ABSTRACT
A novel method of catalytic hairpin assembly (CHA) induced dual signal enhancement is developed for rapid detection of miRNA based on fluorescence light-up silver nanocluster (Ag NC). By the hybridization of a hairpin DNA and a single-stranded DNA, a unique probe is firstly designed. In the terminals of this probe, DNA-Ag NCs can be formed and display very weak fluorescence. In the presence of the target miRNA, the reaction of CHA can be triggered, forming two kinds of double-stranded complexes, in which the terminal DNA-Ag NCs are in close proximity to G-rich overhangs and the fluorescent signal can be dramatically enhanced. Compared with many other enzyme-based amplification strategies, this one exhibits distinct advantages of simplicity in experimental operation and a rapid detection process (within 1â¯h). Moreover, this assay exhibits an excellent selectivity and is successfully applied in the detection of miRNAs in complex biological media, which confirms the reliability and practicality of this protocol.
Subject(s)
DNA Probes/chemistry , DNA/chemistry , Fluorescence , Light , Metal Nanoparticles/chemistry , MicroRNAs/analysis , Silver/chemistry , Catalysis , DNA Probes/chemical synthesis , Humans , Spectrometry, Fluorescence , Tumor Cells, CulturedABSTRACT
Telomerase has been regarded as a biomarker for cancer diagnosis as well as the clinical treatment and the reliable detection of intracellular telomerase activity is of great significance. By developing a telomere elongation-based DNA-catalytic amplification strategy, a novel surface-enhanced Raman scattering (SERS) method is proposed for the assay of telomerase activity. In the presence of telomerase and nucleotide mixture dNTPs, the telomerase substrate (TS) primer extended and generated a long single-strand DNA (ssDNA) containing the telomere repeat units (TTAGGG)n, which could catalyze the entropy-driven circuit reaction (EDCR). One of the products of EDCR was ingeniously used as the catalyst of catalytic hairpin assembly (CHA) occured on magnetic beads (MBs). As a result, a large amount of ROX-labeled Raman probes could be anchored on the surface of MBs and used for SERS detection. Using this strategy, the assay can detect telomerase activity from cell extracts equivalent down to single HeLa cell.
Subject(s)
Biosensing Techniques/methods , DNA, Catalytic/metabolism , Spectrum Analysis, Raman/methods , Telomerase/metabolism , Cell Line, Tumor , Drug Evaluation, Preclinical , Enzyme Assays/methods , Humans , Nucleic Acid Amplification Techniques/methods , Substrate Specificity , Telomerase/antagonists & inhibitors , Telomere/metabolismABSTRACT
A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10(-4)UmL(-1). This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase.
