Ultrasensitive Multiplexed Immunoassay for Tumor Biomarkers Based on DNA Hybridization Chain Reaction Amplifying Signal

In this work, a novel electrochemical immunoassay protocol has been reported for simultaneous determination of multiple tumor biomarkers based on DNA hybridization chain reaction (HCR) for signal amplification. Alpha-fetoprotein (AFP) and prostate specific antigen (PSA) were selected as model biomarkers. The immunoassay protocol contained primary antibodies immobilized on gold nanoparticles (Au NPs), secondary antibodies conjugated with DNA concatemer from HCR of primer, auxiliary probe, and signal probe labeled with signal molecules (methyleneblue (MB) and ferrocene (Fc)). In the presence of target biomarkers, the sandwich immunocomplex was formed between the primary antibodies and secondary antibodies bioconjugates carrying numerous signal molecules. As a result, two well-resolved reduction peaks, one was at −0.35 V (corresponding to MB) and other was at 0.33 V (corresponding to Fc; both vs SCE), were obtained in differential pulse voltammetry, and peak currents changed were related to the level of biomarkers. Under optimal conditions, the electrochemical immunoassay exhibited a wide linear response range (0.5 pg mL–1 to 50 ng mL–1) and low detection limits (PSA, 0.17 pg mL–1; AFP, 0.25 pg mL–1) (at S/N = 3). In addition, the immunoassay was evaluated by analyzing simulate human serum sample, and the recoveries obtained were within 99.4–107.6% for PSA and 97.9–108.2% for AFP, indicating the immnuoassay could be applied to the simultaneous detection of AFP and PSA in human serum samples.