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Sensitive and Specific Analysis of miRNAs in Single Tumor-Derived Extracellular Vesicles Using CRISPR-Based Nanoflow
Fangzhou Lan1, Aipeng Chen1, Yue Ding2
1School of Pharmacy, State Key Laboratory of Advanced Drug Formulations for Overcoming Delivery Barriers, Fudan University, Shanghai 201203, China.
Analytical Chemistry
|November 26, 2025
Summary
This study introduces a new CRISPR/Cas13a nanoflow cytometry platform for ultrasensitive detection of tumor-derived extracellular vesicle microRNAs. The method shows high accuracy for prostate cancer diagnosis using liquid biopsies.
Area of Science:
- Biotechnology
- Molecular Biology
- Nanotechnology
Background:
- Tumor-derived extracellular vesicle (TEV) microRNAs (miRNAs) are valuable cancer biomarkers.
- Low abundance and sequence homology of miRNAs pose detection challenges.
- Need for ultrasensitive and specific detection methods for TEV miRNAs.
Purpose of the Study:
- To develop a CRISPR/Cas13a-based nanoflow cytometry (nFCM) platform for ultrasensitive, single-particle level detection of TEV miRNAs.
- To integrate a DNA-guided orthogonal membrane fusion strategy for specific TEV capture and detection.
- To evaluate the diagnostic performance of the platform for prostate cancer (PCa) detection.
Main Methods:
- Development of an orthogonal barcode-anchored TEV (Orth-TEV) using aptamers for CD63 and EpCAM markers.
- Preparation of cholesterol-tagged DNA probe-modified liposomes with CRISPR/Cas13a components (Tags-CRISPR/Cas13a@Lipo).
- Utilizing DNA hybridization for specific membrane fusion between Orth-TEV and Tags-CRISPR/Cas13a@Lipo for in situ miRNA detection via nFCM.
Main Results:
- The platform achieved ultrasensitive detection of three PCa-associated miRNAs (miR-153, miR-183, miR-940) in single TEVs with low limits of detection.
- Significantly elevated miRNA signals were observed in TEVs from PCa patients' plasma compared to healthy donors.
- High diagnostic performance was demonstrated with ROC analysis (AUCs up to 0.931) and a PCA-LDA model achieving 91.3% accuracy.
Conclusions:
- The CRISPR/Cas13a-based nFCM platform enables robust and accurate single-vesicle miRNA profiling.
- The orthogonal membrane fusion strategy effectively reduces interference and enhances specificity.
- This platform holds significant potential for liquid biopsy-based cancer diagnosis, particularly for prostate cancer.
