Toward Single-Enzyme Molecule Electrochemistry: [NiFe]-Hydrogenase Protein Film Voltammetry at Nanoelectrodes
Source abstract
Abstract
We have scaled down electrochemical assays of redox-active enzymes enabling us to study small numbers of molecules. Our approach is based on lithographically fabricated Au nanoelectrodes with dimensions down to ca. 70 x 70 nm(2). We first present a detailed characterization of the electrodes using a combination of scanning electron microscopy, cyclic voltammetry, and finite-element modeling. We then demonstrate the viability of the approach by focusing on the highly active [NiFe]-hydrogenase from Allochromatium vinosum immobilized on polymyxin-pretreated Au. Using this system, we successfully demonstrate a distinct catalytic response from less than 50 enzyme molecules. These results strongly suggest the feasibility of using bioelectrochemistry as a new tool for studying redox enzymes at the single-molecule level.
Source: openalex_inverted_index · Quality: baseline
Research context
OpenAlex topics: Metalloenzymes and iron-sulfur proteins · Electrocatalysts for Energy Conversion · Advanced battery technologies research
SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.
Affiliations
Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands
Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands
References (26)
Show source-provided references
1. Walter N. G. · Nat. Methods · 2008 · 5 · 47510.1038/nmeth.1215 ↗
2. Léger C. · Chem. Rev. · 2008 · 108 · 237910.1021/cr0680742 ↗
3. Armstrong F. A. · Chem. Soc. Rev. · 1997 · 26 · 16910.1039/cs9972600169 ↗
4. Léger C. · J. Phys. Chem. B · 2002 · 106 · 1305810.1021/jp0265687 ↗
5. Bard A. J. · 2000
6. Arrigan D. W. M. · Analyst · 2004 · 129 · 115710.1039/b415395m ↗
7. Vincent K. A. · Chem. Rev. · 2007 · 107 · 436610.1021/cr050191u ↗
8. De Lacey A. L. · Chem. Rev. · 2007 · 107 · 430410.1021/cr0501947 ↗
9. Lubitz W. · Chem. Rev. · 2007 · 107 · 433110.1021/cr050186q ↗
10. Jones A. K. · Chem. Commun. · 2002 · 86610.1039/b201337a ↗
11. Pershad H. R. · Biochemistry · 1999 · 38 · 899210.1021/bi990108v ↗
12. Hoeben F. J. M. · Langmuir · 2008 · 24 · 592510.1021/la703984z ↗
13. Volbeda A. · J. Am. Chem. Soc. · 1996 · 118 · 1298910.1021/ja962270g ↗
14. Kellers P. · Acta Crystallogr. F · 2008 · 64 · 71910.1107/S1744309108019945 ↗
15. Since the dry etch was found to proceed slower through the small trench than to the much larger contact pads, we did not succeed in creating electrodes with trenches smaller than 100 nm because the discrepancy in etching speed became so big that either the contact pads were lost or the nanoelectrode simply could not be liberated. This may be avoided by using separate lithography steps for the trench and contact pads.
16. Zhang W. · Electroanalysis · 2003 · 15 · 40910.1002/elan.200390047 ↗
17. A similar calculation is possible for H+reduction, which we have primarily measured; however, the value ofKMis unknown for H+reduction byAvH2ase.
18. Fontecilla-Camps J. C. · Chem. Rev. · 2007 · 107 · 427310.1021/cr050195z ↗
19. Jähne B. · J. Geophys. Res. · 1987 · 92 · 1076710.1029/JC092iC10p10767 ↗
20. Léger C. · Biochemistry · 2002 · 41 · 1573610.1021/bi026586e ↗
21. Jones A. K. · J. Am. Chem. Soc. · 2003 · 125 · 850510.1021/ja035296y ↗
22. De Lacey A. L. · Coord. Chem. Rev. · 2005 · 249 · 159610.1016/j.ccr.2005.03.009 ↗
23. This is slower than what we previously observed; it is therefore possible that some reductive reactivation is initially needed after introduction of a small trace of O2during sample injection.AvH2ase not bound to PM-treated Au was not flushed out and therefore still present in solution; we do not expect any perturbation of the measured curves because of this.
24. The relatively large error in fractional enzymatic surface coverage is the consequence of the large uncertainty in the experimentally determined value for the maximum turnover number forAvH2ase (kmax= 1500 to 9000 s−1).(11)Furthermore, these numbers have been determined at 30 °C and pH = 7, whereas we are working at room temperature (∼20 °C) and pH = 5.7. We believe that lowering the temperature to 20 °C (approximately halving the enzymatic activity)(7)while working at pH = 5.7 (approximately doubling the H+reduction activity)(7)does not introduce any significant discrepancy.
25. Alternatively, using the maximum enzymatic coverage of 5.5% established for PM-pretreated Au, ∼14 enzyme molecules would be present on a 100 × 100 nm2electrode, givingkmax= 4900 s−1.
26. Coremans J. M. C. C. · Biochim. Biophys. Acta · 1992 · 1119 · 15710.1016/0167-4838(92)90386-R ↗