Cacciotti_2020_Mater.Sci.Eng.C.Mater.Biol.Appl_111_110744

Herein we report a multiplated and biopolymeric-based optical bioassay for organophosphate detection based on the use of acetylcholinesterase (AChE) as biocomponent and biopolymeric electrospun fibrous mats as eco-designed supports for AChE immobilisation. The principle of the detection relays on the decrease of enzymatic activity due to the capability of the organophosphorus pesticides to irreversibly inhibit AChE, which is optically detected using Ellman colorimetric method. The proposed bioassay consists in a novel, cost-effective, and multiplex-based 96-well system, in combination with customised biopolymeric membranes modified with AChE, with the aim to deliver a sustainable analytical tool. Indeed, the designed set-up should provide and guarantee several advantages, including: i) the re-use of plastic multi-plate with the only replacement of polymer dishes in the case of inhibition absence; ii) the exploiting of the properties of the immobilised enzyme, i.e. multiple analysis using the same amount of enzyme, reducing the AChE amount for analysis. In detail, three different biopolymers (i.e. polylactic acid (PLA), polycaprolactone (PCL), and poly-hydroxybutyrate-co-hydroxyvalerate (PHBV)) were investigated and morphologically characterised, as supports for enzyme immobilisation, to identify the optimal one. Among them, PHBV was selected as the best support to immobilise AChE by cross-linking method. The analytical features of the bioassay were then assessed by measuring standard solutions of paraoxon in a range of concentrations between 10 and 100 ppb, achieving a linear range up to 60 ppb and a detection limit of 10 ppb. Thus, the suitability of this sustainable bioassay to detect organophosphate at ppb level was demonstrated.