An enzyme cascade-based electrochemical immunoassay using a polydopamine-carbon nanotube nanocomposite for signal amplification.

By coupling tyrosinase (Tyr) and β-galactosidase (Gal) into one redox-cycling scheme, an enzyme cascade-based electrochemical immunosensor with boosted selectivity and sensitivity was constructed using polydopamine-functionalized multiwalled carbon nanotube (MWCNTs-PDA) nanohybrid modified electrodes. The MWCNTs-PDA nanohybrid presented a 5 times enhanced capability for antibody conjugation, which was responsible for signal amplification. In the proposed enzyme cascade scheme, Gal was captured on the immunosensor surface by a sandwiched immunoreaction, which catalyzed phenyl β-d-galactopyranoside (P-GP) into phenol based on a hydrolysis reaction. The resulting phenol was used as a substrate of Tyr, which was catalyzed to catechol and subsequently to o-quinone. The o-quinone was then electrochemically reduced to catechol, forming a redox cycle between catechol and o-quinone. The enzyme cascade-based immunoassay not only significantly amplified the electrochemical signal, but also led to a high selectivity. Taking the detection of CEA as an example, the enzyme cascade-based electrochemical immunosensor showed a detectable range of 10 pg mL to 10 ng mL and a low detection limit of 8.39 pg mL (S/N = 3), which was superior/comparable to those using other methodologies in previous reports. The selectivity of the enzyme cascade-based immunosensor was 44-80% higher than that of a single enzyme-based immunosensor. This work shows great potential of the coupling enzyme cascade in immunosensing for clinical diagnosis with boosted selectivity and sensitivity.