Single Enzyme Electrochemistry – SEE

The development of new biotechnologies is tightly bound to our understanding of biological systems and our capacity to efficiently integrate biomolecules such as redox enzymes in electronic devices. The actual trend toward miniaturization of bio-electronic devices reduces considerably the number of enzymes contained within one device. At the nanoscale this number approaches one enzyme! In this particular case our knowledge obtained from large ensemble measurements is insufficient for a correct understanding and development of nano-sized devices. It is thus critical to develop new tools for studying the activity of one or few enzymes.
The objectives of project SEE is developing a new approach based on electrochemistry to monitor the catalytic activity of single redox enzymes. From the electrical current produced by a single redox enzyme we will learn if an enzyme functions continuously or intermittently, if the activity of an enzyme varies smoothly or abruptly, what is the lifetime of an enzyme et By answering these questions project SEE will enlarge our fundamental knowledge of redox enzymes and hence open new avenues for nanoscale biotechnologies.
Project SEE presents two innovations. First, we propose to replace singe molecule fluorescence microscopy, a method restricted to only a couple of fluorogenic redox enzyme and substrates, with a more versatile technique, electrochemistry. In project SEE we propose to investigate the activity of a glucose dehydrogenase - an ubiquitous enzyme found in biofuel cells and glucose sensors - that is not compatible with single molecule fluorescence measurements. Beside its versatility electrochemistry presents also the advantage of being compatible with the design of low-cost portable sensors and thus favors the dissemination of academic knowledge to societal applications.
The second innovation found in project SEE is a robust protocol to deposit an exact and well-controlled number of enzyme starting from one unit. This unprecedented degree of control will constitute a major progress in surface modification science and enable the reproducible production of nano-scale devices. A precise knowledge of the number of immobilized enzyme is also necessary to determine accurately the enzymatic activity (the catalytic current is expected to be directly proportional to the number of enzyme on the electrode).
Our experimental approach to deposit individual enzymes and measure electrochemically their activity is based on the combination of several state-of-the-art techniques and concepts mastered and/or developed by the members of project SEE. In a nutshell, individual enzymes will be immobilized on gold NPs and the ensemble will be captured by electrochemical collision on a passivated ultra-microelectrode. The ensemble, which behaves like a tunnelling nanoelectrode, will be used for single enzyme activity readout. Recent results obtained by our group as well as the group of S.G. Lemay evidence that the technological lock of measuring electrochemically single enzyme activity (expected current ˜ 1 fA) can be overcome with carefully designed instrumentation and electrodes (such as tunnelling nanoelectrodes). The principle investigator of this project is expert in single particle collision on ultra-microelectrodes and bio-electrochemistry while the host group developed a theory for understanding (and thus designing) tunnelling nanoelectrodes.
Principle outcomes and impacts of the project SEE include: (i) Depositing an exact and well-controlled number of enzymes on an electrode ? drastic improvement of wet surface-modification techniques; (ii) Electrocatalytic activity readout of single enzymes ? improved fundamental understanding and guideline for biotech industries willing to develop miniaturized sensors; (iii) Offer an alternative solution to the extremely sensitive but not widely applicable fluorescence microscopy ? increase the number of redox molecules observable at the single entity level.