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), provided 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, collaborated 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 on July 14th. 

In this study, a novel 2D MOF nanosensor was integrated into a droplet microfluidic flow cytometer (Nano-DMFC), achieving in situ, multiplex, and quantitative analysis of miRNAs in single CTC living cells with high throughput. The 2D MOF-based fluorescent resonance energy transfer (FRET) nanosensors were 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 were precisely microinjected into each single-cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano-DMFC platform successfully detected dual miRNAs at single-cell resolution in 10 mixed positive MCF-7 cells out of 10000 negative epithelial cells in serum biomimic samples.  

"This Nano-DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples," said Prof. CHEN Yan, "which demonstrated 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. 

Figure. A) The scheme for the fabrication of MOF-PEG-peps nanocomposite-based nanosensor for simultaneous detection of miR-21 and miR-10a. B) The scheme of Nano-DMFC with sample processing unit and miRNA detection unit to realize the encapsulation of single cell in droplet, microinjection of nanosensors, and fluorescence detection of multi-miRNA in single CTC cell. (Figure by Prof. CHEN Yan)