Highly sensitive rapid chemiluminescent immunoassay using the DNAzyme label for signal amplification

Abstract

A novel trace tag for chemiluminescent (CL) immunoassay was designed by using DNAzyme to functionalize antibody-labeled Au nanoparticles (AuNPs). The trace tag showed an excellent ability to catalyze the oxidation of luminol by hydrogen peroxide, leading to strong CL emission. By coupling the trace tag with a passive mixing accelerated immunoreaction system, a highly sensitive rapid flow-through CL immunoassay method was proposed. Using carcinoembryonic antigen (CEA) as a model analyte, the capture antibody for CEA was immobilized on paramagnetic microspheres, and DNAzyme-anti-CEA antibody functionalized AuNPs were prepared as trace tag. A three-dimensional helical glass tube kept at 37 °C in a water bath was used for passively mixing immunoreagents in a two-step sandwich immunoassay, with which each immunoreaction step could be finished within 150 s. With the help of a magnet, the immunocomplex could conveniently be separated from reactants. Compared with the horseradish peroxidase-based tag, the newly designed trace tag showed obvious signal amplification due to its strong catalytic ability and high loading ratio of DNAzyme on each AuNP. The proposed method showed a linear calibration range from 0.005 to 0.5 ng mL⁻¹ for CEA detection with a detection limit of 4.1 pg mL⁻¹ at a signal-to-noise ratio of 3 and acceptable detection reproducibility. The assay results of clinical serum samples were in acceptable agreement with the reference values. The designed immunoassay system with ultrahigh sensitivity provided a programmable and low-cost approach for high-throughput clinical application.