Single Cell Fluorescence In Situ Hybridization In Microfluidic Droplets

Nuanced variations at the molecular, cellular and physiological level among individuals require designing personalized treatments, which are tailored to their unique characteristics. Cellular heterogeneity and the need for personalized therapeutic interventions provide a strong rationale to perform analysis at single-cell level. Emerging technologies, including genome sequencing, proteomics, and imaging protocols are useful techniques to characterize molecular information at single-cell scale. However, some of these techniques such as qRT-PCR and RNA sequencing rely on the extraction of genetic material and do not enable retention of the intracellular spatial resolution of the transcriptome. To preserve spatial information, in situ hybridization methods such as single-molecule fluorescence in situ hybridization (smFISH), which are powerful tools in detecting individual transcripts, have been developed. However, current smFISH methods have certain limitations including, being time-consuming and costly, requiring a high level of expertise, and being constrained to fixed cells and tissues. Hence, the development of automated, affordable single-cell tools for analysis of non-adherent cells is needed to reveal new insights into tumor heterogeneity and therapeutic resistance, elucidate mechanisms of immune response and immunotherapy, and enhance personalized treatment. 

Researchers at Northeastern developed an on-chip single-molecule fluorescence in situ hybridization microfluidic droplet platform, which performs both in adherent and non-adherent cells. This platform uses a dual-array or multiple-array device capable of generating aqueous microdroplets containing cells within a biocompatible matrix. Moreover, using integrated droplet generation and droplet merging approach in this platform enable simultaneous detection of transcriptome and proteome. Each microdroplet contains one or more cells that are incubated by fluorescent-labeled probes, such as oligonucleotides and antibodies, and then visualized through fluorescent imaging microscopy. This platform is capable of quantitative analysis of gene expression or protein biomarkers in at least 1000 microdroplets (i.e. 1000 groups of two or more interacting cells) within less than 10 hours. Thus, the invention allows assessment of cell activity and the correlation of cell activity with nucleic acid expression and/or protein expression in a rapid manner and at single-cell resolution.

 

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