The overall effectiveness of enzyme-based electrochemical devices such as biosensors and fuel cells are heavily dependent on the ability of the molecules that attach enzymes to the electrode to successfully harvest and transport charges from the outer oxidizing point (enzyme active-site) to the inner electrode surface. Current technology uses a series of ancillary molecules (with the correct prosthetic groups) to tether the essential electron mediator and the enzyme complex on to the surface of an electrode. Unfortunately, the inherently low conductivity of presently known organic tethering molecules makes the electron transport process highly constrained; therefore, contributing to fuel cells with low power density and sensors with low sensitivity. The lack of an effective molecular wiring system that can allow unimpeded charge transport is a significant problem and hinders harnessing the tremendous potential bioelectronics devices have to offer.
The overall goal of this proposal is to evaluate whether it is possible to replace the current complex wiring system with a single molecule that has the ability to mediate electron shuttling between the coenzyme and a metal surface, and that has the correct prosthetic groups to latch simultaneously onto the metal surface and the enzyme complex.
Inspired by the natural electron transport mechanism that occurs in the mitochondrial surface, we hypothesize that the glucose dehydrogenase-nicotinamide adenine dinucleotide (GDH-NAD) apoenzymecoenzyme system could be directly attached onto a gold electrode using synthetic iron-sulfur [Fe-S] complexes. Iron-sulfur complexes are well known to transport electrons generated by glucose oxidation in eukaryotic cells. The rationale is that the thiol groups of the [Fe-S] complex would attach onto the gold surface via covalent linkages while iron will coordinate with heterocyclic nitrogen atoms in imidazolediazine moieties of NAD attaching the apoenzyme-coenzyme complex on to the electrode.
The hypothesis will be tested via the following Specific Objectives:
1. Evaluate the effectiveness of tethering GDH-NAD+ complex onto a gold electrode via [Fe-S] surrogates. This will be done by synthesizing simple forms of [Fe-S] complexes, evaluating the effectiveness of binding the S end onto the Au electrode and the Fe end to the NAD via quartz crystal microbalance (QCM) studies, and elucidating the current-voltage (CV) behavior of a developed electrode via potentiometric and current sensing atomic force microscopy (CS-AFM) studies.
2. Elucidate electrochemical phenomena occurring at the electrode surface in the presence of the fuel oxidant. The electrochemical response of the novel simplified electrode will be compared with the conventionally wired electrode in the presence of glucose. The parameters studied will include CV response and fuel-consumption/electrode kinetics.
Broader Impacts and the Appropriateness of the Project for EAGER Funding:
The proposed research is high-risk since direct attachment of NAD to a metal surface via a surrogate known to act as a redox mediator, to the best of our knowledge, has never been attempted before. If successful, the research will provide a transformative impact on the bioelectronics area by alleviating one of the most intricate bottlenecks impeding development of effective bio-electronic devices, i.e., constrained charge transport. Moreover, if successful, this research will lay the foundation for mimicking the first step of the electron transport chain which in turn may take us a step closer to developing a synthetic version of the most effective power plant known ?mitochondria!
A high-risk proposition of this nature will not fit into a regular proposal due to lack of preliminary data. However, a potentially high-payoff project of this caliber is a ?good fit? for an EAGER
Overview: The overall effectiveness of enzyme-based electrochemical devices such as biosensors and fuel cells are heavily dependent on the ability of the molecules that attach enzymes to the electrode to successfully harvest and transport charges from the outer oxidizing point (enzyme active-site) to the inner electrode surface. Current technology uses a series of ancillary molecules (with the correct prosthetic groups) to anchor the essential electron mediator and the enzyme complex on to the surface of an electrode. Unfortunately, the lengthy wiring scheme used today for this purpose makes the electron transport process highly constrained; therefore, contributing to fuel cells with low power density and sensors with low sensitivity. The lack of an effective molecular wiring system that can allow unimpeded charge transport is a significant problem and hinders harnessing the tremendous potential bioelectronics devices have to offer. The overall goal of this proposal was to evaluate whether it is possible to replace the current complex wiring system with a single molecule that has the ability to mediate electron shuttling between the coenzyme and a metal surface, and that has the correct prosthetic groups to anchor the enzyme complex onto the electrode. Intellectual Merits: Inspired by the natural electron transport mechanism that occurs in the mitochondrial surface, we hypothesized that an iron-sulfur-based based electrode system will yield a better current/voltage response to varying glycerol concentrations as opposed to an electrode formed with the conventional pyrroloquinoline quinone (PQQ)-based wiring system. Iron-sulfur complexes are well known to transport electrons generated by glucose oxidation in eukaryotic cells. The rationale is that the thiol groups of the iron-sulfur moiety would attach onto the gold surface via covalent linkages while iron will coordinate with heterocyclic nitrogen atoms in imidazole-diazine moieties of NAD attaching the apoenzyme-coenzyme complex on to the electrode. To test this hypothesis, a novel iron (II) sulfide (FeS) based molecular wiring system was developed; characterized; and tested for its ability to transport charges as compared to a conventional wiring system. As a result of this work, we were able to successfully fabricate enzyme bound gold electrodes using the conventional PQQ-cystamine links as well as novel FeS links. We were able to verify the formation of gold-sulfur links as well as individual self-assembled monolayers. Amperometric and potentiometric analyses with glycerol dehydrogenase-based model electrodes confirmed the ability of this single-molecule to remarkably amplify, on average seven-fold increase in current and up to 24% increase in voltage outputs, as compared to electrodes fabricated with the conventional Pyrroloquinoline quinone-based molecular wiring system. FeS seems to achieve the dual purpose of anchoring the enzyme to the gold electrode while also mediating electron shuttling between coenzyme and the electrode surface. This dual functionality allows usage of a single-molecular wire to foster electrical communication between the enzyme and the electrode instead of the conventional multi-molecular wiring system and in turn reducing the internal resistance of the electrode. Broader Impacts: As a result of this work, a NAD-dependant glycerol dehydrogenase enzymatic electrode was fabricated by using iron(II)sulfide as the only linking agent (that anchors the enzyme system onto the gold electrode surface). The electrode was able to reduce overpotential issues significantly while increasing the current response as compared to a conventionally wired electrode (using the cystamine-PQQ route). This work sheds light onto the possibility of significantly improving transport issues that bio-based electrodes face by using simple linker molecules that have multiple functionalities (electron mediating ability as well as the ability to anchor an enzyme system on to the metal electrode surface); and if proven feasible to be compatible with a broader range of coenzymes and enzymes, the impact this resarch can have on bioelectronics industry is significant.
