A general method to develop excitation ratiometric neuropeptide sensors with enhanced sensitivity for in vivo applications
- 1Peking University School of Life Sciences
Abstract
Neuropeptides regulate a wide range of physiological processes throughout the nervous and endocrine systems, yet their spatiotemporal dynamics in vivo remain poorly understood. While recently developed intensiometric sensors can detect neuropeptides, their use in vivo is often confounded by artifacts induced by hemodynamic changes, pH fluctuations, and motion. Here, we present a general strategy for engineering dual-excitation ratiometric (Ex-ratiometric) neuropeptide sensors by tuning the excited-state proton transfer properties of the fluorescent reporter. Dual excitation at 405 nm and 488 nm produces a ratiometric signal that intrinsically corrects fluorescence fluctuations unrelated to ligand binding. Using an AlphaFold3-guided in silico design approach combined with experimental validation, we developed a suite of Ex-ratiometric sensors. As a representative example, Ex-NTS2.0 enables robust detection of neurotensin (NTS) in vivo while remaining largely resistant to hemodynamics, pH, and motion-induced artifacts. Overall, these findings establish a scalable and mechanistically grounded platform for developing new Ex-ratiometric tools and provide a broadly applicable strategy for sensitive detection of neuropeptide dynamics in living systems.
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BibTeX
@misc{wang2026general,
title = {{A general method to develop excitation ratiometric neuropeptide sensors with enhanced sensitivity for in vivo applications}},
author = {H. Wang and Y. Yan and Y. Zhao and D. Wang and C. Huang and Y. Li},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.17.752296},
url = {https://www.biorxiv.org/content/10.64898/2026.09.17.752296v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A general method to develop excitation ratiometric neuropeptide sensors with enhanced sensitivity for in vivo applications
AU - H. Wang
AU - Y. Yan
AU - Y. Zhao
AU - D. Wang
AU - C. Huang
AU - Y. Li
PY - 2026
DA - 2026/09/17
PB - bioRxiv
DO - 10.64898/2026.09.17.752296
UR - https://www.biorxiv.org/content/10.64898/2026.09.17.752296v1
AB - Neuropeptides regulate a wide range of physiological processes throughout the nervous and endocrine systems, yet their spatiotemporal dynamics in vivo remain poorly understood. While recently developed intensiometric sensors can detect neuropeptides, their use in vivo is often confounded by artifacts induced by hemodynamic changes, pH fluctuations, and motion. Here, we present a general strategy for engineering dual-excitation ratiometric (Ex-ratiometric) neuropeptide sensors by tuning the excited-state proton transfer properties of the fluorescent reporter. Dual excitation at 405 nm and 488 nm produces a ratiometric signal that intrinsically corrects fluorescence fluctuations unrelated to ligand binding. Using an AlphaFold3-guided in silico design approach combined with experimental validation, we developed a suite of Ex-ratiometric sensors. As a representative example, Ex-NTS2.0 enables robust detection of neurotensin (NTS) in vivo while remaining largely resistant to hemodynamics, pH, and motion-induced artifacts. Overall, these findings establish a scalable and mechanistically grounded platform for developing new Ex-ratiometric tools and provide a broadly applicable strategy for sensitive detection of neuropeptide dynamics in living systems.
ER -