Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT
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 permute...
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Editorial Office of Journal of Shanghai Jiao Tong University (Medical Science)
2025-05-01
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| Series: | Shanghai Jiaotong Daxue xuebao. Yixue ban |
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| Online Access: | https://xuebao.shsmu.edu.cn/article/2025/1674-8115/1674-8115-2025-45-5-529.shtml |
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| author | XU Mufan ZHANG Kun WANG Jingyi GAO Xinke CHENG Aobing ZHANG Peng |
| author_facet | XU Mufan ZHANG Kun WANG Jingyi GAO Xinke CHENG Aobing ZHANG Peng |
| author_sort | XU Mufan |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-e618a33d67a94c13995f7f5d5f56333e |
| institution | OA Journals |
| issn | 1674-8115 |
| language | zho |
| publishDate | 2025-05-01 |
| publisher | Editorial Office of Journal of Shanghai Jiao Tong University (Medical Science) |
| record_format | Article |
| series | Shanghai Jiaotong Daxue xuebao. Yixue ban |
| spelling | doaj-art-e618a33d67a94c13995f7f5d5f56333e2025-08-20T02:07:27ZzhoEditorial Office of Journal of Shanghai Jiao Tong University (Medical Science)Shanghai Jiaotong Daxue xuebao. Yixue ban1674-81152025-05-0145552953910.3969/j.issn.1674-8115.2025.05.0011674-8115（2025）05-0529-11Optimization of a genetically encoded fluorescent sensor for the detection of 5-HTXU Mufan0ZHANG Kun1WANG Jingyi2GAO Xinke3CHENG Aobing4ZHANG Peng5Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, ChinaDepartment of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, ChinaDepartment of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, ChinaDepartment of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, ChinaDepartment of Anesthesiology, Guangzhou First People′s Hospital, Guangdong Province, Guangzhou 510180, ChinaDepartment of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, ChinaObjective·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.https://xuebao.shsmu.edu.cn/article/2025/1674-8115/1674-8115-2025-45-5-529.shtmlgenetically encoded sensor5-hydroxytryptamine (5-ht)circularly permuted superfolder gfpperiplasmic binding protein |
| spellingShingle | 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 Shanghai Jiaotong Daxue xuebao. Yixue ban genetically encoded sensor 5-hydroxytryptamine (5-ht) circularly permuted superfolder gfp periplasmic binding protein |
| title | Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT |
| title_full | Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT |
| title_fullStr | Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT |
| title_full_unstemmed | Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT |
| title_short | Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT |
| title_sort | optimization of a genetically encoded fluorescent sensor for the detection of 5 ht |
| topic | genetically encoded sensor 5-hydroxytryptamine (5-ht) circularly permuted superfolder gfp periplasmic binding protein |
| url | https://xuebao.shsmu.edu.cn/article/2025/1674-8115/1674-8115-2025-45-5-529.shtml |
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