An extracellular vesicle microRNA–initiated 3D DNAzyme motor for colorectal cancer diagnosis

MicroRNA is regarded as a significant biomarker for cancer diagnosis, disease process evaluation and therapeutic guidance, and dual–parameter measurement may contribute to a more accurate and realistic assessment. To meet the urgent need for simultaneous detection of multiple biomarkers, we combined three–dimensional DNAzyme motors with single molecule imaging technique to construct a convenient, intuitive, and sensitive approach for the simultaneous detection of dual miRNAs in free state or in extracellular vesicles. The quantification of target miRNAs can be realized through the detection of amplified fluorescence signals generated by the target miRNA–initiated cleavage of fluorescent substrate strands by DNAzyme motors. The practicability was systematically validated with microRNA–21–5p and microRNA–10b–5p as targets, acquiring a satisfactory sensitivity sufficient to detect low abundance targets of 0.5 or 1 pM to 100 pM. Besides, the extracellular vesicular miRNAs can be conveniently detected without extraction. The clinical applicability was verified with a parallel of extracellular vesicles from clinical samples, which exhibited good distinguish ability between colorectal cancer patients and healthy donors. In addition to the advantages of good specificity and high sensitivity, the system is potential to be easily adapted by minor alternation of the DNA sequences and fluorophore sets for multiple detection of various miRNAs and even other types of biomarkers such as proteins. Therefore, it is promising to be widely applicated in various fields such as early diagnosis of cancer and its prognostic assessment.