Selective In Situ Analysis of Mature microRNAs in Extracellular Vesicles Using a DNA Cage-Based Thermophoretic Assay.

Mature microRNAs (miRNAs) in extracellular vesicles (EVs) are involved in different stages of cancer progression, yet it remains challenging to precisely detect mature miRNAs in EVs due to the presence of interfering RNAs (such as longer precursor miRNAs, pre-miRNAs) and the low abundance of tumor-associated miRNAs. By leveraging the size-selective ability of DNA cages and polyethylene glycol (PEG)-enhanced thermophoretic accumulation of EVs, we devised a DNA cage-based thermophoretic assay for highly sensitive, selective, and in situ detection of mature miRNAs in EVs with a low limit of detection (LoD) of 2.05 fM. Our assay can profile EV mature miRNAs directly in serum samples without the interference of pre-miRNAs and the need for ultracentrifugation. A clinical study showed that EV miR-21 or miR-155 had an overall accuracy of 90 % for discrimination between breast cancer patients and healthy donors, which outperformed conventional molecular probes detecting both mature miRNAs and pre-miRNAs. We envision that our assay can advance EV miRNA-based diagnosis of cancer.

Cascaded microfluidic circuits for pulsatile filtration of extracellular vesicles from whole blood for early cancer diagnosis.

Tumor-derived extracellular vesicles (EVs) hold the potential to substantially improve noninvasive early diagnosis of cancer. However, analysis of nanosized EVs in blood samples has been hampered by lack of effective, rapid, and standardized methods for isolating and detecting EVs. To address this difficulty, here we use the electric-hydraulic analogy to design cascaded microfluidic circuits for pulsatile filtration of EVs via integration of a cell-removal circuit and an EV-isolation circuit. The microfluidic device is solely driven by a pneumatic clock pulse generator, allowing for preprogrammed, clog-free, gentle, high-yield, and high-purity isolation of EVs directly from blood within 30 minutes. We demonstrate its clinical utility by detecting protein markers of isolated EVs from patient blood using a polyethylene glycol-enhanced thermophoretic aptasensor, with 91% accuracy for diagnosis of early-stage breast cancer. The cascaded microfluidic circuits can have broad applications in the field of EV research.

Poly(ethylene oxide) Concentration Gradient-Based Microfluidic Isolation of Circulating Tumor Cells.

Circulating tumor cells (CTCs) have emerged as promising circulating biomarkers for non-invasive cancer diagnosis and management. Isolation and detection of CTCs in clinical samples are challenging due to the extreme rarity and high heterogeneity of CTCs. Here, we describe a poly(ethylene oxide) (PEO) concentration gradient-based microfluidic method for rapid, label-free, highly efficient isolation of CTCs directly from whole blood samples. Stable concentration gradients of PEO were formed within the microchannel by co-injecting the side fluid (blood sample spiked with 0.025% PEO) and center fluid (0.075% PEO solution). The competition between the elastic lift force and the inertial lift force enabled size-based separation of large CTCs and small blood cells based on their distinct migration patterns. The microfluidic device could process 1 mL of blood sample in 30 min, with a separation efficiency of >90% and an enrichment ratio of >700 for tumor cells. The isolated CTCs from blood samples were enumerated by immunofluorescence staining, allowing for discrimination of breast cancer patients from healthy donors with an accuracy of 84.2%. The concentration gradient-based microfluidic separation provides a powerful tool for label-free isolation of CTCs for a wide range of clinical applications.

One-Step Thermophoretic AND Gate Operation on Extracellular Vesicles Improves Diagnosis of Prostate Cancer.

Circulating extracellular vesicles (EVs) have emerged as a valuable source of cancer biomarkers. However, the high degree of EV heterogeneity and the complexity of clinical samples pose a challenge in the sensitive identification of tumor-derived EVs. Here we introduce a one-step thermophoretic AND gate operation (Tango) assay that integrates polyethylene glycol (PEG)-enhanced thermophoretic accumulation of EVs and simultaneous AND gate operation on EV membranes by dual-aptamers recognition. By using the Tango assay to detect tumor-derived EVs with co-presence of EpCAM and PSMA directly from serum in a homogeneous, separation-free format, we can discriminate prostate cancer (PCa) patients from benign prostatic hyperplasia (BPH) patients in the diagnostic gray zone with an accuracy of 91 % in 15 min. Our approach streamlines EV enrichment and AND gate operation on EVs in a single assay, providing a rapid, straightforward, and powerful method for precise and non-invasive diagnosis of cancer.