复旦大学王璐团队报道了基于荧光罗丹明的监测细胞NADPH动力学的化学生物传感器。相关研究成果于2024年7月22日发表于国际一流学术期刊《美国化学会杂志》。

采用Frster共振能量转移（FRET）的比率生物传感器能够实时跟踪代谢物动力学。

该文中，研究人员介绍了一种生成基于FRET的生物传感器的方法，其中表观FRET效率的变化取决于罗丹明的分析物控制的荧光性，而不是供体-受体荧光团之间常用的距离变化。基于荧光、罗丹明的化学遗传生物传感器（FOCS）依赖于一种合成的、蛋白质连接的FRET探针，其中作为FRET受体的罗丹明以分析物依赖的方式，从黑暗状态切换到荧光状态。从而能够对分析物浓度进行比率传感。

研究人员使用这种方法来生成烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的化学遗传生物传感器。FOCS-NADPH对NAPDH表现出快速可逆的反应，具有良好的动态范围、选择性和pH不敏感性。FOCS-NADPH能够实时监测氧化应激期间或药物暴露后，活细胞中细胞质NADPH的波动。研究人员还使用FOCS-NADPH通过葡萄糖代谢的戊糖磷酸途径，研究了NADPH稳态调节。

FOCS-NADPH是研究NADPH代谢的有力工具，也是未来荧光生物传感器发展的蓝图。

附：英文原文

Title: Fluorogenic Rhodamine-Based Chemigenetic Biosensor for Monitoring Cellular NADPH Dynamics

Author: Huimin Chang, Simon Clemens, Pingting Gao, Quanlin Li, Hanqing Zhao, Lehua Wang, Jingye Zhang, Pinghong Zhou, Kai Johnsson, Lu Wang

Issue&Volume: July 22, 2024

Abstract: Ratiometric biosensors employing Frster Resonance Energy Transfer (FRET) enable the real-time tracking of metabolite dynamics. Here, we introduce an approach for generating a FRET-based biosensor in which changes in apparent FRET efficiency rely on the analyte-controlled fluorogenicity of a rhodamine rather than the commonly used distance change between donor–acceptor fluorophores. Our fluorogenic, rhodamine-based, chemigenetic biosensor (FOCS) relies on a synthetic, protein-tethered FRET probe, in which the rhodamine acting as the FRET acceptor switches in an analyte-dependent manner from a dark to a fluorescent state. This allows ratiometric sensing of the analyte concentration. We use this approach to generate a chemigenetic biosensor for nicotinamide adenine dinucleotide phosphate (NADPH). FOCS-NADPH exhibits a rapid and reversible response toward NAPDH with a good dynamic range, selectivity, and pH insensitivity. FOCS-NADPH allows real-time monitoring of cytosolic NADPH fluctuations in live cells during oxidative stress or after drug exposure. We furthermore used FOCS-NADPH to investigate NADPH homeostasis regulation through the pentose phosphate pathway of glucose metabolism. FOCS-NADPH is a powerful tool for studying NADPH metabolism and serves as a blueprint for the development of future fluorescent biosensors.

DOI: 10.1021/jacs.3c13137

Source: https://pubs.acs.org/doi/abs/10.1021/jacs.3c13137