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Proximity hybridization triggered rolling-circle amplification for sensitive electrochemical homogeneous immunoassay

Abstract

A new homogeneous electrochemical immunoassay strategy was developed for ultrasensitive detection of carcinoembryonic antigen (CEA) based on target-induced proximity hybridization coupled with rolling circle amplification (RCA). The immobilization-free detection of CEA was realized by the use of uncharged peptide nucleic acids (PNAs) probe labeled with ferrocene (Fc) as the electroactive indicator on negatively charged indium tin oxide (ITO) electrode. In the presence of a target protein and two DNA-labeled antibodies, the proximate complex formed in homogeneous solution could unfold the molecular beacon, and the part of the unfolded molecular beacon as a primer hybridized with the RCA template to initiate the RCA process. Subsequently, the detection probe modified Fc (Fc-PNAs) hybridized with the long amplified DNA products. The consumption of freely diffusible Fc-PNAs (neutrally charged) resulted in a significant reduction of Fc signal due to the fact that long amplified DNA/Fc-PNAs products were electrostatically repelled from the ITO electrode surface. The reduction of the electrochemical signal (signal-off) could indirectly detect the CEA concentration. Under the optimal conditions, CEA detection was implemented in a wide range from 1 pg mL−1 to 10 ng mL−1, with a low detection limit of 0.49 pg mL−1. The proposed strategy exhibited advantages of good selectivity, high sensitivity, acceptable accuracy, and favorable versatility of analytes. Moreover, the practical application value of the system was confirmed by assay of CEA in human serums with satisfactory results.

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Supplementary files

Publication details

The article was received on 30 Aug 2017, accepted on 26 Sep 2017 and first published on 27 Sep 2017


Article type: Paper
DOI: 10.1039/C7AN01434A
Citation: Analyst, 2017, Accepted Manuscript
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    Proximity hybridization triggered rolling-circle amplification for sensitive electrochemical homogeneous immunoassay

    F. Gao, F. Zhou, S. Chen, Y. Yao, J. Wu, D. Yin, D. Geng and P. Wang, Analyst, 2017, Accepted Manuscript , DOI: 10.1039/C7AN01434A

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