We have designed here a label-free direct electrochemical immunosensor for the detection of cytochrome c (cyt c), a heme containing metalloprotein using its specific monoclonal antibody. Two nanocomposite-based electrochemical immunosensor platforms were evaluated for the detection of cyt c; (i) self-assembled monolayer (SAM) on gold nanoparticles (GNP) in polypyrrole (PPy) grafted screen printed electrodes (SPE) and (ii) carbon nanotubes (CNT) integrated PPy/SPE. The nanotopologies of the modified electrodes were confirmed by scanning electron microscopy. Electrochemical impedance spectroscopy and cyclic voltammetry were employed to monitor the stepwise fabrication of the nanocomposite immunosensor platforms. In the present method, the label-free quantification of cyt c is based on the direct electron transfer between Fe (III)/Fe (II)-heme redox active site of cyt c selectively bound to anti-cyt c nanocomposite modified SPE. GNP/PPy and CNT/PPy nanocomposites promoted the electron transportation through the conductive pore channels. The overall analytical performance of GNP/PPy based immunosensor (detection limit 2 nM; linear range: 2 nM to 150 µM) was better than the anti-cyt c/CNT/PPy (detection limit 10 nM; linear range: 10 nM to 50 µM). Further, the measurement of cyt c release in cell lysates of cardiomyocytes using the GNP/PPy based immunosensor gave an excellent correlation with standard ELISA.
Keywords: Cytochrome c; ELISA; Electrochemical immunosensors; Label-free detection; Screen printed electrodes.
© 2013 Published by Elsevier B.V.