文献类型：期刊文献

英文题名：Simultaneous quantification of intracellular pH and lactate at the single-cell level using a genetically encoded fluorescence lifetime biosensor

作者：Zhou, Huangmei[1];Zou, Yejun[2,3,4];Song, Yike[2];Zhang, Zhuo[2,4];Chen, Kai[1];Deng, Lunhua[1];Li, Xie[2,4];Zhang, Sanjun[1,5,6];Zhao, Yuzheng[2,4,7]

机构：[1]East China Normal Univ, State Key Lab Precis Spect, 500 Dongchuan Rd, Shanghai 200241, Peoples R China;[2]East China Univ Sci & Technol, Optogenet & Synthet Biol Interdisciplinary Res Ctr, Shanghai Frontiers Sci Ctr Optogenet Tech Cell Met, Sch Pharm,State Key Lab Bioreactor Engn, 130 Mei Long Rd, Shanghai 200237, Peoples R China;[3]Tongji Univ, Shanghai Matern & Infant Hosp 1, Shanghai Inst Maternal Fetal Med & Gynecol Oncol, Shanghai Key Lab Maternal Fetal Med,Sch Med, Shanghai 200092, Peoples R China;[4]Chinese Acad Med Sci, Res Unit New Tech Live Cell Metab Imaging, Beijing 100730, Peoples R China;[5]Shanxi Univ, Collaborat Innovat Ctr Extreme Opt, Taiyuan 030006, Shanxi, Peoples R China;[6]NYU Shanghai, NYU ECNU Inst Phys, 3663 North Zhongshan Rd, Shanghai 200062, Peoples R China;[7]Shanghai Inst Stem Cell Res & Clin Translat, Shanghai 200120, Peoples R China

年份：2025

卷号：441

外文期刊名：SENSORS AND ACTUATORS B-CHEMICAL

收录：;EI(收录号：20252118452780);WOS:【SCI-EXPANDED(收录号:WOS:001498916600003)】；

基金：This work was supported by National Key Research and Development Program of China (2024YFA0917700 to Y. Zhao, 2023YFA1802000 and 2024YFA1803500 to X.L., 2023YFC3404800 and 2025YFC3408600 to Y. Zou, 2024YFC3406403 to S.Z.) , National Natural Science Foundation of China (12174114 to S.Z., 32030065 and 92457301 to Y. Zhao, 32471361 and 32394001 to X.L., 32201230 to Y. Zou) , Shanghai Science and Technology Innovation Program (22520712500 to S.Z., 24HC2810700 to Y. Zhao) , Natural Science Foundation of Shanghai (23ZR1415800 to X.L.) , the Fundamental Research Funds for the Central Universities (to S.Z., Y. Zhao and X.L.) , the Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism (Y. Zhao) , Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai, and we also thank Dr. Weikang Wang of the Materials Characterization Center of East China Normal University Multifunctional Platform for Innovation for his helpful discussions and assistances in live cell imaging.

语种：英文

外文关键词：Genetically encoded fluorescent biosensors; Fluorescent proteins; Fluorescence lifetime imaging; Lactate metabolism; pH homeostasis

摘要：Metabolism plays an essential role in supporting physiological functions and maintaining a stable intracellular environment, including pH homeostasis. It is of biological significance to dynamically track metabolites and pH simultaneously in living cells. Genetically encoded fluorescent protein biosensors are extensively used to monitor metabolites; however, their fluorescence intensity-the commonly used readout-is often responsive to pH fluctuations, rendering analyte quantification challenging. In this study, we report a novel method that involves the steady-state fluorescence, time-resolved fluorescence and occupancy of the biosensor, called biosensor occupancy-enabled absolute quantification (BOEAQ) method, for simultaneous measurement of pH and analyte concentrations. We first demonstrate that FiLa (a recently reported lactate biosensor) can serve as a high performance fluorescence lifetime biosensor for lactate with a large intensity-weighted lifetime response (similar to 1.1 ns and similar to 1.2 ns at 405 nm and 470 nm excitation, respectively). In a proof-of-principle, we simultaneously quantify pH and lactate concentration with our BOEAQ method both in vitro and in living cells at the single-cell resolution. In principle, the BOEAQ methodology holds promise for applications to other metabolite biosensors, thereby expanding the possibilities for multiplexed imaging.