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

Pinto R, Amaral AL, Ferreira EC, Mota M, Vilanova M, Ruel K, Gama M - BMC Biotechnol. (2008)

Bottom Line: This result does not imply the existence of several adsorbed protein layers.It was verified that CBDs were able to penetrate the fibres, according to confocal microscopy and TEM-immunolabelling analysis.The surface concentration of adsorbed CBDs was greater on amorphous fibres (phosphoric acid swollen) than on more crystalline ones (Whatman CF11 and Sigmacell 20).

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Affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. joaoricardo@yahoo.com

ABSTRACT

Background: Cellulose Binding Domains (CBD) were conjugated with fluorescein isothiocyanate (FITC). The surface concentration of the Binding Domains adsorbed on cellulose fibres was determined by fluorescence image analysis.

Results: For a CBD-FITC concentration of 60 mg/L, a coating fraction of 78% and 110% was estimated for Portucel and Whatman fibres, respectively. For a saturating CBD concentration, using Whatman CF11 fibres, a surface concentration of 25.2 x 10-13 mol/mm2 was estimated, the equivalent to 4 protein monolayers. This result does not imply the existence of several adsorbed protein layers.

Conclusion: It was verified that CBDs were able to penetrate the fibres, according to confocal microscopy and TEM-immunolabelling analysis. The surface concentration of adsorbed CBDs was greater on amorphous fibres (phosphoric acid swollen) than on more crystalline ones (Whatman CF11 and Sigmacell 20).

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Whatman CF11 images. The characteristic curled structure of the fibres (circle).
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Figure 2: Whatman CF11 images. The characteristic curled structure of the fibres (circle).

Mentions: In a previous work [21], we have shown that CBDs affect the technical properties of paper fibres (secondary fibres from the paper mill Portucel). The concentration of CBDs, used in those experiments, was in the range of 1–2 mg of CBD per gram of fibres. It is arguable whether this relatively low amount of protein is sufficient to cause modifications in the fibres' interfacial properties. This would probably imply a substantial coating of the fibres by CBDs. In this work, we analyzed fibres from Portucel treated with CBDs conjugated to FITC, and attempted to estimate the percentage of surface coverage. Fibres treated with only CBDs didn't present any fluorescence. As may be observed in Figure 1, the fibres do not display a uniform distribution of fluorescence. This may be due to the chemical heterogeneity (lignin/hemicellulose) and/or to the variable crystallinity [24]. The regions with less intense fluorescence in the picture were selected for CBD-FITC quantification, since these regions are expected to be more crystalline (Pinto et al., unpublished). The detected fluorescence is produced by CBDs adsorbed on both sides of the fibres (top and bottom). Indeed, the fluorescent radiation crosses the fibres with just a slight reduction in intensity [25]. Figure 2 shows a Whatman CF11 fibre, both on bright field and fluorescence microscopy. As it can be observed in the circled area, this cellulose has a rather smooth surface. The extremities of the fibres are expected to have a higher number of fissures and loosen microfibrils, increasing the available area, and consequently the adsorbing sites for CBDs [12], as indicated by the higher fluorescence emission (Fig. 2). The adsorption of CBDs on Whatman CF11 fibres was analyzed, using a protein concentration of 2 mg/gfibres. The estimated surface concentrations of CBDs adsorbed on Portucel and Whatman CF11 fibres (Fig. 1) are shown in Figure 3.


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

Pinto R, Amaral AL, Ferreira EC, Mota M, Vilanova M, Ruel K, Gama M - BMC Biotechnol. (2008)

Whatman CF11 images. The characteristic curled structure of the fibres (circle).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254392&req=5

Figure 2: Whatman CF11 images. The characteristic curled structure of the fibres (circle).
Mentions: In a previous work [21], we have shown that CBDs affect the technical properties of paper fibres (secondary fibres from the paper mill Portucel). The concentration of CBDs, used in those experiments, was in the range of 1–2 mg of CBD per gram of fibres. It is arguable whether this relatively low amount of protein is sufficient to cause modifications in the fibres' interfacial properties. This would probably imply a substantial coating of the fibres by CBDs. In this work, we analyzed fibres from Portucel treated with CBDs conjugated to FITC, and attempted to estimate the percentage of surface coverage. Fibres treated with only CBDs didn't present any fluorescence. As may be observed in Figure 1, the fibres do not display a uniform distribution of fluorescence. This may be due to the chemical heterogeneity (lignin/hemicellulose) and/or to the variable crystallinity [24]. The regions with less intense fluorescence in the picture were selected for CBD-FITC quantification, since these regions are expected to be more crystalline (Pinto et al., unpublished). The detected fluorescence is produced by CBDs adsorbed on both sides of the fibres (top and bottom). Indeed, the fluorescent radiation crosses the fibres with just a slight reduction in intensity [25]. Figure 2 shows a Whatman CF11 fibre, both on bright field and fluorescence microscopy. As it can be observed in the circled area, this cellulose has a rather smooth surface. The extremities of the fibres are expected to have a higher number of fissures and loosen microfibrils, increasing the available area, and consequently the adsorbing sites for CBDs [12], as indicated by the higher fluorescence emission (Fig. 2). The adsorption of CBDs on Whatman CF11 fibres was analyzed, using a protein concentration of 2 mg/gfibres. The estimated surface concentrations of CBDs adsorbed on Portucel and Whatman CF11 fibres (Fig. 1) are shown in Figure 3.

Bottom Line: This result does not imply the existence of several adsorbed protein layers.It was verified that CBDs were able to penetrate the fibres, according to confocal microscopy and TEM-immunolabelling analysis.The surface concentration of adsorbed CBDs was greater on amorphous fibres (phosphoric acid swollen) than on more crystalline ones (Whatman CF11 and Sigmacell 20).

View Article: PubMed Central - HTML - PubMed

Affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. joaoricardo@yahoo.com

ABSTRACT

Background: Cellulose Binding Domains (CBD) were conjugated with fluorescein isothiocyanate (FITC). The surface concentration of the Binding Domains adsorbed on cellulose fibres was determined by fluorescence image analysis.

Results: For a CBD-FITC concentration of 60 mg/L, a coating fraction of 78% and 110% was estimated for Portucel and Whatman fibres, respectively. For a saturating CBD concentration, using Whatman CF11 fibres, a surface concentration of 25.2 x 10-13 mol/mm2 was estimated, the equivalent to 4 protein monolayers. This result does not imply the existence of several adsorbed protein layers.

Conclusion: It was verified that CBDs were able to penetrate the fibres, according to confocal microscopy and TEM-immunolabelling analysis. The surface concentration of adsorbed CBDs was greater on amorphous fibres (phosphoric acid swollen) than on more crystalline ones (Whatman CF11 and Sigmacell 20).

Show MeSH