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Exosomes are emerging as one of the most promising biomarkers for early disease diagnosis and prognosis. The significant challenges facing the available methods include improving the detection specificity and sensitivity in complex biological samples. Herein, a fluorescence assay was established based on a combination of immunomagnetic separation and a two-step signal amplification strategy for direct isolation and subsequent detection of exosomes. First, immunomagnetic beads capture and enrich the exosomes via antibody-antigen reactions. Second, bivalent cholesterol (BC) anchors spontaneously insert into the lipid bilayer of bead-captured exosomes, forming a "one to many" amplification effect. The simultaneous recognition of the surface protein and the lipid bilayer structure of the exosome significantly eliminates the interference risk from free proteins. The detection of exosomes converts to the detection of BC-anchors. Finally, the sticky end of the BC-anchor acts as the initiator to trigger the enzyme-free DNA circuits for secondary signal amplification. Under the optimal conditions, highly sensitive and selective detection of exosomes was achieved ranging from 5.5 × 103 to 1.1 × 107 particles/μL with a limit of detection of 1.29 × 103 particles/μL. Moreover, this method allows the isolation and quantitative analysis of exosomes in several biological fluids with satisfactory recovery rates (92.25-106.8%). Thus, this approach provides a sensitive, anti-interference platform for isolating and detecting exosomes.

MicroRNAs (miRNAs) are promising targets for disease diagnosis. However, miRNA detection requires rapid, sensitive, and selective detection to be effective as a diagnostic tool. Herein, a miRNA-initiated exponential strand-displacement amplification (SDA) assay was reported. With the Klenow fragment, nicking enzyme Nt.AlwI, and two primers, the miRNA target can trigger two cycles of nicking, polymerization, and displacement reactions. These reaction cycles amplified the target miRNA exponentially and generated dsDNAs detectable with SYBR Green I in real-time PCR. As low as 16 zmol of the target miRNA was detected by this one-pot assay within 90 min, and the dynamic range spanned over 9 orders of magnitude. Negligible impact from the complex biological matrix was observed on the amplification reaction, indicating the assay's capability to directly detect miRNAs in biofluids. © 2013 American Chemical Society.