Benjamin Chu

Protein-driven energy transduction across polymeric biomembranes

Block copolymer-based membrane technology enables the development of a versatile class of nanoscale materials in which biomolecules, such as membrane proteins, can be reconstituted. These active materials possess a broad applicability in areas such as the enhancement of existing technologies or production of current-generating films for power sources. For example, these active materials can be integrated with fuel cell ion transport membranes such as Nafion® in order to improve the ability of Nafion® to retain leaking protons. Also, the demonstration of protein-driven current production across these membranes represents a possible alternative power source that is both highly efficient and light in weight. Our work has demonstrated the fabrication of large-area copolymer biomembranes that are functionalized by bacteriorhodopsin (BR) and cytochrome c oxidase (COX) ion transport proteins. Among their many advantages over conventional lipid-based membrane systems, block copolymers can mimic natural cell biomembrane environments in a single chain, enabling large-area membrane fabrication using methods such as Langmuir–Blodgett (LB) deposition. Following the large-scale insertion of proteins into block copolymer LB films, we have demonstrated significant pH changes based upon light-actuated proton pumping. Protein activity across the BR and COX-functionalized membrane has also been observed using impedance spectroscopy as well as direct current measurement.

Fabrication of biomolecule-copolymer hybrid nanovesicles as energy conversion systems

This work demonstrates the integration of the energy-transducing proteins bacteriorhodopsin (BR) from Halobacterium halobium and cytochrome c oxidase (COX) from Rhodobacter sphaeroides into block copolymeric vesicles towards the demonstration of coupled protein functionality. An ABA triblock copolymer-based biomimetic membrane possessing UV-curable acrylate endgroups was synthesized to serve as a robust matrix for protein reconstitution. BR-functionalized polymers were shown to generate light-driven transmembrane pH gradients while pH gradient-induced electron release was observed from COX-functionalized polymers. Cooperative behaviour observed from composite membrane functionalized by both proteins revealed the generation of microamp-range currents with no applied voltage. As such, it has been shown that the fruition of technologies based upon bio-functionalizing abiotic materials may contribute to the realization of high power density devices inspired by nature.

Hybrid protein-polymer biomimetic membranes

Protein-functionalized polymers retain dramatically increased stability over lipid membranes and the unique ability to be deposited on solid substrates in the ABA complex. Furthermore, since these polymers can mimic hydrophilic/hydrophobic biological environments in a single molecular chain, direct adsorption of protein-functionalized biomembrane films is enabled, which is a significant advantage over conventional lipid systems. Following the demonstration of protein mutagenesis and nanoscale biomimetic membrane fabrication, monolayer arrays of pore proteins have been deposited onto silicon wafers for applications in sensing nanomolecules such as conjugated quantum dots and colloidal gold beads. Furthermore, we have characterized monolayer surface properties of custom tailored polymers with varied block length for biomimetic membrane applications, as well as developed a multiwell microelectromechanical-system-based membrane testing platform for enhanced versatility in film deposition. We have successfully demonstrated the reconstitution of a genetically engineered OmpF porin in block copolymer-based biomembranes, fabrication of large-area hybrid protein-polymer Langmuir-Blodgett films, as well as protein insertion via macromolecule detection using protein-polymer active materials with the goal of buildup toward a multicomponent microsystem while preserving inherent molecular function.

Hybrid protein/polymer biomimetic membranes

Protein-functionalized polymers retain dramatically increased stability over lipid membranes and the unique ability to be deposited on solid substrates in the ABA complex. Monolayer arrays of pore proteins have been deposited onto silicon wafers for applications in sensing nanomolecules such as conjugated quantum dots and colloidal gold beads. We have successfully demonstrated the reconstitution of the OmpF porin in triblock copolymer membranes, fabrication of large-area hybrid Langmuir-Blodgett Films, as well as protein insertion via analyte detection using protein/polymer active materials with the goal of buildup towards a multicomponent microsystem while preserving inherent molecular function.

