Available online at www.sciencedirect.com
ScienceDirect Procedia Technology 27 (2017) 238 – 239
Biosensors 2016
Collision-based electrochemistry for investigation of direct electron transfer of a single enzyme molecule Alina N. Sekretaryova*, Mikhail Yu. Vagin, Anthony P.F. Turner, Mats Eriksson Linköping University, Department of Physics, Chemistry and Biology (IFM), SE-58183 Linköping, Sweden
Abstract Electron transfer between a biorecognition element and an electrode is an essential element of bioelectrocatalytic systems, such as biosensors and biofuel cells. The number of working systems based on direct electron communication is limited and detailed investigations of the mechanism of the process are still required. Here, we present the use of a novel approach of collision-based bioelectrocatalysis to monitor electrocatalytic currents from individual redox enzyme molecules. This approach allowed us to calculate the individual turnover rates of these molecules and investigate the influence of the applied potential, pH and additions of inhibitor on the observed rates of direct electron transfer. © 2017 2016The TheAuthors. Authors.Published Published Elsevier © byby Elsevier Ltd.Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of Biosensors 2016. Peer-review under responsibility of the organizing committee of Biosensors 2016 Keywords: direct electron transfer; bioelectrocatalysis; single molecule; laccase; collisions; ultramicroelectrode.
Modern science witnesses a burgeoning interest in single molecule detection and investigation. Of particular importance are studies of biological macromolecules such as proteins. Electrochemical approaches have been used for single-molecule investigations, but their use is, so far, limited compared to mechanical and spectroscopic measurements. The main obstacle for electrochemical studies of single molecules is the tiny currents produced in typical electrochemical reactions. In order to detect these currents some means of current amplification is needed. This amplification can be achieved by catalysis. Such an approach was recently introduced by Bard for investigation of catalytic nanoparticles during collisions with microelectrodes (1). Current amplification by a few enzyme molecules was first reported by Lemay and co-workers (2), where catalytic currents produced by a [NiFe]hydrogenase film modified nanoelectrode were monitored. Li et al. used the collision-based approach to monitor
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2212-0173 © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of Biosensors 2016 doi:10.1016/j.protcy.2017.04.101
Alina N. Sekretaryova et al. / Procedia Technology 27 (2017) 238 – 239
catalytic currents from several enzyme molecules assembled on the surface of graphene oxide flakes (3). In a very recent publication our group, for the first time, presented experimental data indicating that electrocatalytic currents can be measured during collisions of single laccase enzyme molecules with a gold ultramicroelectrode (4). Later Zhan and co-workers reported similar work on the detection of catalytic currents from single horseradish peroxidase enzyme molecules during their collisions on gold nanoelectrodes (5). In the work presented here the enzyme laccase was used as a model catalytic macromolecule. The basic idea is that if a laccase globule hits a gold ultramicroelectrode (AuUME) in the correct orientation, then the electrode will turn over the enzyme and allow catalysis of the substrate, i.e. oxygen (Fig. 1a). The catalytic reaction is accompanied by direct electron transfer (DET) between the electrode and the active site of the enzyme, giving rise to current spikes in an amperometric measurement (Fig. 1b). Control experiments with and without macromolecular catalysts and with and without the catalyst’s substrate was performed to confirm that it is the catalytic reaction that gives rise to the currents. These control experiments demonstrated that the current spikes were only observed when both the enzyme and oxygen are present at the same time. The experimental data provided information for the calculation of turnover rates for single enzyme molecules and investigation of the factors influencing the efficiency of DET, such as applied overpotential, pH and the presence of inhibitors.
Fig. 1. (a) Scheme showing the orientation dependence of the catalytic current during adsorption of a single enzyme molecule. (b) Amperometric measurement with current spikes registered as a result of enzyme molecule collisions on a gold microelectrode (diam. 12 μm) at an applied potential of +0.21 V vs NHE, 0.1 U mL-1 enzyme solution in 0.1 M acetate buffer at pH 5.0, T=20°C.
The investigation demonstrated the possibility to monitor electrocatalytic events from single laccase molecules and the influence of various factors on the efficiency of direct electron transfer. The methodology is applicable to other redox enzymes with high catalytic activity and allows single enzyme molecules to be studied electrochemically. The suggested approach can give new insight into electron transfer mechanisms and determine parameters that will facilitate DET in bioelectrocatalytic systems. Acknowledgements The authors would like to thank The Swedish research council Formas (245-2010-1062), the research center Security Link (VINNOVA 2009-00966) and the Centre in Nano Science and Technology (CeNano, Linkoping University) for financial support. References 1. Xiao X, Bard AJ. Observing single nanoparticle collisions at an ultramicroelectrode by electrocatalytic amplification. Journal of the American Chemical Society. 2007;129(31):9610-2. 2. Hoeben FJ, Meijer FS, Dekker C, Albracht SP, Heering HA, Lemay SG. Toward single-enzyme molecule electrochemistry:[NiFe]hydrogenase protein film voltammetry at nanoelectrodes. Acs Nano. 2008;2(12):2497-504. 3. Li D, Liu J, Barrow CJ, Yang W. Protein electrochemistry using graphene-based nano-assembly: an ultrasensitive electrochemical detection of protein molecules via nanoparticle–electrode collisions. Chemical Communications. 2014;50(60):8197-200. 4. Sekretaryova AN, Vagin MY, Turner AP, Eriksson M. Electrocatalytic Currents from Single Enzyme Molecules. Journal of the American Chemical Society. 2016;138(8):2504-7. 5. Han l, Wang W, Nsabimana J, Yan J-W, Ren B, Zhan D. Single molecular catalysis of redox enzyme on nanoelectrode. Faraday Discussions. 2016.
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