Delivered by Ingenta to: Indian Association for the Cultivation of Science (IACS) IP : 202.54.54.240 Sat, 04 Apr 2009 09:21:21 RESEARCH ARTICLE Copyright © 2009 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Nanoscience and Nanotechnology Vol. 9, 2956–2964, 2009 Fabrication of Ovalbumin–Phospolipid Thin Film with Minimal Protein Aggregation by Different Self-Assembly Methods Tapanendu Kamilya, Prabir Pal, Mrityunjoy Mahato, and G. B. Talapatra ∗ Department of Spectroscopy, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India This paper describes a new approach for the preparation of Ovalbumin (OVA)-phospholipids (DPPC) thin film, where we minimize the aggregation among protein molecules. A comparative study on the films fabricated by self-assembly (SA) of protein-lipid mixed vesicle on hydrophilic glass substrate and SA of protein alone on prefabricated Langmuir-Blodgett (LB) film of DPPC is being carried out. Far UV circular dichroism spectroscopy, FTIR analysis of amide bands and surface morphology study by FE-SEM imaging is being used to examine the extent of conformational change as well as aggregation of protein. Protein-lipid thin film prepared by the former method provides the secondary structure of individual OVA molecules with increment of -helical segments. In this process, OVA molecules do not show larger aggregation among themselves. However, in the second method, we find unfolded larger aggregated structure of OVA with increment of -structure of OVA. Keywords: Ovalbumin, DPPC, Vesicle, Self-Assembly, Aggregation, Thin Film. 1. INTRODUCTION 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 appli- cations and devices fabrication. 1–11 Proteins or enzymes are easily denatured by some changes in their surrounding environments, such as exposure to the hydrophobic envi- ronment. The immobilization of protein without denatura- tion on solid support, or on lipid membrane, is extremely valuable and is the elementary step for scheming stable and sensitive biosensing devices. The functionality of the biomolecules is affected by specific intermolecular inter- action with neighboring molecules, mainly driven from the intramolecular reorganization. 12 Unfolding and irreversible aggregates are the main outcome of this process. For these reasons, a foremost challenge is to immobilize the protein molecules on appropriate substrate without aggregates. Self-assembly (SA) and Langmuir-Blodgett (LB) methods 13–23 are typical techniques to immobilize bioma- terials on to solid surfaces. However, there are some dif- ficulties in forming Langmuir films of proteins, mainly, proteins are soluble in the water subphase 24 and partially ∗ Author to whom correspondence should be addressed. denature at air/water interface. There are several studies found in the literature on protein monolayer, which indi- cate the surface induced irreversible denaturation of pro- tein at air/water interface. 24–26 However, the presence of a lipid monolayer at air/water interface reduces the surface induced denaturation of protein. 20 Our earlier studies on LB film deposition technique show that protein preserves their conformation in neutral lipid monolayer rather than anionic or cationic lipid monolayers. 16–18 27 However, the extent of aggregation is found to be less in zwitterionic lipid but cannot be minimized by the LB film deposition technique. This work is concerned with two new approaches of SA. One is the SA of the protein, the ovalbumin (OVA), on pre- fabricated LB monolayer film of a phospholipid (DPPC) dipped into OVA aqueous solution. The other one is the SA on hydrophilic glass slide dipped into OVA-DPPC mixed vesicular solution. High-resolution field emission scanning electron microscope (FE-SEM) is used to study the struc- tural aspects of these films. In addition, the conformational changes of OVA in different DPPC matrices were analyzed and compared by the far UV circular dichroism (CD) and FTIR studies of these films. Results are discussed in the context of aggregation and conformational change of OVA. A comparative study between these two methods is also made. 2956 J. Nanosci. Nanotechnol. 2009, Vol. 9, No. 5 1533-4880/2009/9/2956/009 doi:10.1166/jnn.2009.dk23