Ratiometric Afterglow Luminescent Imaging of Matrix Metalloproteinase-2 Activity via an Energy Diversion Process

Ratiometric afterglow luminescent (AGL) probes are attractive for in vivo imaging due to their high sensitivity and signal self-calibration function. However, there are currently few ratiometric AGL probes available for imaging enzymatic activity in living organisms. Here, we present an energy diversion (ED) strategy that enables the design of an enzyme-activated ratiometric AGL probe (RAG-RGD) for in vivo afterglow imaging. The ED process provides RAG-RGD with a radiative transition for an 'always on' 520-nm AGL signal (AGL₅₂₀) and a cascade three-step energy transfer (ET) process for an 'off-on' 710-nm AGL signal (AGL₇₁₀) in response to a specific enzyme. Using matrix metalloproteinase-2 (MMP-2) as an example, RAG-RGD shows a significant ~11-fold increase in AGL₇₁₀/AGL₅₂₀ toward MMP-2. This can sensitively detect U87MG brain tumors through ratiometric afterglow imaging of MMP-2 activity, with a high signal-to-background ratio and deep imaging depth. Furthermore, by utilizing the self-calibration effect of ratiometric imaging, RAG-RGD demonstrated a strong negative correlation between the AGL₇₁₀/AGL₅₂₀ value and the size of orthotopic U87MG tumor, enabling accurate monitoring of orthotopic glioma growth in vivo. This ED process may be applied for the design of other enzyme-activated ratiometric afterglow probes for sensitive afterglow imaging.