Abstract
A novel chemiluminescent (CL) immunoassay protocol for the sensitive determination of Gibberellic acid (GA) using DNA-based hybridization chain reaction (HCR) is described. The carboxyl terminated magnetic beads (MBs) were modified with GA antibody and the gold nanoparticles (AuNPs) signal probes were labeled with the GA antibody and DNA initiator strands. In the presence of target GA, the sandwiched immunocomplex was formed between the immobilized antibody on the MBs and the signal antibody on the AuNPs. When the two complementary stable species of DNA hairpins, which are G-rich DNA, were added the hybridization events were happened, thereby resulting forming the long nicked double-helix. Numerous double G-rich DNA were formed on the AuNPs, each of which produced a CL signal within the applied CL reaction. The CL signal was obtained via the instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxyl-phenylglyoxal (TMPG), and the G-rich DNA on immunocomplex. Under optimal conditions, GA can be detected in the concentration ranged from 0.01ng/mL to 70ng/mL, and the limit of detection was 0.003ng/mL. The reproducibility and selectivity of the developed method were also investigated. In addition, the real samples were assayed by using the developed immunoassay, and the recovery was 96.0–102.0%.
Published Version
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