A label-free and PCR-free electrochemical assay for multiplexed microRNA profiles by ligase chain reaction coupling with quantum dots barcodes

• An electrochemical method is presented for multiplexed evaluation of miRNAs.
• Two miRNAs were concurrently detected in a single-tube without label or PCR.
• The detection limits were fM, with linear ranges of 5 and 8 orders of magnitude.
• The proposed method fulfilled the assay in less than 70 min.
• Practical application in biological matrix showed acceptable testing recoveries.

The profiling of microRNAs (miRNAs) is greatly significant for cellular events or disease diagnosis. Electrochemical methods for miRNAs analysis mostly can only measure one kind of miRNA, which is unambiguous to indicate the disease type and state. Here a label-free and PCR-free electrochemical method is presented for multiplexed evaluation of miRNAs in a single-tube experiment. The method is based on the combination of the high base-mismatch selectivity of ligase chain reaction (LCR) and the remarkable voltammetric signature of electrochemical QDs barcodes. Two reporting probes of RP1 and RP2 were labeled with PbS and CdS quantum dots (QDs) to prepare PbS–RP1 and CdS–RP2 conjugates, and two capture probes of CP1 and CP2 were co-immobilized on magnetic beads (MBs) to fabricate MB–CP1CP2 conjugate. The miRNAs samples were simply incubated with MB–CP1CP2, PbS–RP1, and CdS–RP2 conjugates, and then added with T4 DNA ligase. After release of the disjoined QDs barcodes from the MB-conjugates, two target miRNAs of miR-155 and miR-27b were simultaneously detected by square wave voltammetry with linear ranges of 50 fM–30 pM and 50 fM–1050 pM, and limits of detection (LODs) of 12 fM and 31 fM (S/N=3). The method fulfilled the assay in less than 70 min, and showed acceptable testing recoveries for the determination of miRNAs in biological matrix. Currently there are rare reports about electrochemical multiplexed quantification of miRNAs. The method is likely to provide a new platform for identification of multiple miRNAs in a simple way.

Based on the combination of the high base-mismatch selectivity of ligase chain reaction and the remarkable voltammetric signature of electrochemical quantum dots barcodes, a label-free and PCR free electrochemical method is developed for multiplexed evaluation of miRNAs in a single-tube experiment.Figure optionsDownload as PowerPoint slide