Amperometric flow injection determination of putrescine and putrescine oxidase

Flow injection methods for sensitive, accurate and rapid determination of diamines, mainly putrescine (PUT) and cadaverine (CAD), and diamine oxidase (DAO) are described. Putrescine determination is based on an immobilized enzyme reactor (IER) loaded with putrescine oxidase and amperometric detection of the produced hydrogen peroxide at a platinum electrode. Putrescine oxidase (PUO) was assayed by simple injection of putrescine oxidase solution into a carrier buffer containing a fixed concentration of putrescine followed by similar detection of hydrogen peroxide. In PUT measurements peak currents were linearly related to putrescine concentration in the range from 10 μM to 1 mM with sample frequency of ~70 samples/hour. No change in sensitivity and detection limit were observed after more than 1000 injections over a two week period. PUO was determined in the range of 50-2500 mU/ml, with sample frequency of ~40 samples/hour. The lower detection limit of the present method (1.5 mU/ml) is suitable for determining the expected low levels of PUO in the amniotic fluid as a possible marker for premature rupture of membranes (PROM). The effects of flow rates, sample sizes, and substrate concentrations in the detection limits and assay efficiencies are given in the present work.