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

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.

Attomolar determination of coumaphos by electrochemical displacement immunoassay coupled with oligonucleotide sensing.

Coumaphos, an organophosphorus pesticide (OP) used worldwide, has raised serious public concerns due to its positive association with major types of cancer. Herein, a novel method for attomolar coumaphos detection was developed on the basis of an electrochemical displacement immunoassay coupled with oligonucleotide sensing. An optimized displacement immunoassay was constructed to improve the binding efficiency of an antigen-antibody pair, and a guanine-rich single-strand DNA label, in combination with oligonucleotide sensing, was used to amplify the detection signal with "direct" relationship to the analyte. As a result, coumaphos was sensitively determined from the enhanced catalytic cycle of guanine-Ru(bpy)(3)(2+) by chronoamperometry. The limit of detection (LOD) was down to 0.18 ng L(-1) (S/N = 3), which is equal to 49.6 amol in a sample solution of 100 µL. In comparison with conventional methods, the proposed method has the lowest LOD and better accessibility to high-throughput sensing systems. Besides, it can complete the whole analysis process in under 50 min and exhibits good performance of excellent selectivity to the OPs. With regard to the advantages of rapidity, convenience, low cost, and ease of operation, the proposed method has provided a promising platform capable of fast and in-field OP detection, which may make the system promising for potential applications in the detection of other small molecules.