ADC Reviews

Research progress of CTC single-cell miRNA in situ multiplex detection technology

CTC

In liquid biopsy studies, circulating tumor cell (CTC) detection strategies based on surface epithelial markers (EpCAM and CK) are widely used, but there are limitations. Studies have shown that tumor-associated miRNAs in CTCs are highly correlated with the occurrence and development of cancer, and have the potential to serve as markers for tumor characterization and identification. At present, high-throughput in situ analysis of single CTCs at the living cell level and further simultaneous analysis of multiple miRNAs are challenging tasks. However, new two-dimensional nanomaterials, metal-organic frameworks (MOFs), have provided researchers with new ideas for living cell probe carriers due to their controllable structure and diverse functions.​​

Recently, Chen Yan’s team from Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, together with Tan Ying’s team from Tsinghua University Shenzhen International Graduate School and Yang Mo’s team from Hong Kong Polytechnic University, proposed a new 2D MOF nanosensor-integrated droplet microfluidic flow cytometer ( Nano-DMFC), which can be applied to in situ multiplex detection of CTC single-cell miRNA. The related research results were published in Small with the title of 2D MOF Nanosensor-Integrated Digital Droplet Microfluidic Flow Cytometry for In Situ Detection of Multiple miRNAs in Single CTC Cells.​​

In this study, a novel 2D MOF nanosensor-integrated droplet microfluidic flow cytometer (Nano-DMFC) was developed, which broke through the technical bottleneck of in situ analysis of nucleic acids in living cells, and realized high-throughput in-situ analysis of nucleic acids in samples. In situ, multiplex, and quantitative analysis of miRNAs in single CTC live cells. In this nanosensing scheme, metal-organic framework MOFs are used as quenchers and DNA probes labeled with dual-color fluorescent dyes are used as donors, and biofunctionalized MOF fluorescence resonance energy transfer (FRET) nanoprobes for dual miRNAs detection are synthesized for the first time. The 2D MOF nanosensor was modified with two breast cancer-targeting polypeptide sequences to increase tumor cell targeting and endosomal escape capabilities. A digital droplet microfluidic flow cytometer integrating 2D MOF nanosensors enables in situ detection of dual miRNA markers (miRNA-21 and miRNA-10a) in single breast cancer cells.​​

The nanosensor-integrated droplet microfluidic flow cytometer consists of three parts—a single-cell droplet generator, a nanoprobe microinjection unit, and a droplet fluorescence detection unit. The Nano-DMFC system first generates single-cell droplets, and then the 2D MOF nanosensors are precisely microinjected into each single-cell droplet, enabling dual miRNA characterization in single tumor cells at the live-cell level. In the presence of the target miRNA within a single tumor cell, the dye-labeled ssDNA on the MOF nanosheet forms a hybrid double-stranded DNA (dsDNA) with its target, and the interaction between dsDNA and MOF is weakened, allowing the dsDNA to separate from the MOF surface, ultimately triggering the fluorescence recover. Different types of miRNAs produce different fluorescent signals in a single cell. Finally, a fiber-optic-integrated droplet flow detection device was used to detect and analyze the signals in the droplets after nanoprobe incubation, thereby realizing the detection of dual miRNAs in single cells. The experimental results showed that the Nano-DMFC platform was able to detect 10 positive CTC cells in a biomimetic sample (containing 10,000 negative epithelial cells) targeting dual miRNAs, and showed good reproducibility in the recovery experiment of spiked blood samples. . This platform validates a new strategy for CTC detection using miRNA as a marker. As a miniaturized, highly integrated, and easy-to-operate live cell miRNA analysis platform, the Nano-DMFC system provides a new idea for exploring tumor cell heterogeneity and identifying cell subtypes, and has early cancer diagnosis and postoperative monitoring in clinical research. potential.