Development and evaluation of a highly sensitive rapid response enzymatic nanointerfaced biosensor for detection of putrescine

Putrescine (1,4-diaminobutane) a biologically active diamine has been found to be a valuable analyte for several clinical and analytical purposes. The present work deals with diamine oxidase immobilized on iron oxide nanoparticles for quantifying the amount of putrescine produced, by the decarboxylation of ornithine, which is converted into hydrogen peroxide by the enzyme diamine oxidase (DAO). This reaction can be quantified using electrochemical techniques, which forms the basis of this work. Iron oxide (Fe3O4) nanoparticles, synthesized using thermal co-precipitation, were chosen for immobilization of DAO due to its simple preparation procedure, high surface area and cost-effectiveness. The size of the particles was in the range of 25-35 nm and the enzyme was linked covalently by carbodiimide activation and confirmed using FT-IR. For detecting the hydrogen peroxide released in the reaction, a glassy carbon-working electrode coated with enzyme linked iron oxide nanoparticles was poised at +0.4 V versus an Ag/AgCl reference electrode and a platinum wire was used as the counter electrode. A step-wise increase in current was observed and linearity was obtained in the range of 2-8 nM, with 0.65 nM as the minimum detection limit and the response time was found to be 0.3 seconds. Ascorbic acid, a common interfering molecule in biological samples, did not interfere with the measurements indicating the high degree of specificity of the diamine oxidase-based nano-interfaced biosensor.