Immobilization of casein micelles for probing their structure and interactions with polysaccharides using scanning electron microscopy (SEM) Anneke H. Martin, H. Douglas Goff, Alexandra Smith, Douglas G. Dalgleish * Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 7 June 2005; revised 29 August 2005; accepted 31 August 2005 Abstract A new method is described for probing the structure of casein micelles and their interactions with k-carrageen and l-carrageenan using scanning electron microscopy (SEM). Immobilization of casein micelles at pH 6.7 to a gold substrate by means of covalent binding resulted in a homogeneous distribution of isolated, spherical casein micelles on the substrate. Detailed images of the casein micelle structure were obtained. Electron micrographs of casein micelle–carrageenan mixtures revealed aggregates, which became larger with increasing carrageenan concentration. Because of the ability of k-carrageenan to form a gel, in contrast to l-carrageenan, larger and more aggregates were found with k- carrageenan at similar carrageenan concentration. Detailed images showed strands of k-carrageenan to which casein micelles were attached, which was not observed for l-carrageenan. q 2005 Elsevier Ltd. All rights reserved. Keywords: Casein micelles; Immobilization; Gold; Scanning electron microscopy (SEM); Polymer interactions; Carrageenan 1. Introduction To study biomolecules (e.g. proteins or polysaccharides) and their interactions by techniques of microscopy such as Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) or Scanning Tunneling Microscopy (STM), immobilization of the biomolecule to the surface is essential. Whereas proteins can be physically adsorbed (physisorbed) via electrostatic interactions or chemically adsorb (chemisorb) to solid substrates, immobilization of proteins by the formation of covalent bonds results in a more stable protein layer, which can withstand pressure, such as that exerted by AFM tips, or washing procedures (Wadu-Mesthrige, Amro, & Liu, 2000). The surfaces most commonly used as a substrate for immobilization of biomolecules are gold and glass (silica). Gold appears to be a suitable substrate for covalent binding of proteins because it can be functionalized, typically through a thiol (–SH) group (Leung, Xirouchaki, Berovic, & Palmer, 2004). If the protein contains a free cysteine group or even a disulfide bridge, direct immobilization on the gold coated substrate is possible (Cavalleri, Natale, Stroppolo, Relini, Cosulich and Thea, 2000; Leung et al., 2004). For all proteins, self-assembled monolayers (SAMs) of small mol- ecules can be used, if covalent coupling can occur between the free end of the molecules in the SAM and the protein by using a linker (Uricanu, Duits, & Mellema, 2004). An advantage of SAMs is that they can shield proteins from direct contact with the solid surfaces, and thus reduce considerably the possible denaturation and spreading of adsorbed proteins (Wadu-Mes- thrige et al., 2000). Among SAMs, the most widely studied systems have been those formed by chemisorption of alkanethiols [CH 3 (CH 2 ) nK1 –SH] onto gold or a gold coated substrate (Cavalleri et al., 2000; O’Dwyer, Gay, de Lesegno, & Weiner, 2004). Depending on the pH and the surface functionality of the alkanethiol (especially the end group, e.g. methyl, carboxylic acid), proteins may physisorb directly to the substrate (Wadu-Mesthrige et al., 2000). However, using a carboxylic acid terminated alkanethiol, proteins can be covalently bonded through carbodiimide chemistry (Lahiri, Isaacs, Tien, & Whitesides, 1999; Ostuni, Yan, & Whitesides, 1999; Patel, Davies, Heaton, Roberts, Tendler and Williams, 1998; Uricanu et al., 2004; Veiseh, Zareie, & Zhang, 2002). This reaction involves the formation of a reactive ester to which amino groups within the protein can be linked. In this way, multiple protein-SAM linkages can occur for a single protein or Food Hydrocolloids 20 (2006) 817–824 www.elsevier.com/locate/foodhyd 0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2005.08.004 * Corresponding author. Tel.: C1 519 824 4120x56929; fax: C1 519 824 6631. E-mail address: ddalglei@uoguelph.ca (D.G. Dalgleish).