Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT

doi:10.3969/j.issn.1674-8115.2025.05.001

Journal of Shanghai Jiao Tong University (Medical Science) ›› 2025, Vol. 45 ›› Issue (5): 529-539.doi: 10.3969/j.issn.1674-8115.2025.05.001

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Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT

XU Mufan¹, ZHANG Kun¹, WANG Jingyi¹, GAO Xinke¹, CHENG Aobing²(), ZHANG Peng¹()

^1.Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.Department of Anesthesiology, Guangzhou First People′s Hospital, Guangdong Province, Guangzhou 510180, China

Received:2024-11-06 Accepted:2024-12-31 Online:2025-05-28 Published:2025-05-28
Contact: CHENG Aobing, ZHANG Peng E-mail:eychengaobing@scut.edu.cn;benzhp@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(82102184);Shanghai High-level Local University Construction Project(PT21011);Shanghai 2023 “Science and Technology Innovation Action Plan” Natural Science Foundation Project(23ZR1436800);NATCM′s Project of High-level Construction of Key TCM Disciplines(ZYYZDXK-2023070);Alzheimer′s Association Research Fellowship(AARF-19-619387);Shanghai High-Level Local Universities Construction Project 2024 Integrated Traditional Chinese and Western Medicine Research Platform′ Construction Project(ZXY24006)

Abstract

Abstract:

Objective ·To optimize iSeroSnFR, a genetically encoded 5-hydroxytryptamine (5-HT) fluorescent sensor based on bacterial periplasmic binding proteins (PBPs), to enhance its performance for both in vivo and in vitro 5-HT detection. Methods ·iSeroSnFR1.2 was engineered by replacing the circularly permuted superfolder green fluorescence protein (cpsfGFP) sequence in iSeroSnFR1.0 with that from the acetylcholine sensor iAChSnFR using Gibson assembly. The fluorescence response and kinetic properties of iSeroSnFR1.0 and iSeroSnFR1.2 were compared by overexpressing the sensors in HEK293 cells and puffing with exogenous 5-HT. Additionally, to mimic physiological conditions, cultured mouse cortical neurons infected with Sindbis virus carrying each sensor were electrically stimulated to induce endogenous 5-HT release and further evaluate sensor performance. Results ·iSeroSnFR1.2 showed significantly improved performance over iSeroSnFR1.0. In HEK293 cells, it exhibited a 1.5-fold increase in fluorescence response (ΔF/F₀) to exogenous 5-HT, along with faster kinetics (rise time: 36.3 ms vs 44.9 ms; decay time: 1 003.6 ms vs 1 730.4 ms). In cortical neurons, it demonstrated a 2.7-fold increase in response to endogenously released 5-HT, with rise and decay times reduced by 44.0% and 26.7%, respectively. Notably, iSeroSnFR1.2 showed increased basal fluorescence, enabling better imaging in high-background environments. Conclusion ·The optimized iSeroSnFR1.2 sensor offers a markedly improved fluorescent response and temporal resolution for 5-HT detection, providing an advanced tool for studying 5-HT dynamics in neuroscience and psychiatric research.

Key words: genetically encoded sensor, 5-hydroxytryptamine (5-HT), circularly permuted superfolder GFP, periplasmic binding protein

CLC Number:

R915

XU Mufan, ZHANG Kun, WANG Jingyi, GAO Xinke, CHENG Aobing, ZHANG Peng. Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(5): 529-539.

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Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT

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