Adsorption of liposomes on charged polyelectrolyte substrates (Collaborative with Worden and Lee)

One of the problems with some constructions of biomimetic interfaces using lipid bilayers is that no cushion is provided between the bilayer and the substrate to allow for full insertion of membrane proteins, enable their lateral mobility, and provide for ionic reservoirs on either side of the membrane. The deposition of polyelectrolyte multilayers provides ideal cushions for this purpose, and therefore has potential in biosensor applications. In this project, we are studying the adsorption of fluorescently labeled vesicles on charged polyelectrolyte multilayers (PDAC, PAH, PEG, SPS) deposited on glass slides, using total internal reflection fluorescence microscopy (TIRFM) and fluorescence recovery after pattern photobleaching (FRAPP) to estimate the mobility and lateral diffusion coefficients of the lipids in the bilayer. The resulting interfaces were also characterized by cyclic voltammetry (CV) and quartz crystal microbalance (QCM). This work is being done in collaboration with the research groups of Dr. Ilsoon Lee and Dr. RM Worden.

Catalytic transformation of biorenewables to petrochemicals

An important global challenge is the need to eventually replace finite fossil fuels with viable renewable resources. Currently, there are many significant initiatives on converting biomass to alternative fuels, but much less activity on using renewables for petrochemicals production. There is much knowledge on the transformation of crude oil to fuels and chemical feedstocks. However, due to significant differences between crude oil and biomass, these well-tested transformation pathways are not suitable for biomass conversion. In this project, we are developing generic methods for synthesizing catalytic nanoparticles (NPs) and surface modification to attach molecular catalysts (MCs), developing protocols for physical and/or chemical immobilization of catalytic NPs and NP-MC complexes in microfluidic channels, and assessing and optimizing catalytic NPs in both classical and microfluidic reactors. We will use the conversion of lactic acid to glycerol as the model reaction. Fatty acids from plant oils represent another important class of abundant biorenewables. To convert these to petrochemicals, we will anchor molecular catalysts to catalytic bimetallic nanoparticles to obtain NP-MC hybrid (NMH), and assess NMH catalyst effectiveness in microreactors using the hydrogenation of selected fatty acids to petrochemicals as model reactions. This is a collaborative project with Professor Obare (Department of Chemistry at UNCC; adjunct in CHEMS).

Interactions of nisin with selected proteins at the liquid-liquid interface

Nisin is a small (3510 Da), positively charged amphipathic polypeptide; it is produced by Lactococcus lactis and contains dehydrated residues and lanthionine or thioether rings. It has antimicrobial activity against a wide range of gram-positive bacteria and has been widely used as a food preservative in the dairy industry. Its antimicrobial activity stems primarily from permeabilization/pore formation of the cytoplasmic membrane of target organisms, making it a promising candidate for use in formulations that could effectively transfer drugs across cellular membranes and significantly reduce microbial resistance to antibiotics. The goal of this project is to characterize the adsorption dynamics of nisin at a model oil-water interface, and to study its interactions with other macromolecules in sequential and competitive adsorption experiments, using a diverse group of proteins of varying molecular weight.

Nanostructured Biomimetic Interfaces

Cell membranes efficiently perform many molecular-scale processes, including catalysis, molecular transport, recognition, signal transduction, and energy generation. These processes can be mimicked in the laboratory using biomimetic interfaces that consist of synthetic lipid bilayers with embedded membrane proteins. For this approach to be commercially exploited, biomimetic interfaces must be developed that are versatile, for use with diverse classes of proteins; customizable, for use in diverse applications; robust, for long lifetime; and mass-producible, for inexpensive fabrication. Multidisciplinary research involving several science and engineering laboratories is underway with the following objectives: (1) Express and purify membrane proteins, including dehydrogenase enzymes, Major Facilitator Subfamily (MFS) transporter proteins, and gated ion channels; (2) Develop functional and nanostructured biomimetic interfaces containing these proteins; (3) Apply physical, electrical, and optical methods to measure, and optimize, performance properties of the biomimetic interfaces; (4) Develop practical devices and processes based on the biomimetic interfaces, including biosensor arrays and biocatalytic systems.

Opto-electrochemical characterization of bilayer-embedded biomacromolecules in TIR geometry

Membrane-bound enzymes and proteins can serve as ion channels and/or catalyze a variety of biological processes. These processes can be characterized by a variety of techniques, including optics and electrochemistry. In this project, we are developing surface chemistries to enable us to use ITO as an effective electrode on which bilayer lipid membranes can be deposited to provide an electrode platform for simultaneous optical and electrochemical measurements of protein activity. We also have capabilities for physical characterization of biomacromolecules (fluidity and mobility measurements) on planar as well as tethered bilayers by fluorescence recovery after pattern photobleaching (FRAPP) experiments, enabling us to do a complete study of many enzymes and proteins of medical interest.

