The goal of this Collaborative Project is the development of self-powered enzymatic biosensors using direct electron transfer between novel enzymes and novel nano-structured electrode materials. The self-powering is achieved by means of constructing a fuel cell with enzyme-catalyzed electrodes that generates response to the environmental change without the need of an external power source. The research plan is focused on improving selectivity and sensitivity of sensors utilizing inhibition and activation-based self-powered sensing. This is a multidisciplinary research project between University of Utah and University of New Mexico who will be working together in order to understand and develop the materials systems needed for self powered biosensors/biofuel cells. The two universities will utilize this interest to train high school students, undergraduate students, and graduate students in the interdisciplinary sciences of bio-electrochemistry, bio-nanotechnology, biosensing and bio-inspired energy conversion. The immediate involvement of students from underrepresented minorities is expected and will be aggressively sought.
Proposal number: 1158936 PI Plamen Atanassov, University of New Mexico The goal of this project was to investigate and integrate both fundamental and innovative applied research in order to provide detailed understanding of the catalytic and bio-catalytic processes occurring at the bio-organic-inorganic interfaces and thus provide insights into biological activity of enzymes outside of their normal, cellular environment, creating a basis for implementation of self-powered enzymatic electrodes. PQQ-dependent dehydrogenases were explored as model enzymes due to their ability to oxidize a given fuel and carry out direct electron transfer. Extremely novel combined experimental and computational approach was developed under the project to determine important parameters that govern enzyme/molecule and molecule/nanomaterials interactions, providing insight into the mechanism of biocatalysis and the conformational and structural changes accompanying these processes. Some of the parameters that explored included enzyme affinity, molecular recognition mechanism, molecule position with respect to the enzyme active center, adsorption energy, charge transfer, redox potential, electron-transfer rate, and surface coverage. Theoretical approaches, such as quantum and molecular mechanics calculations were used to gain understanding of the operational mechanisms of the developed enzymatic systems on one hand and to predict the systems behavior based on theoretically discovered dependencies on the other hand, which set the basis for a smart pre-selection of chemistries of interest bridging and facilitating the enzyme/nanomaterials electron transfer. This combined approach provides a new avenue for fundamental and applied research that will impact the general area of bio-nano-interfaces: science and technology developed at the confluence of biochemistry & biotechnology and nanoscience & nanotechnology. As a result of this approach, new bio-inspired methods for enhancing the enzyme/nanomaterial interactions were developed based on the specificity of enzyme-substrate recognition and the organization of various enzymatic active centers at a macromolecule level. Strategically selected chemistries with predicted electrochemical activity was shown to bring the redox active center of the enzyme close to the conductive surface, facilitating the interfacial electron transfer. The described methods were applied towards acceleration of the bio-nano interface reactions when PQQ-dependent glucose dehydrogenase (PQQ-GDH) is utilized. The first method relied on natural coupling of glucose oxidation carried out by this enzyme with reduction of a final electron acceptor – ubiquinone or its functional analogues and the specificity of substrate-enzyme interactions. Positioning of the final electron acceptor on nanomaterial surface led to proper enzyme orientation along with a mediation of the electron transfer at the bio-nano interface. The outcome of this method expressed in increased kinetics of electrocatalysis and improved efficiency of energy conversion (current density of 70 μA/cm2). The second method developed was inspired by the structure of quinohemoproteins. These enzymes have PQQ and one or multiple hemes as cofactors. During substrate oxidation fast and efficient internal electron transfer is being carried out from PQQ towards and along the hemes. In a similar fashion nanomaterials can be modified in a way to have heme on their surface, which in combination with PQQ-dependent glucose dehydrogenase can mimic the natural internal electron transfer pathway in quinohemoproteins. Procedures for covalent binding of natural quinoprotein redox mediator (artificial analogue of heme b) on nanomaterials and coupling of these with PQQ-GDH have been demonsrated. Covalently bound hemin dramatically improved the electrocatalytic response of the PQQ-GDH anode in all potential regions reaching current densities of 480 μA/cm2, the highest value achieved for these type of enzymes. Besides the demonstrated practical benefits (patent application "Highly Efficient Enzymatic Bioanodes and Biocathodes" 2013-049), the project significantly contributed to the fundamental understanding of the electrochemical processes involving bio-catalytic systems and engineering of bio-inspired devices and thus will contribute to the development of new technologies in medical, defense, and environmental field. It is expected that the described herein approach will have a direct impact over the whole biotechnology spectrum. Both of the methods developed under this project are applicable not only for the optimization of PQQ-dependent anodes but towards all types of enzymatic electrodes taking into account the specificity of the given enzymes, their natural substrates and internal electron transfer pathways. The predictive power of computational modeling as demonstrated would provide the right direction towards other modification approaches, which could provide stability, improve reproducibility, and lead to an increase in the overall electrochemical output of the designed systems.
