Orthometric multicolor encoded hybridization chain reaction amplifiers for multiplexed microRNA profiling in living cells

Multiplexed microRNA (miRNA) profiling of more than four types in living cells is challenging due to fluorescent spectral overlap, representing a significant limitation in studying the complex interactions related to the occurrence and development of diseases. Herein, we report a multiplexed fluorescent imaging strategy based on an orthometric multicolor encoded hybridization chain reaction amplifier named multi-HCR. The targeting miRNA can trigger this multi-HCR strategy due to the specific sequence recognition, and then its self-assembly to amplify the programmability signals. We take the four-colored chain amplifiers, showing that the multi-HCR can form 15 combinations simultaneously. In a living process of hypoxia-induced apoptosis and autophagy under complicated mitochondria and endoplasmic reticulum stress, the multi-HCR demonstrates excellent performance in detecting eight different miRNA changes. The multi-HCR provides a robust strategy for simultaneously profiling multiplexed miRNA biomarkers in studying complicated cellular processes.