title: Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations.
abstract: For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.
authors.affiliation: Department of Chemistry, Syracuse University, Syracuse, NY 13244, USA.
authors.forename: Hewen
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Department of Chemistry, Syracuse University, Syracuse, NY 13244, USA.
authors.forename: Ivan V
authors.initials: IV
authors.lastname: Korendovych
authors.affiliation: Department of Chemistry, Syracuse University, Syracuse, NY 13244, USA; Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY 13244, USA; Department of Biology, Syracuse University, Syracuse, NY 13255, USA. Electronic address: yluk@syr.edu.
authors.forename: Yan-Yeung
authors.initials: YY
authors.lastname: Luk
mesh_terms: D000464:Alginates / Q000032:analysis* / Q000737:chemistry; D002118:Calcium / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005840:Gentian Violet / Q000737:chemistry; D020723:Glucuronic Acid / Q000032:analysis / Q000737:chemistry; D006603:Hexuronic Acids / Q000032:analysis / Q000737:chemistry; D007477:Ions / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000464:Alginates; D002118:Calcium; D016650:Fluorescein-5-isothiocyanate; D005840:Gentian Violet; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D007477:Ions; D011073:Polyamines; D000071228:Polyelectrolytes
keywords: Aggregation; Alginate; Increased scattering; Mucoid Pseudomonas aeruginosa; Multivalent binding