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.
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