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  • In Situ Detection of Multiple miRNAs in Single CTC Cells

    In liquid biopsy studies, circulating tumor cells (CTCs) detection strategies based on surface epithelial markers are widely used. However, they suffer from low specificity in distinguishing between CTCs and epithelial cells in hematopoietic cell population. Tumor-associated miRNAs within CTCs are emerging as new biomarkers due to their high correlation with tumor development and progress. Currently, it is still very challenging to perform in-situ analysis of multiple miRNAs of single CTCs in living cells. In this work, the novel two-dimensional nanomaterial, metal organic framework (MOF), provides new ideas for live cell probes due to its controllable structure and diverse functions.

    Recently, a research team led by Prof. CHEN Yan from Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, collaborating with Prof. TAN Ying from Tsinghua Shenzhen International Graduate School and Prof. MO Yang from Hong Kong Polytechnic University, developed a novel digital droplet microfluidic flow cytometry technique for in-situ analysis of multiple miRNAs in Single CTC Cells. The study was published in Small online on July 14th (Small 2022, 2201779).

    In this study, a novel 2D MOF nanosensor was integrated into a droplet microfluidic flow cytometer (Nano-DMFC), achieving in situ, multiplex, quantitative analysis of miRNAs in single CTC living cells with high throughput. The 2D MOF-based fluorescent resonance energy transfer (FRET) nanosensors are established by conjugating dual-color fluorescence dye-labeled DNA probes on MOF nanosheet surface. Two breast cancer targeting peptide sequences were used to modify the nanosensors to increase tumor cell targeting and endosomal escape capabilities. The Nano-DMFC enables in situ detection of dual miRNA markers (miRNA-21 and miRNA-10a) in individual breast cancer cells.

    The Nano-DMFC consists of three components: a single-cell droplet generator, a nanoprobe microinjection unit, and a droplet fluorescence detection unit. In the Nano-DMFC, 2D MOF-based nanoprobes are precisely microinjected into each single-cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano-DMFC platform successfully detects dual miRNAs at single-cell resolution in 10 mixed positive MCF-7 cells out of 10000 negative epithelial cells in serum biomimic samples. Moreover, this Nano-DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples, which demonstrate the high potential for CTC-based cancer early diagnosis and prognosis. The Nano-DMFC platform successfully demonstrated a new strategy for CTC detection using miRNAs as biomarkers. This miniaturized, highly integrated and easy-to-operate platform for miRNA analysis in live cells may provide a useful tool for clinical research.

  • Precise isolation and molecular analysis of CTCs and fusion cells

    A century ago, German pathologist Otto Aichel discovered that tumor cells could fuse with immune cells, forming fusion cells with both immune cell motility and tumorigenic ability of tumor cells, which are more likely to spread and metastasize through the circulatory system. Due to the limitation of currently available approaches, such fusion cells are difficult to be isolated and analyzed thoroughly.

    A new study published in Lab on a Chip on Jun 28th proposed a novel hydrodynamic structure to achieve one-step and label-free isolation of circulating tumor cells (CTCs) and fusion cells (CFCs) from whole blood. The integrated microfluidic platform isolated rare CTCs and CFCs with high purity and high cell viability, enabling direct downstream analysis with single-cell RNA sequencing. The study was led by Dr. CHEN Yan's group from Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences.

    In this study, researchers proposed a filter deterministic lateral displacement (filter-DLD) concept to achieve one-step and label-free CTCs and CFCs isolation. The novel hydrodynamic structure is designed and simulated by multiphysics finite element analysis, which enables precise manipulation of cell motion. The filter-DLD structure not only has a lower critical cell separation size than conventional DLD designs, but also achieves a higher depletion rate of smaller red blood cells, which make up the largest proportion of blood. By combining the filter-DLD concept and the cascaded chip design, researchers fully explored the tremendous potential of rare cell sorting based on physical properties. The integrated microfluidic platform demonstrated excellent performance for size-based cell separation, and achieved high separation efficiency (>96%), high cell purity (WBC removal rate 99.995%), high cell viability (>98%) and high processing rate (1mL/min).

    Using this platform, researchers analyzed samples from non-small cell lung cancer patients. CTCs and tumor cell-leukocyte fusion cells (CFCs) were efficiently collected, and changes in CTCs levels were used for treatment response monitoring. Due to the extremely high viability of the enriched cells, downstream analysis can be performed directly with high-throughput single-cell RNA sequencing to study cancer driver genes and heterogeneity. The results showed that there were more CFCs than CTCs in the peripheral blood of cancer patients. CFCs are expected to reveal new mechanisms of tumor evolution and metastasis, and provide a novel marker for early diagnosis, prognosis and monitoring of tumors.

    In summary, the novel label-free rare cell isolation strategy represents a powerful tool for liquid biopsy, and offers great promise for cancer diagnostics and therapeutics.

  • A compact fiber-integrated optofluidic platform for highly specific microRNA detetion

    MicroRNAs (miRNAs) have attracted extensive interest as promising biomarkers for the profiling of diseases. However, quantitative measurement of miRNAs presents a significant challenge in biochemical studies. In this work, we developed an innovative optofluidic platform to perform rapid, simple, quantitative and high-specificity miRNA assay using Förster resonance energy transfer (FRET) principle. A novel three-way junction FRET probe was proposed to enable rapid and enzyme-free miRNA detection. Using this platform, we performed one-step, amplification-free miRNA detection with simple device operation and achieved miRNA identification at a low concentration. The detection system could achieve high specificity of discrimination of three-base mismatches, and the sample volume was significantly reduced, favorable for low-level miRNA detection in material-limited samples. The establishment of a compact, low-cost, highly sensitive and selective miRNA analysis platform provides a valuable tool for point-of-care diagnosis.

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