Development of an amperometric biosensing method for the determination of l-fucose in pretreated urine

• Published in: Biosensors & bioelectronics 2004-10, Vol.20 (3), p.620-627
• Main Authors: Tsiafoulis, Constantinos G., Prodromidis, Mamas I., Karayannis, Miltiades I.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biosensing Techniques - instrumentation; Biosensing Techniques - methods; Biosensors; Biotechnology; Carbohydrate Dehydrogenases - chemistry; Electrochemistry - instrumentation; Electrochemistry - methods; Enzymes, Immobilized - chemistry; Ferricyanide; Fucose - urine; Fucose biosensing method; Fucose dehydrogenase; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Lead dioxide; Methods. Procedures. Technologies; NADP - chemistry; Polyvinyl alcohol–styrylpyridinium; Pretreated urine; Urinalysis - instrumentation; Urinalysis - methods; Various methods and equipments
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2004.03.012

The first amperometric biosensing method for the determination of l-fucose is described. l-Fucose is the objective of much current research, as it is considered as a potential marker for various pathologic disorders. Recombinant l-fucose dehydrogenase, having as cofactor β-nicotinamide adenine dinucleotide phosphate (NAD +P), was cross-linked in a water-soluble photosensitive polymer matrix, that is, polyvinyl alcohol (PVA) modified with styrylpyridinium (SbQ), in the presence of BSA and glutaraldehyde. The resulting membrane was sandwiched between two polycarbonate membranes and was mounted in an amperometric cell. The oxidation of the enzymatically produced NADPH was monitored at a platinum anode at +0.25 V versus a silver pseudoreference electrode in the presence of ferricyanide. The system was fully optimized with respect to various analytical parameters. Regarding to the mechanical properties of the membrane and the storage stability of the immobilized enzyme, various parameters were also optimized. Several methods for the pretreatment of urine samples were investigated. Treatment of the samples with PbO 2 found to eliminate the interference effect of various electroactive species exist in urine; optimum incubation time was determined since at prolonged incubation times l-fucose is also affected. Calibration curves for the direct and the mediated monitoring of NADPH were liner over the concentration ranges 0.04–1.0 mM ( r 2=0.9995) and 0.03–3.0 mM ( r 2=0.9997) fucose, respectively. The detection limits (S/N 3) were 2 and 1.5 μM fucose, respectively. The R.S.D. of the mediated biosensor is better than 1.5% ( n=10, 0.5 mM fucose). The proposed biosensor correlates well with a reference enzymatic method and exhibits very good working and storage stability.