Mo Wang,Bingchen Li,Qing Zhou,Huanshun Yin,Yunlei Zhou,Shiyun Ai
In this work, we proposed a selective and sensitive biosensor for microRNA detection based on the high specificity and affinity of anti-DNA:RNA hybrids antibody (S9.6 antibody) and alkaline phosphatase catalytic signal amplification. Briefly, after the hybridization of probe DNA and the target microRNA, the S9.6 antibody can be captured on the electrode surface through antigen-antibody immunoreaction. Then, alkaline phosphatase labeled goat anti-mouse IgG (ALP-IgG) was further captured on the electrode surface through the specific recognition effect between the primary antibody and the secondary antibody. Finally, ALP catalyzed the hydrolysis reaction of p -nitrophenyl phosphate to generate p -nitrophenol, resulting in a electrochemical oxidation signal. The simple signal amplification assay performed a successful linear range from 0.5-500 fM with a detection limit of 0.40 fM. Moreover, this biosensor exhibited high selectivity with discriminating only single-base mismatched microRNA sequence. Additionally, the simplicity of this method hold a great promise for further investigation of the role of microRNAs in phytohormone signaling transduction.
Bladder cancer (BC) occurrence and progression are accompanied by alterations in microRNAs (miRNAs) expression levels. Simultaneous detection of multiple miRNAs contributes to the accuracy and reliability of the BC diagnosis. In this work, wrinkled silica nanoparticles (WSNs) were applied as the microreactor for multiplex miRNAs analysis without enzymes or nucleic acid amplification. Conjugated on the surface of WSNs, the S9.6 antibody was adopted as the universal module for binding DNA/miRNA duplexes, regardless of their sequence. Furthermore, single-stranded DNA (ssDNA) was labeled with quantum dots (QDs) for identifying a given miRNA to form QDs-ssDNA/miRNA, which enabled the specific capture of the corresponding QDs on the wrinkled surface of WSNs. Based on the detection of fluorescence signals that were ultimately focused on WSNs, target miRNAs could be sensitively identified to a femtomolar level (5 fM) with a wide dynamic range of up to 6 orders of magnitude. The proposed strategy achieved high specificity to obviously distinguish single-base mutation sequences and possessed multiplex assay capability. Moreover, the assay exhibited excellent practicability in the multiplex detection of miRNAs in clinical serum specimens.
危险品化学品经营许可证（带存储）