Block Copolymer-Based Biomembranes Functionalized with Energy Transduction Proteins

Block copolymer-based membranes can be functionalized with energy transducing proteins to reveal a versatile family of nanoscale materials. Our work has demonstrated the fabrication of protein-functionalized ABA triblock copolymer nanovesicles that possess a broad applicability towards areas like biosensing and energy production. ABA triblock copolymers possess certain advantages over lipid systems. For example, they can mimic biomembrane environments necessary for membrane protein refolding in a single chain (hydrophilic(A)- hydrophobic(B)-hydrophilic(A)), enabling large-area membrane fabrication using methods like Langmuir-Blodgett (LB) deposition. Furthermore, the robustness of the polymer molecules/structure result in spontaneous and rapid protein-functionalized nano-vesicle formation that retains structure as well as protein functionality for up to several months, compared to one to two weeks for the lipid systems (e.g. POPC). The membrane protein, Bacteriorhodopsin (BR), found in Halobacterium Halobium , is a light-actuated proton pump that develops gradients towards the demonstration of coupled functionality with other membrane proteins, such as the production of electricity through Bacteriorhodopsin activity-dependent reversal of Cytochrome C Oxidase (COX), found in Rhodobacter Sphaeroides . Protein-functionalized materials have the exciting potential of serving as the core technology behind a series of fieldable devices that are driven completely by biomolecules.

Coupled-protein functionality for energy conversion in biomimetic systems

In nature, proteins commonly function as energy transducing machinery, converting solar energy to chemical or electrical energy. In its natural forward reaction, cytochrome c oxidase accepts an electron from cytochrome c to form water from the translocation of protons to combine with oxygen. We have shown that by coupling cytochrome c oxidase with bacteriorhodopsin, a light-dependent proton pump, in a biomimetic triblock copolymer membrane, we can partially reverse cytochrome c oxidase activity to serve as an electron donor to oxidized cytochrome c. We have demonstrated nanoampere-range current switching in response to exposure to light

Reconstituting Membrane Proteins Into Artificial Membranes and Detection of Their Activities

We have successfully purified BR from purple membrane of Halobacterium Salinarium and Cox from the genetically engineered plasmid inserted in Rhodobacter Sphaeroides. The activities of the purified enzymes have shown in lipid vesicles as well as in polymer vesicles and planar membranes. Phosphatidylcholine derived lipid vesicles created the most nature like environment for the enzymes. Triblock copolymer membrane was the alternative choice for membrane protein reconstitution since polymers are more durable, ideal for industrial applications and support enzyme activities better. We also demonstrated the backward function of Cox in vitro by changing proton concentration in the surrounding medium. Langmuir-Blodgett method was used to reconstitute the enzymes into the planar lipid or polymer membranes. The enzyme activities of the enzymes in planar membrane system were tested by impedance spectroscopy.Copyright

Protein-Functionalized Proton Exchange Membranes

Protein-functionalized biomimetic membranes, based upon a triblock copolymer simulating a natural lipid bilayer in a single chain, serves as a core technology for applications in bioenergetics. Monolayers of block copolymer, which simulates the hydrophilic-hydrophobic-hydrophilic chain of a natural cell membrane, can be formed by Langmuir-Blodgett (LB) deposition and provides a favorable environment for protein refolding. Large-scale membrane formation is achieved using LB deposition on a variety of substrates, such as gold, quartz, silicon, and Nafion®. We have successfully inserted membrane proteins, such as the light-activated proton pump, bacteriorhodopsin (BR) and the pH/voltage-gateable porin, Outermembrane Protein F (OmpF), into large-area LB monolayers. We have also established sustained protein functionality in films through the measurement of light-activated proton transport.Copyright

Fabrication of Hybrid Bionanodevices Based on Coupled Protein Functionality

Block copolymer-based membrane technology represents a versatile class of nanoscale materials in which biomolecules, such as membrane proteins, can be reconstituted. Among its many advantages over conventional lipid-based membrane systems, block copolymers can mimic natural cell biomembrane environments in a single chain, enabling large-area membrane fabrication using methods like Langmuir-Blodgett deposition, or spontaneous protein-functionalized nanovesicle formation. Based on this unique membrane property, a wide variety of membrane proteins possessing unique functionalities including pH/voltage gatable porosity, photon-activated proton pumping, and gradient-dependent production of electricity have been successfully inserted into these biomimetic systems.Copyright