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Unique biosensing system for the simultaneous multiplex fluorescent determination of catecholamines and metabolites.Derivatization of analytes using benzylamine and 1,2-diphenylethylenediamine in the presence of peroxidase as a catalyst.First-order derivative fluorescence spectroscopy for simultaneous multiplex determination of catecholamines and metabolites.Rapid screening of catecholamines and metabolites in 96-wells microplate.Fluorescent analysis of urine and blood plasma with minimal sample treatment.A novel original biosensing system for the simultaneous multiplex determination of general markers of catecholamine-producing diseases – catecholamines (dopamine, epinephrine, norepinephrine) and their metabolites (homovanillic and vanillylmandelic acids) in biological liquids without preliminary separation of analytes, in the absence of specific antibodies and receptors and with minimum pretreatment of a samples has been developed. This outstanding approach includes the unique combination of obtaining highly fluorescent derivatives of the analytes as a result of their interaction with two different amines - benzylamine and 1,2-diphenylethylenediamine in the presence of peroxidase as a catalyst, with the application of first-order derivative fluorescence spectroscopy for the resolution of their spectra. Fluorescence is measured in 96-well microplates, which wells contain a bio-recognizing film consisted of horseradish peroxidase immobilized in the polymer chitosan. Spectra of the solutions are recorded in the range 400–500 nm (λex ˜ 305–356 nm). The proposed procedures provide sensitive (in the range of 3–200 nM), selective, and reproducible (RSDs ≤ 1%, n = 6) multiplex determination of the catecholamines and their metabolites in biological liquids were successfully applied for the rapid simultaneous (20 samples per 15–30 min) screening of human urine and mice blood plasma. The validated results showed good linearity, precision, accuracy and selectivity of this method.