Numerous long single-stranded DNAs produced by dual amplification reactions for electrochemical detection of exosomal microRNAs

Abstract

Exosomal microRNAs (miRNAs) have been explored as an extremely promising biomarker of liquid biopsy for the diagnosis, treatment and prognosis of diseases such as cancer, in which sensitive and selective detection is significant. Herein, we describe the construction and testing of an electrochemical biosensor for the sensitive detection of exosomal miRNAs. It is based on synthetizing numerous long single-stranded DNAs (ssDNAs), which are produced by dual amplification reactions of target-triggered cyclic strand displacement reaction (TCSDR) and primer exchange DNA amplification reaction (PEDAR). In the first signal amplification step, target miRNAs are captured by the hairpin DNA strands (capture probes, Cp) that are immobilized on electrode. After strand unfolding with target capture, primer probes (Pp) enable to hybridize with Cp. And then target miRNAs were displaced for starting the TCSDR process that enable the introduction of numerous primers in Pp. In the second signal amplification step, the primers associated with PEDAR produce copious amounts of elongated ssDNAs. These ssDNAs absorb abundant quantities of methylene blue (MB) that enables the highly sensitive and label-free detection of exosomal miRNAs. This dual amplification process is characterized by a low limit of detection (LOD) of 3.04 aM. In addition, the electrochemical biosensor exhibits good selectivity for miR-21 detection, and shows benefits of simple operation, low cost, portability. Overall, the electrochemical biosensor provides a promising platform for the early diagnosis and screening of tumor biomarkers and the development of devices for point-of-care testing (POCT).