Three-Dimensional In Situ Sensing and Spatiotemporal Imaging Analysis of Dual Targets in Steatotic Liver Disease

Accurate monitoring of steatotic liver disease (SLD) remains challenging, particularly in capturing the spatial distribution of different types of SLD targets within a tissue-like complex physiological matrix. In this study, we developed a novel hydrogel-based three-dimensional (3D) in vitro fatty liver mimic model enabling simultaneous in situ detection and imaging of key SLD biomarkers. This model was constructed by integrating nucleic acid probes into a gelatin methacrylate photo-cross-linkable hydrogel, which served as a biomimetic scaffold for fatty liver cells and their secreted targets. Within this 3D matrix, vaspin binding restored FAM fluorescence via aptamer displacement, while miRNA-122 initiated a hybridization chain reaction to amplify Cy3 signals, enabling dual-target spatiotemporal imaging. The assay achieved simultaneous detection in both solution and the 3D scaffold, with broad linear ranges of 10 pM-1 μM for vaspin and 1 pM-1 μM for miRNA-122, and limits of detection of 4.3 pM and 0.1 pM, respectively. A key novel finding in this study revealed a distinct difference in the spatial diffusion dynamics between protein and miRNA targets within a 3D cellular microenvironment. Our research indicated that vaspin diffused extensively into the periphery of the 3D space, while miRNA-122 remained more localized, which is unobservable in conventional 2D cultures. This platform enables in situ 3D dual-target monitoring in SLD, underscoring the critical importance of early in situ detection across multiple dimensions, including target source, 3D imaging, and information analysis. This integrated system provided a powerful preclinical tool for early SLD monitoring and targeted screening.