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Cellulose Binding Domains (CBD) were conjugated with fluorescein isothiocyanate (FITC).

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- Quantification of the CBD-FITC conjugates surface coating on cellulose fibres

Due to its high sensitivity and specificity, fluorescence analysis has been extensively used in microarray technology [1], gene expression monitoring [2], protein diffusion [3] or in vivo chemical elements uptake and localization studies [4]. Another area to benefit from fluorescence based techniques is protein quantification. The use of either non-covalent [5] or covalent labelling [6,7] increased the detection sensitivity to as low as 40 ng/mL.

Cellulose-Binding Domains (CBD) are modules present in most celulases, being responsible for their high affinity to cellulose crystalline surfaces [8,9]. The CBD used in this work, produced by limited proteolysis, belongs to cellobiohydrolase I (CBHI) of Trichoderma reesei, as shown in a previous work [10]. Three tyrosine residues define a flat surface, which may be responsible for the affinity to cellulose [11]. This protein has a single amine, the N-terminal of the linker region, which allows a specific reaction with fluorescein isothiocyanate (FITC). The conjugation with FITC does not affect the CBD interaction with cellulose, since the N-terminal is isolated from the cellulose interacting part of the protein. Indeed, the conjugation of FITC does not modify the CBD adsorption isotherms [12,13]. Since there is only one amine group present in the CBD, the stoichiometry of the conjugation reaction is 1:1. The FITC fluorophore has been attached to antibodies [14,15], to microparticles [16] or to other binding domains [12,17]. Several recombinant CBDs, fused to different proteins, have been produced, as recently reviewed by Shoseyov et al.[18].

Cellulose-Binding Domains (CBD) have been used to target functional molecules to cellulose-containing materials [19], to improve pulp properties [20] or as an additive for paper recycling [21]. Bearing in mind that these applications are related to surface effects, in this work we attempted to quantify the CBD surface coverage of cellulose fibres, using the approach based on the use of CBD-FITC previously developed. Our aim was to quantify the protein adsorbed on cellulose fibres and, more specifically, the surface concentration of CBD. This value could, alternatively, be estimated by measuring the specific surface area, by means of the BET isotherm [22]. However, the BET approach is not ideal for porous materials [23]. The presence of CBDs in the interior of the fibres was also investigated.

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