Department of Laboratory Medicine , Chongqing General Hospital , Chongqing 400014 , China.
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering , Southwest University , Chongqing 400715 , People's Republic of China.
ACS Appl Mater Interfaces. 2019 Aug 28;11(34):31427-31433. doi: 10.1021/acsami.9b12428. Epub 2019 Aug 13.
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)], 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.
在这项工作中，我们构建了一种基于夹心免疫测定诱导的目标转化辅助催化发夹组装（CHA）扩增的新型电致化学发光（ECL）策略，以富半胱氨酸蛋白 61（CCN1）为模型进行超灵敏生物分析。首先，基于特异性夹心免疫测定，目标 CCN1 可以均等转化为标记在检测抗体上的特异性寡核苷酸（引发子 I）。此外，在存在二茂铁标记的发夹 1（Fc-H1）和发夹 2（H2）的情况下，引发子 I 触发了高效的非酶 CHA 扩增，以产生大量含有二茂铁标记粘性末端的杂交体（Fc-H1-H2）。最后，Fc-H1-H2 可以通过 Fc-H1-H2 的粘性末端与 ssDNA 之间的杂交固定在捕获探针单链 DNA（ssDNA）修饰的电极上，并且由于二茂铁和 ECL 指示剂钌（II）三（4,4'-二羧酸-2,2'-联吡啶）[Ru（dcbpy）]之间的有效猝灭效应，可以获得显著猝灭的 ECL 信号，固定在电极表面上，这与目标 CCN1 的浓度有关。正如预期的那样，所提出的 ECL 生物传感器在 10 fg/mL 至 100 ng/mL 的线性范围内表现出相对较低的检测限 3.9 fg/mL。这种 ECL 策略激发了生物标志物 CCN1 的临床检查，为癌症的早期诊断和恶性监测提供了潜在的应用。
