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Updated: Jun 20, 2026
Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Published on: April 14, 2015
Lab on a single microbead: An enzyme-free strategy for the sensitive detection of microRNA via efficient localized
Yan Qi1, Jiangtao Yu1, Ming Lou2
1Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China.
Background:
Accurate quantification of microRNA (miRNA) is of great significance because it provides opportunities for the accurate early diagnosis of a series of human diseases including cancers. Currently, complicated nucleic acid amplification technologies are always required for the highly sensitive miRNA detection. The introduction of nucleic acid signal amplification coupled with various enzymes will inevitably lead to tedious work and increase the complexity of the analysis process. It is still urgently desired to develop enzyme-free yet sensitive assays that enable the sensitive analysis of miRNA in complicated biological samples.
Results:
A single microbead (MB)-based localized catalytic hairpin assembly (CHA) strategy is proposed for the sensitive analysis of microRNA (miRNA). This rationally designed CHA strategy allows target miRNA to walk only on a single MB which can create a micro-amplification zone, initiating a highly efficient localized CHA reaction, generating a large number of fluorescent DNA duplexes on the surface of single MB. The efficient localized CHA on single MB can not only greatly suppress the nonspecific reaction between two hairpin probes, thus decreasing the background signal, but also greatly enhance the brightness of MB owing to the highly-concentrated fluorescence enrichment on only one MB. Therefore, highly sensitive quantification of miRNA has been achieved by measuring the fluorescence signal on MB using a confocal fluorescence microscope. This new strategy exhibits a detection limit of 1.09 pM for let-7a detection, and enables high specificity of distinguishing homologous miRNA family members.
Significance:
This is the first report by only using one single MB as a carrier to conduct localized CHA, rendering highly-concentrated fluorescence enrichment on only one MB and a dramatic increase in sensitivity. This single MB-based localized CHA strategy has been successfully applied to the accurate analysis of miRNA target in complex biological sample.
