Single Cell Quantification of Let-7a with an Electrophoresis-Assisted Tandem Signal Amplification Strategy based on Intracellular Catalytic Hairpin Assembly-Branched Hybridization Chain Reaction-MNAzyme

The microRNA let-7a plays a pivotal role in tumor suppression by regulating RAS and other oncogenes. However, due to its extremely low abundance in individual cells, quantitative analysis of let-7a at the single-cell level remains challenging. In this study, we developed an electrophoresis-assisted intracellular catalytic hairpin assembly branched hybridization chain reaction-MNAzyme tandem signal amplification strategy. By integrating multimodal cascade signal amplification with microchip electrophoresis separation and laser-induced fluorescence detection, we achieved a detection limit of 8 × 10 –16 M, which is sufficient for single-cell quantification of let-7a. The levels of let-7a in cell lysates and in 20 individual cells from three cell lines were quantitatively analyzed with this method on a microfluidic chip electrophoresis platform. The results revealed marked differences in let-7a expression not only among distinct cell types but also between individual cells of the same type. The average copy numbers of let-7a per cell were determined to be 3258 in HL-7702 cells, and 1408 and 200 in MCF-7 and A549 cancer cells, respectively, underscoring the characteristic low expression of let-7a in various tumor cells, likely associated with its tumor-suppressive function. This method provides a powerful tool for single-cell biological studies of let-7a.