Quantitation of biomolecular coverages at the liquid-liquid interface

We revisit work done earlier in our laboratory on estimating interfacial coverages of proteins at the oil-water interface, using total internal reflection fluorescence microscopy (TIRFM) and fluorescence photobleaching recovery (FPR). This method, first proposed by Zimmerman et. al (J. Colloid Interface Sci. 1990, 139, 268-280) for estimating surface coverages at the solid-liquid interface, is based on the principle that macromolecular interfacial coverages are related to the ratio of bulk and interfacial contributions to the total fluorescence emission detected. Interfacial coverages obtained for the adsorption of bovine serum albumin (BSA) at the oil-water interface using this technique are in the range of 0.02 to 0.3 mg/m2 in the bulk concentration region of 0.2 mg/ml to 3.5 mg/ml. These values are lower than those typically reported in the literature for similar proteins, most likely due to the low shear rates (<0.5 s-1) used in our experiments. We are investigating the effect of low shear rates on protein relaxation and spreading, and associated implications on ultimate coverages at fluid-fluid interfaces. We are also using this technique to obtain interfacial coverages at the oil-water interface for human plasma fibronectin, a large glycoprotein that has a rod-like structure and exhibits considerable flexibility. As part of this work, we are investigating fluorescence lifetimes of dye-labeled proteins at the liquid-liquid interface using two-photon excitation spectroscopy. This segment of the project is being done in collaboration with Dr. Gary J. Blanchard of the Department of Chemistry at MSU.

Reviewed Journal Publications

Lapinski, Monique M., Castro-Forero, Angelines, Greiner, Aaron J., Ofoli, Robert Y., and Blanchard, Gary J. Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore, Langmuir 23 (23): 11677-11683 (2007).

Kohli, N.; Vaidya, S.; Ofoli, R.Y.; Worden, R.M.; Lee, I. "Arrays of Lipid Bilayers on Patterned Polyelectrolyte Templates," Journal of Colloid and Interface Science 301, 461-469, (2006).

Kohli, N.; Hassler, B.L.; Parthasarathy, L.; Richardson, R.J.; Ofoli, R.Y.; Worden, R.M.; Lee, I. "Tethered Lipid Bilayers on Electrolessly Deposited Gold for Bioelectronic Applications," Biomacromolecules 7, 3327-3335, (2006).

Vaidya, S.; Ofoli, R.Y. "Adsorption and Interaction of Fibronectin and Human Serum Albumin at the Liquid-Liquid Interface," Langmuir 21, 5852-5858, (2005).

Vaidya, S.; Orta-Ramirez, A.; Smith, D.M.; Ofoli, R.Y. "Influence of thermal exposure on the fluorescence emission of R-phycoerythrin," Biotechnology and Bioengineering, 83(4): 465-473 (2003).

Books, Chapters, Monographs

Wilkinson, Bruce W., Lira, Carl T., Bender, Timothy O, Ofoli, Robert Y., Manual on Laboratory Practice and Statistical Analysis (Student laboratory manual for CHE 316, junior-level experiential course), (January 2007).

Conference Proceedings

Kohli, N.; Vaidya, S.; Ofoli, R.Y.; Worden, R.M.; Lee, I. "3-D Bionanocomposite Surfaces and Interfaces: Fabrication and Characterization," Polymer Preprints 45(1), 124-125, (2004).

Kohli, N.; Kim, P.; Mason, A.; Lee, I.; Ofoli, R.Y.; Worden, R. M. "Nanoscale Sensing for Biological and Environmental Applications," Proceedings of the Sensors Topical Conference, Annual Meeting of the AIChE, San Francisco, CA, Nov. 16-21, 2003, AIChE:New York, (2003).

Kim, P.; Kohli, N.; Hassler, B.; Dotson, N.; Mason, A.; Worden, R. M.; Ofoli, R. "An Electrochemical Interface for Integrated Biosensors," Proc. IEEE Int. Conf. on Sensors, Toronto, Canada, October (2003).

Kohli, N.; Hassler, B.; Vaidya, S.; Parthasarathy, L.; Ofoli, R.Y.; Lee I.; Worden, R.M. "Biomimetic Interfaces for Integrated Biosensor Arrays," Proceedings of Sensors Topical proceedings of 2003 Annual Meeting of the American Institute of Chemical Engineers."