Genetically Tailoring Pilin-Based Nanowires Expressed with E. Coli for Enhanced Electronic Functions

Pilin-based electrically conductive protein nanowires (e-PNs) are sustainably produced ‘green’ electronic materials that have served as the functional components of novel electronic devices for sensing, neuromorphic memory, and the generation of electricity from atmospheric humidity. An <i>E. coli</i> chassis for the expression of e-PNs, grown on the biodiesel waste-product glycerol, enables sustainable e-PN mass production. It is a simple matter to genetically tailor e-PN properties, such as conductivity, and their sensitivity and selectivity for sensing analytes of biomedical and/or environmental interest. Thus, the possibility of incorporating e-PNs in a water-stable polymer while maintaining e-PN function was evaluated to further expand fabrication and applications options.<br/><br/>Mixing e-PNs and polyvinylbutryal (PVB) yielded transparent, electrically conductive composites. e-PNs were evenly distributed throughout the composite. Composite conductivity increased with increasing weight percent e-PNs in a manner consistent with percolation theory. The conductivity of the e-PNs was tuned by modifying the abundance of aromatic amino acids encoded in the pilin genes. Incorporating e-PNs with different conductivities resulted in the changes in the conductivities of the e-PN/PVB composites that were consistent with the changes made in pilin aromatic amino acid composition.<br/><br/>Electronic sensors with e-PN/PVB composites as the sensing unit were incorporated into a microfluidic system that continuously passed water over the sensor, enabling high throughput analysis of different analyte concentrations. Flexible wearable sensors for detecting ammonia in sweat or other bodily fluids are of interest because ammonia concentrations are diagnostic of certain aspects of the body’s physiological state and the presence of several diseases. The conductivity of the e-PN/PVB composites changed in direct response to the concentration of ammonia dissolved in the water, consistent with previous demonstrations that the conductivity of thin films of e-PNs differently responded to gaseous ammonia concentrations.<br/><br/>Previous studies demonstrated that modifying pilin genes to encode specific sequences of six-to-nine amino acids at the carboxyl end of the pilin protein yielded e-PNs with the added amino acids displayed on the e-PN outer surface. Pilin genes were modified to determine whether displaying amino acid sequences known to specifically bind analytes of interest would enhance the e-PN/PVB sensor response to the target dissolved analytes. Sensor response was dramatically improved. For example, displaying a peptide known to bind ammonia increased sensor sensitivity 10-fold. There was negligible sensor response to other common sweat components. Modifying pilin genes to display different amino acid sequences on the e-PN outer surface specifically increased sensitivity to other target analytes.<br/><br/>Conductive composites for effective sensing were also made by mixing whole cells of <i>E. coli</i>, with e-PNs still attached, with PVB. This method eliminates the need for e-PN harvesting and purification, the most labor-intensive step in producing e-PN-based electronics.<br/><br/>These results demonstrate for the first time the fabrication of electrically conductive, water-stable e-PN/polymer composites and show that it is possible to tune the conductivity and sensing capabilities of these composites with simple modifications of the pilin gene sequence. It is well-known that it is possible to design short amino acid sequences to specifically bind a diversity of biomedical and environmentally relevant analytes. Therefore, our findings demonstrate the ability to exquisitely genetically tailor e-PN properties to inexpensively and sustainably fabricate a broad array of flexible e-PN/polymer composites, each with the potential to specifically sense in real-time one of a broad diversity of medically and/or environmentally relevant analytes.