Sensitive amperometric biosensor for the determination of biogenic and synthetic amines using pea seedlings amine oxidase: a novel approach for enzyme immobilisation.
We prepared a new inorganic sorbent based on modified triazine (2-[4,6-bis (aminoethylamine)-1,3,5-triazine]-Silasorb; BAT-Silasorb) which binds pea seedlings amine oxidase (PSAO) very tightly without loss of its catalytic activity. This unique feature as well as the wide substrate specificity of PSAO was successfully utilised in the construction of an amperometric biosensor based on a carbon paste electrode for the fast and sensitive detection of various amines at a formal potential 0 mV versus Ag/AgCl reference electrode. The reaction layer of the biosensor is created by the direct immobilisation of PSAO at the electrode surface via affinity carrier BAT-Silasorb. Used arrangement facilitates a simple restoration of the inactive biosensor. An amperometric signal results from horseradish peroxidase catalysed reduction of H2O2, a secondary product of the oxidative deamination of amines, catalysed by PSAO. The sensor was used for the basic characterisation of 55 biogenic and synthetic amines, from numerous mono-, di- and polyamines to various hydroxy-, thio-, benzyl- and aromatic derivatives in order to establish its suitability as a postcolumn detector. Its high sensitivity to putrescine 20.0 +/- 0.64 mA l-1 per mol (636.9 +/- 2.03 mA l-1 per mol per cm2), a limit of detection of 10 nmol l-1 (determined with respect to a signal-to-noise ratio 3:1), a linear range of current response to 0.01-100 mumol l-1 concentration of substrate and good reproducibility all indicate that the sensor could be applied to future industrial and clinical analyses.
Wimmerová
M
M
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic. michaw@chemi.muni.cz
Macholán
L
L
D006631:Amine Oxidase (Copper-Containing); D000588:Amines / Q000032:analysis* / Q000737:chemistry; D001679:Biogenic Amines / Q000032:analysis* / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods* / Q000706:statistics & numerical data; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005069:Evaluation Studies as Topic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D018532:Pisum sativum / Q000201:enzymology; D010944:Plants / Q000201:enzymology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D000588:Amines; D001679:Biogenic Amines; D004800:Enzymes, Immobilized; D006631:Amine Oxidase (Copper-Containing)