8-9th August, 2008 School of Materials Science & Nanotechnology,Jadavpur University
Langmuir Blodgett and self assembly methods to fabricate Ovalbumin–Phospolipids thin film with minimal protein aggregation
Tapanendu Kamilya, Prabir Pal, Mrityunjoy Mahato and G. B. Talapatra*
Department of Spectroscopy, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700032, India
The fabrication of protein as well as protein-lipid mixed films on appropriate substrates is the subject that has received considerable attention in recent years due to their prospective biotechnological and biomedical applications and devices fabrication. A foremost challenge is to immobilize the protein molecules on appropriate substrate without aggregates. Self-assembly (SA) and Langmuir-Blodgett (LB) methods are typical techniques to immobilize biomaterials on to solid surfaces. Our main aim is to prepare a protein- lipid mixed film with minimal aggregation of protein by using LB and SA technique.
We have used zwitterionic (DPPC) [1-2], cationic ODA [3], anionic SA [4] lipids and a protein OVA to prepare a protein lipid mixed film by LB technique using a computerized, Teflon-bar-barrier type LB trough (model 2000C, Apex Instruments Co. India). Pressure-area isotherm, surface pressure growth kinetics, fluorescence studies show that DPPC-OVA mixed film is better to prepare a protein-lipid mixed film by LB technique. FE-SEM analysis shows the squeezing out of protein from the monolayer at higher surface pressure. In situ compressibility analysis is used to transfer a film with almost smooth surface morphology below the squeezing out point. Moreover, nanometer to micrometer aggregates were found in case of ODA-OVA [Fig.1], SA-OVA [Fig. 2] mixed film. DPPC-OVA mixed film shows the aggregates with 60-80 nm in dimension [Fig. 3]. Therefore, we have controlled the aggregation and squeezing out of protein in protein-lipid mixed film by using appropriate zwitterionic lipid [Fig.4]. Next our aim is to minimize the aggregation of protein in protein lipid mixed film.
To fulfill our aim we have done self assembly of OVA on prefabricated DPPC LB film. It shows nanometer to micrometer aggregates with unfolding of OVA, studied by
FE-SEM [Fig.5], CD and FTIR spectroscopy. Hence, this method is not good to prepare a protein –lipid mixed film. LB method is far better than this technique.
Finally, we invented a simple but novel technique to prepare a protein-lipid mixed film. This is self-assembly of DPPC-OVA vesicle on hydrophilic glass substrate. FE-SEM analysis shows that distribution of whitish small globules through out the film [Fig.6]. The dimensions of these globules are in range of 8-10 nm in diameter, similar to the dimension of OVA monomer (7 nm x 4.5 nm x.5 nm) [5]. Therefore, these granules are expected to be the individual OVA monomeric structure and they preserve their individual structure in vesicle matrix. Here, DPPC may form membrane by fusion of vesicle on hydrophilic glass slide. CD and FTIR analysis of this film show that the increment of α-helix due to hydrophobic interaction in vesicle matrix preserves the folding of the OVA molecules and prevents the formation of larger aggregates [6]. Immobilization of protein on vesicle and subsequent self-assembly on hydrophilic substrate may be useful for the preparation of protein-lipid mixed film with a minimum aggregation of protein.
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References: [1] T. Kamilya, P. Pal, G. B. Talapatra, J. Phys. Chem. B 11, 1199, 2007 [2] T. Kamilya, P. Pal, G. B. Talapatra, Asian J. Physics. (In press) [3] T. Kamilya, P. Pal, G. B. Talapatra, J. Colloid Interface Sci. 315, 464, 2007 [4] T. Kamilya, P. Pal, G. B. Talapatra, Colloids Surf. B: Biointerfaces 58, 137, 2007. [5] Protein data bank [6] T. Kamilya, P. Pal, M. Mahato, G. B. Talapatra, J. Nanosci. Nanotech. (Communicated). Acknowledgement: We thank DST, Government of India (Project No. - SR/S2/CMP-0051/2006) for partial financial support. MM also thanks to CSIR, Government of India for providing the CSIR-NET fellowship.
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