A general method to develop excitation ratiometric neuropeptide sensors with enhanced sensitivity for in vivo applications

Neuropeptides play a crucial role in various physiological processes across the nervous and endocrine systems, yet their real-time dynamics in living organisms are not well understood. Recent intensiometric sensors have shown promise in detecting neuropeptides, but their application in vivo is often hindered by artifacts caused by hemodynamic changes, pH fluctuations, and motion.

In this study, researchers introduce a novel method for creating dual-excitation ratiometric (Ex-ratiometric) neuropeptide sensors. By manipulating the excited-state proton transfer properties of the fluorescent reporter, the researchers were able to develop a ratiometric signal that inherently corrects for fluorescence fluctuations unrelated to ligand binding. This correction eliminates the need for external compensation for artifacts.

The researchers employed an AlphaFold3-guided in silico design approach, followed by experimental validation, to create a range of Ex-ratiometric sensors. As a demonstration of their effectiveness, they developed a representative sensor called Ex-NTS2.0. This sensor is capable of accurately detecting neurotensin (NTS) in vivo while showing remarkable resistance to artifacts induced by hemodynamics, pH changes, and motion.

The findings presented in this research pave the way for the development of new Ex-ratiometric tools, offering a scalable and mechanistically grounded platform for studying neuropeptide dynamics in living systems. This strategy has the potential to provide more accurate and reliable measurements of neuropeptide levels, thereby advancing our understanding of their roles in various physiological processes.