An ultrasensitive immunosensor based on potentiometric ELISA for the detection of a cardiac biomarker, troponin I-T-C (Tn I-T-C) complex, was developed. The sensor fabrication involves typical sandwich ELISA procedures, while the final signal readout was achieved using open circuit potentiometry (OCP). Glassy carbon (GC) working electrodes were first coated with emulsion-polymerized polyaniline/dinonylnaphthalenesulfonic acid (PANI/DNNSA) and the coated surface was utilized as a transducer layer on which sandwich ELISA incubation steps were performed. An enzymatic reaction between o-phenylenediamine (OPD) and hydrogen peroxide (H2O2) was catalyzed by horseradish peroxidase (HRP) labeled on the secondary antibodies. The polymer transducer charged state was mediated through electron (e(-)) and charge transfers between the transducer and charged species generated by the same enzymatic reaction. Such a change in the polymer transducer led to potential variations against an Ag/AgCl reference electrode as a function of Tn I-T-C complex concentration during incubations. The sequence of OPD and H2O2 additions, electrochemical properties of the PANI/DNNSA layer and non-specific binding prevention were all crucial factors for the assay performance. Under optimized conditions, the assay has a low limit of detection (LOD) (< 5 pg/mL or 56 fM), a wide dynamic range (> 6 orders of magnitude), high repeatability (coefficient of variance < 8% for all concentrations higher than 5 pg/mL) and a short detection time (< 10 min).
Keywords: ELISA; Polyaniline; Potentiometry; Troponin I–T–C; Ultrasensitive immunosensor.
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