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 an uncharged peptide nucleic acid (PNA) probe labeled with ferrocene (Fc) as the electroactive indicator on a 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 a 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 the Fc signal due to the fact that long amplified DNA/Fc-PNA products were electrostatically repelled from the ITO electrode surface. The reduction of the electrochemical signal (signal-off) could indirectly provide the CEA concentration. Under the optimal conditions, CEA detection was implemented in a wide range from 1 pg mL to 10 ng mL, with a low detection limit of 0.49 pg mL. 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 the assay of CEA in human serums with satisfactory results.

Single-step and ultrasensitive detection of carcinoembryonic antigen based on an aptamer transduction-mediated exonuclease III-assisted dual-amplification strategy

“…† In addition, this single-step detection strategy had a dramatically shorter incubation time in comparison with most of the previously reported amplication strategies. 26,27,[35][36][37] Specicity and reproducibility of this developed strategy…”

Electrochemistry/Photoelectrochemistry-Based Immunosensing and Aptasensing of Carcinoembryonic Antigen

“…After the RCA process, a large In recent years, nucleic acid-based immunosensing strategies have been proved as powerful tools for protein identifications via the integration of DNA strands and antibodies to improve analytical performances [42,43]. Among the aforementioned methods, proximity hybridization-assisted amplification generally utilizes a couple of specific antibody-labeled DNA strands (also called proximity probes) to synchronously recognize the target antigen, followed by the hybridization of proximity probes for the stimulation of detection signals [44,45]. According to this consideration, Xiong and coworkers exploited an ultrasensitive electrochemical CEA immunosensor by the intelligent use of proximity hybridization-stimulated, three-layer cascade amplification [46].…”

Suppressing Non-Specific Binding of Proteins onto Electrode Surfaces in the Development of Electrochemical Immunosensors

“…According to the IUPAC, a biosensor is an analytical device that incorporates a biological element immobilized on the surface of a physicochemical transducer, which recognizes the target molecule or analyte present in a sample and, by means of the transducer, this event of biorecognition is transduced into a discrete or continuous electric signal, directly proportional to the amount of analyte in the sample [7,8,9,10]. Finally, the electrical signal is shown as a response in a computer work station (Figure 1).…”