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 2x10(-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 7x10(-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.
Departamento de Química Física y Analítica, Universidad de Oviedo, 33006, Oviedo, Spain.
Pablo
P
Fanjul-Bolado
María Begoña
MB
González-García
Agustín
A
Costa-García
D000469:Alkaline Phosphatase; D001426:Bacterial Proteins / Q000032:analysis; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005240:Feasibility Studies; D008722:Methods; D009596:Nitrophenols / Q000032:analysis*; D013301:Streptolysins / Q000032:analysis
D001426:Bacterial Proteins; D009596:Nitrophenols; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; D000469:Alkaline Phosphatase; C024836:4-nitrophenol