Metastable DNA hairpins driven isothermal amplification for in situ and intracellular analysis

Highlights: • Summarizing classical and recent studies of intracellular analysis using Mh-IA. • Mh-IA applying in detecting diverse analytes without cell destruction are reviewed. • We compare mainstream delivery strategies for Mh-IA efficient detection. Intracellular substance analysis is critical for understanding cellular physiological mechanisms and predicting disease progression. Isothermal amplification technologies have been raised to accurately detect intracellular substances due to their low abundance, which is significant for the mechanism analysis and clinical application. However, traditional isothermal method still needs to cell destruction and extraction, resulting in fluctuant results. Moreover, it only works on dead cells. Therefore, non-destructive analysis based on isothermal amplification deserves to be studied, which directly reveals the content and position of relevant molecules. In recent years, metastable DNA hairpins-driven isothermal amplification (Mh-IA) blazes a trail for analysis in living cells. This review tracks the recent advances of Mh-IA strategy in living cell detection and highlights the potential challenges regarding this field, aiming to improve in vivo isothermal amplification. Also, challenges and prospects of Mh-IA for in situ and intracellular analysis are considered.