cases the CNTs act as a molecular wire, allowing electrical communication between the underlying electrode and a redox species [7], [12]. For well-ordered or vertically aligned CNT arrays, fast charge transfer has been demonstrated and is a significant advantage compared to randomly dispersed CNTs [5]. This has lead to the development of highly sensitive, reagentless sensing devices [7], where direct ET between the active redox-centre and an electrode surface occurs without the need for mediators. Due to the presence of a large working surface area and easy access of an analyte to the immobilized sensing probe [6], well-ordered or vertically aligned carbon nanotube electrodes have been loaded with molecular sensitive materials such as DNA [6], [13], Cu nanoparticles [1] and anti-immunoglobulin G [11].

This has led to the detection of a wide range of analytes with improved sensitivity and selectivity [2]. For example, Guo et al. [13] recently reported a horizontally aligned CNT genosensor consisting of single-stranded DNA bridging a gap between SWCNTs, which was subsequently sensitive to complementary-stranded DNA. A well-matched DNA duplex was shown to exhibit resistance in the order of 1 MΩ, which in the presence of a GT or CA mis-match was increased ∼300-fold. Somenath et al. also demonstrated a cholesterol sensor based upon modification of vertically aligned multi walled CNTs (MWCNTs) with the biocompatible polymer, polyvinyl alcohol, decorated with cholesterol oxidase (ChOx) [14]. In this sensor the use of carbon nanotubes was found to significantly increase sensor sensitivity through increases in the signal to noise ratio, and was directly attributed to an increase in surface area allowing a high loading of ChOx. An almost linear relationship between cholesterol concentration and the response current was observed in a clinical range up to 300 mg dL−1. Flavel et al. [15] also demonstrated a copper ion sensor fabricated from CNT arrays decorated with the tripeptide Gly-Gly-His, capable of detecting concentrations at a micro molarity level. While this limit of detection is higher than previously reported gold-based sensors, where copper detection down to 3 nM was achieved [16], this novel silicon based sensor offers ease of integration into sophisticated electrical and electronic devices, and is of relevance under current water regulations within Australia. Additionally, Gooding et al. utilized vertically aligned glucose oxidase (GOx) modified SWCNTs on a cysteamine modified gold surface, demonstrating a GOx surface concentration of 5.2 × 10−8 mol cm−2 with apparent ET rate of 9 s−1 [12].

As detailed in a recent review from Diao and Liu [17], several methods exist to chemically assemble CNTs on electrode surfaces. However, common to each method is chemical functionalization of the nanotubes, most commonly with carboxylic acid groups [18] and their subsequent covalent linking to a surface via an ester [19], [20] or amide bond [12]. Unfortunately, such chemical functionalization introduces defects in the hexagonally bonded sp2 [21] lattice, which has the effect of disrupting many of the attractive properties of nanotubes, particularly charge transport [22]. We have recently shown that in order to avoid such disruption DWCNT are advantageous, whereby the outer tube can be selectively functionalized [23] with the inner tube retaining its undisrupted sp2 network and hence its intrinsic electronic properties. The ability to achieve this was then demonstrated in a comparison between carboxyl functionalized SWCNTs and DWCNTs immobilized on a self-assembled cysteamine layer on gold [24]. ET was then isolated to the CNTs by introduction of a polystyrene layer, filling the voids between CNT bundles, with DWCNTs showing a higher apparent ET rate constant for diffusion limited redox with Ru(NH3)63+/2+.

In this work the demonstrated benefits of utilizing DWNTs are capitalized upon by covalently linking an electro-active ferrocene terminated α-aminoisobutyric acid peptide (Aib5-Fc), see Fig. 1(a), to vertically aligned arrays of DWCNTs on cysteamine modified gold substrates. As a further demonstration of the advantage of DWCNTs, an analogous system is also fabricated for SWCNTs. It is shown that not only do DWCNTs have an improved redox peptide loading, but also superior ET kinetics compared to SWCNTs.

Section snippets

Synthesis of Aib5-Fc

Fmoc-Aib-OH loaded 2-chlorotrityl chloride resin (GL Biochem Ltd.) was transferred into a sintered funnel. After the Fmoc group was removed by reaction with a solution of 25% piperidine (Merck) in N,N-dimethylformamide (DMF) (Merck) for 30 min, a solution of 0.05 M Fmoc-Aib-OH (GL Biochem Ltd.) in DMF containing 0.2 M 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate methanaminium (HATU) (GL Biochem Ltd.) and 0.2 M diisopropylethyl amine (DIPEA) (Sigma–Aldrich) was added

Results and discussion

In order to determine the packing density of the cysteamine monolayer and thereby the availability of amine terminal groups for carbon nanotube attachment, the surface concentration of the cysteamine was calculated. Three self assembled monolayer (SAM) modified gold electrodes were cycled between 0 and 1.4 V by cyclic voltammetry in 1 mM pH 6.5 phosphate buffer against Ag/AgCl/KCl (3 M). Fig. 2 shows the first three cycles at a scan rate of 100 mV s−1. In the first cycle a large oxidation peak

Conclusion

In this work, two types of electrochemical surfaces, consisting of DWCNTs and SWCNTs, with similar surface topographies were loaded with a ferrocene modified α-peptide. Due to the larger number of defects on the DWCNT outer walls, more of the peptide could be covalently bonded, resulting in a higher surface concentration and hence, a higher electrochemical current was observed. Determination of the apparent ET rate showed this was at no expense to the DWCNT's ET capabilities. This result

Acknowledgements

KEM wishes to thank the Australian Government for an APA scholarship as well as the Playford Memorial Trust for a top up scholarship. This work is supported by the Australian Microscopy and Microanalysis Research Facility (AMMRF). BSF gratefully acknowledges the support of the Alexander von Humboldt Foundation. ADA and JY acknowledge financial support from the Australian Research Council.

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