Flow screen-printed amperometric detection of p-nitrophenol in alkaline phosphatase-based assays

p-Nitrophenyl phosphate is one of the most widely used substrates for alkaline phosphatase in ELISAs because its yellow, water-soluble product, p-nitrophenol, absorbs strongly at 405 nm. p-Nitrophenol is also electroactive; an oxidative peak at 0.97 V (vs. an Ag pseudoreference electrode) is obtained when a bare screen-printed carbon electrode is used. When an amperometric detector was coupled to a flow-injection analysis system the detection limit achieved for p-nitrophenol was 2 x 10(-8) mol L-1, almost two orders of magnitude lower than that obtained by measuring the absorbance of the compound. By use of this electrochemical detection method, measurement of 7 x 10(-14) mol L-1 alkaline phosphatase was achieved after incubation for 20 min. The feasibility of coupling immunoassay to screen-printed carbon electrode amperometric detection has been demonstrated by performing an ELISA for detection of pneumolysin, a toxin produced by Streptococcus pneumoniae, which causes respiratory infections. The method is simple, reproducible, and much more sensitive than traditional spectrophotometry.