Reports: G10

47121-G10 Active Orientation and Encapsulation of Bacteriorhodopsin-Driven Photo-Energy Transduction in Copolymer Shells

Dean Ho, Northwestern University

This study addressed the development of hybrid protein/abiotic material platforms towards a spectrum of applications which included nanomanufacturing and protein packaging, among others. These architectures may be important as they harness the properties of the protein (e.g. energy transduction, protein-protein binding) while potentially prolonging their activity due to the stabilizing properties of the material. In order to realize devices based upon protein function, our laboratory has investigated the integration of proteins with materials such as nanodiamonds and parylene which have been shown to sustain protein activity, and may be applicable towards both protein packaging as well as device fabrication due to the enhanced stabilizing properties of both the proteins and polymers. Nanodiamonds serve as useful materials to facilitate stable protein interactions due to several integrated beneficial properties. For example, as they are carbon-based, nanodiamonds are biocompatible. Furthermore, they are highly scalable to process as primary particles of 4-6nm in diameter can be obtained via ultracentrifugation, ball milling, and acid washing. These processed particles also possess surface bound carboxyl groups due to the acid washing step, which also reduces the presence of contaminants. As such, nanodiamond dispersal in water can be further promoted. It has also been previously been shown that diamond surfaces can promote potent hydration because of alternating electrostatic properties of the nanodiamond facets (e.g. [111] or [100]) which can result in oppositely oriented water dipoles to facilitate the presence of a nano-hydration shell.  Specific areas of relevance represented by the interface of materials such as nanodiamonds with proteins include self-assembled or potential flexible electrodes for biological transduction, among others. Our work will examine 3 nanomaterial examples which include the initial examination of protein adsorption with nanodiamonds, protein-nanodiamond self assembled thin film structures, as well as packaging nanodiamonds in biostable parylene microfilms.

The initial protein-nanodiamond structures were developed using insulin as a model protein. Insulin was adsorbed to the nanodiamonds which was confirmed via transmission electron microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), as well as UV-vis spectrophotometry. These methods demonstrated that the proteins were capable of complexing with the nanodiamonds to form clusters. Dynamic light scattering and zeta potential measurements further examined the cluster particle sizes while zeta potential measurements assisted with confirming protein-nanodiamond interactions. Subsequent MTT assays, among others, demonstrated that the protein remained functional. In addition to demonstrating protein adsorption, this study also showed that protein desorption could be enabled based upon changes to the pH of the surrounding environment. This may realize avenues towards controllable packaging and unpackaging of proteins.

In addition to the initial protein-nanodiamond adsorption studies, protein-based nanodiamond assembly was also shown. For example, the coating of solid surfaces with alternating layers of poly-l-lysine (PLL) proteins and nanodiamond particles revealed a highly ordered assembly process which was also scalable in that the process could be repeated in an alternating fashion. Continuous nanodiamond films could be synthesized using this approach (PLL/Nanodiamond) to build successive layers of nanodiamonds and proteins, which may be applicable towards domains such as sensing and bio-energy (e.g. self-assembling electrodes). These thin films were also shown to be biocompatible and very robust as they were able to withstand multiple stringent washing steps during the layer-by-layer deposition process.

Nanodiamond-polymer architectures were also synthesized and explored using pary-xylene as the packaging polymer. Pary-xylene is a favorable packaging material in that it can be conformally deposited using chemical vapor deposition, yielding a translucent, flexible, and extremely stable film with tunable thickness. The deposition process can take place under room temperature conditions, which supports the packaging of biological compounds such as proteins. Furthermore, parylene is biocompatible due to its highly stable properties, and the shapes and sizes of the devices can be easily tailored.  Nanodiamond-pary-xylene devices were synthesized in 2 different formats, with one being based upon the sandwiching of the nanodiamonds between 2 layers of pary-xylene, with the top layer serving as a ‘release layer’ and the bottom layer serving as a structural layer so that the films could be manually handled. This studied showed that tuning the thickness of the top pary-xylene layer resulted in the ability to collect materials that were released from the packaged nanodiamond clusters. In a second format of the microfilms, an amine-terminated pary-xylene was used for direct conjugation of the nanodiamonds which contained surface bound carboxyl groups. This component of the study reduced microfilm synthesis by one processing step as the top layer of pary-xylene was not present in the device architecture. It is envisioned that both the sandwich and direct conjugation versions of the device will be applicable depending on the agent that is being packaged and potentially released and the specific application being addressed by the devices. This study is important in that previous studies utilized block copolymers to package the proteins being explored. As the block copolymers possess nanoscale thicknesses, the utilization of pary-xylene then enables the devices to package and preserve the properties that exist at the nanoscale (e.g. material stability, protein function) while also being amenable towards manual handling.

Our work has sought to develop novel hybrid particle and thin film platforms for the mediation of biomolecular adsorption, packaging, and desorption for a spectrum of applications. Given the broad range of studies that can be accomplished, this collection of materials provides increased opportunities to fabricate devices based upon biomolecular (e.g. proteins) function.