Tyramine–enzyme conjugate repeats for an interdigitated capacitance immunosensing array in the detection of neuroblastoma biomarker neuron-specific enolase

Abstract

Methods based on enzyme labelling strategies have been widely developed for capacitance immunoassays, but most suffer from low sensitivity and are unfavorable for routine use in the early stages of diagnostics. Herein, we designed a highly efficient capacitance immunosensing method for the low-abundance neuroblastoma biomarker neuron-specific enolase (NSE) using an interdigitated micro-comb electrode. Initially, monoclonal mouse anti-human NSE capture antibodies were immobilized on the interdigitated gold electrodes using bovine serum albumin. Thereafter, a sandwich-type immunoreaction was carried out in the presence of target NSE using horseradish peroxidase (HRP)-labeled secondary antibodies. The labelled HRP subsequently triggered the formation of tyramine–enzyme conjugate repeats with the help of HRP–tyramine and H₂O₂. The concatenated HRP molecules catalyzed the oxidation of 4-chloro-1-naphthol to produce an insoluble precipitate on the interdigitated micro-comb electrode, resulting in a shift in capacitance. Two protocols, with and without tyramine–HRP repeats, were investigated for the detection of NSE, with improved analytical performance achieved through tyramine signal amplification. Under optimum conditions, the interdigitated capacitance immunosensors exhibited good responses to target NSE within a dynamic linear range of 1.0–10 000 pg mL⁻¹, with a low detection limit of 0.78 pg mL⁻¹. An intermediate reproducibility of ≤9.67% was accomplished with batch-to-batch consistency, and good anti-interference capacity against other proteins was acquired. No significant differences at the 0.05 significance level were encountered in the analysis of 12 human serum specimens between the developed capacitance immunosensor and the commercially available enzyme-linked immunosorbent assay (ELISA).