Development of novel carbon black-based heterogeneous oligonucleotide-antibody assay for sulfur mustard detection

Herein, a novel configuration for the detection of a chemical warfare agent, namely sulfur mustard (bis(2-chloroethyl)sulphide) was designed to develop a biosensor that can play a relevant role in the monitoring of such a chemical threat in risk and war areas. In detail, a tailor-made heterogeneous oligonucleotide-antibody biosensor was realised, based on the formation of a sulfur mustard-oligonucleotide adduct on the surface of a screen-printed electrode. Thus, the immunoassay was carried out through a sequential immobilisation process, involving a primary antibody, ad hoc synthesised for the selective recognition of the sulfur mustard-oligonucleotide adduct, followed by an alkaline phosphatase-conjugated anti-mouse anti-IgG as secondary antibody. The detection was performed by adding the substrate 1-naphthyl phosphate and measuring the enzymatic product in differential pulse voltammetry. In order to improve the electroanalytical performances of the novel heterogeneous oligonucleotide-antibody sensor, we have explored the advantageous use of carbon black for the detection of alkaline phosphatase by-products. The electrochemical response of graphite-based screen-printed electrodes modified with three types of carbon black (i.e. N220, N115, and HP160) was thoroughly investigated by using cyclic voltammetry and amperometry for the detection of different enzymatic byproducts namely 1-naphthol, hydroquinone, 4-nitrophenol, and ascorbic acid. This study allowed to shed light on the capacity of carbon black of remarkably enhancing the measurement of alkaline phosphatase's products. Particularly, carbon black N220 and 1-naphthol were selected for developing a highly performant heterogeneous oligonucleotide-antibody assay for the detection of sulfur mustard. Under optimized conditions, the carbon black-based biosensor showed a linear range from 20 mu M to 80 mM and a LOD of 12 mu M in liquid samples, demonstrating high sensitivity as well as selectivity in presence of some possible interfering sources in the war field.