1-Naphthyl phosphate as an enzymatic substrate for enzyme-linked immunomagnetic electrochemistry

Abstract

Abstract We demonstrate substitution of the custom-synthesized alkaline phosphatase (AP) substrate, p -aminophenyl phosphate (pAPP), with the commercially available 1-naphthyl phosphate (1-NP) as applied in the enzyme-linked immunomagnetic electrochemical (ELIME) detection of the pathogenic bacterium, Escherichia coli O157:H7. ELIME entails ‘sandwiching’ bacterial analyte between antibody-coated magnetic beads and an AP-conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multi-well plate format. Enzyme substrate (pAPP or 1-NP) was added and conversion to an electroactive product was measured using Osteryoung square wave voltammetry. Using this technique, quantitative detection of E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ≤4.7×10 3 cells ml −1 in buffer or porcine carcass wash water within ca. 80 min.