A Novel Electrochemiluminescent Immunoassay Based on Target Transformation Assisted with Catalyzed Hairpin Assembly Amplification for the Ultrasensitive Bioassay

In this work, we constructed a novel electrochemiluminescent (ECL) strategy based on sandwich immunoassay-induced target transformation assisted with catalyzed hairpin assembly (CHA) amplification for ultrasensitive bioassay with cysteine-rich protein 61 (CCN1) as a model. First, the target CCN1 could be equally transformed into the specific oligonucleotide (initiator I) labeled on the detection antibody based on the specific sandwich immunoassay. In addition, the initiator I triggered an efficient nonenzymatic CHA amplification in the presence of ferrocene-labeled hairpin 1 (Fc-H1) and hairpin 2 (H2) to produce massive hybrids (Fc-H1-H2) containing a sticky end labeled with ferrocene. Finally, Fc-H1-H2 could be immobilized on the capture probe single-stranded DNA (ssDNA)-modified electrode through the hybridization between the sticky end of Fc-H1-H2 and ssDNA, and a significantly quenched ECL signal could be obtained due to the efficient quench effect between ferrocene and the ECL indicator, ruthenium(II) tris(4,4'-dicarboxylicacid-2,2'-bipyridyl) [Ru(dcbpy)₃²⁺], immobilized on the surface of the electrode, which was related to the concentration of target CCN1. As expected, the proposed ECL biosensor exhibited a relatively low detection limit of 3.9 fg/mL in a linear range from 10 fg/mL to 100 ng/mL. This ECL strategy inspired the clinical examination of the biomarker CCN1, providing potential application in early diagnosis and malignant monitoring of cancer.