Tyramine-enzyme conjugate repeats for interdigitated capacitance immunosensing array with neuroblastoma biomarker of neuron-specific enolase

Abstract

Methods based on enzyme labelling strategies have been widely developed for capacitance immunoassays, but most involve low sensitivity and are unfavorable for routine use at the early stage of diagnostics. Herein, we designed a highly efficient capacitance immunosensing method for low-abundance neuroblastoma biomarker of neuron-specific enolase (NSE) on an interdigitated micro-comb electrode. Initially, monoclonal mouse anti-human NSE capture antibodies were encapsulated on the interdigitated gold electrodes by using bovine serum albumin. Thereafter, a sandwich-type immunoreaction was carried out in the presence of target NSE by using horseradish peroxidase (HRP)-labeled secondary antibody. Following that, the labelled HRP could trigger the formation of tyramine-enzyme conjugate repeats with the help of HRP-tyramine and H2O2. The concatenated HRP molecules catalyzed oxidation of 4-chloro-1-naphethol to produce an insoluble precipitation on the interdigitated micro-comb electrode, thus causing the shift in the capacitance. Two protocols with and without the tyramine-HRP repeats were investigated for detection of NSE, and improved analytical features were achieved with tyramine signal amplification. Under optimum conditions, the interdigitated capacitance immunosensors exhibited good responses toward target NSE within a dynamic linear range of 1.0 – 10000 pg mL-1 at a low detection limit of 0.78 pg mL-1. An intermediate reproducibility of ≤9.67% was accomplished with batch-to-batch identification, and good anti-interferring capacity against other proteins was acqiured. 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).