Space‐Confined Amplification for In Situ Imaging of Single Nucleic Acid and Single Pathogen on Biological Samples

Abstract Direct in situ imaging of nucleic acids on biological samples is advantageous for rapid analysis without DNA extraction. However, traditional nucleic acid amplification in aqueous solutions tends to lose spatial information because of the high mobility of molecules. Similar to a cellular matrix, hydrogels with biomimetic 3D nanoconfined spaces can limit the free diffusion of nucleic acids, thereby allowing for ultrafast in situ enzymatic reactions. In this study, hydrogel‐based in situ space‐confined interfacial amplification (iSCIA) is developed for direct imaging of single nucleic acid and single pathogen on biological samples without formaldehyde fixation. With a polyethylene glycol hydrogel coating, nucleic acids on the sample are nanoconfined with restricted movement, while in situ amplification can be successfully performed. As a result, the nucleic acids are lighted‐up on the large‐scale surface in 20 min, with a detection limit as low as 1 copy/10 cm 2 . Multiplex imaging with a deep learning model is also established to automatically analyze multiple targets. Furthermore, the iSCIA imaging of pathogens on plant leaves and food is successfully used to monitor plant health and food safety. The proposed technique, a rapid and flexible system for in situ imaging, has great potential for food, environmental, and clinical applications.