Near-infrared Fluorescent Probes for Sensitive Detection of NADH in Live Cells

用于灵敏检测活细胞中 NADH 的近红外荧光探针

基本信息

批准号：
10439008
负责人：
Haiying Liu
金额：
$ 46.95万
依托单位：
MICHIGAN TECHNOLOGICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10439008
关键词：
Amino Acids Anions BODIPY Benzopyrans Calibration Cations Cell physiology Cells Chemical Structure Computing Methodologies Coumarins Detection Dyes Electrons Energy Transfer Equilibrium Fluorescence Fluorescent Probes Glucose Glutathione Glutathione Disulfide Glycolysis Pathway Goals Image Light Measurement Metabolism Methyl Ethers Mitochondria Monitor NADH Near-infrared optical imaging Niacinamide Nitrogen Normal Cell Oligonucleotides Optics Oxidants Oxidation-Reduction Oxidative Phosphorylation Oxygen Pathologic Processes Penetration Permeability Pharmacotherapy Physiological Processes Process Property Pyruvate Reaction Rhodamine Role Source Spironolactone Starvation Sulfur Tissues Variant Water absorption aqueous base biomaterial compatibility cancer cell cell injury computational chemistry cytotoxicity design detection limit dihydronicotinamide ethylene glycol fluorophore optical spectra quantum ratiometric response water solubility

项目摘要

The project aims to develop water-soluble near-infrared fluorescent probes for the precise and quantitative analysis of NAD(P)H concentration changes in mitochondria of live cells by incorporating the nicotinamide moiety into different electron-withdrawing fluorophores as electron acceptors such as tetraphenylethylene (TPE), dicyanomethylene-4H-chromene, coumarin, naphthalimide, rhodamine, rhodol, and BODIPY. We will develop ratiometric near-infrared fluorescent probes to overcome systematic errors of intensity-based fluorescent probes by introducing near-infrared BODIPY donors to near-infrared rhodamine and rhodol acceptors, respectively. In order to significantly enhance water solubility and biocompatibility, we will introduce branched oligo(ethylene glycol) residues to the probes. The strong electron-withdrawing properties of the fluorophores such as dicyanomethylene-4H-chromene, coumarin, naphthalimide, rhodamine, rhodol, and BODIPY dyes will facilitate a quick reduction of the nicotinamide moiety by NAD(P)H into an electron-donating dihydronicotinamide moiety and significantly turn on the probe fluorescence. We will use computation chemistry to guide how to design effective fluorescent probes and study the relationship of the optical properties of the probes and their chemical structures such as HOMO and LUMO levels, pKa values, absorption and emission wavelengths, fluorescence quantum yields, and redox reaction between the probes and NAD(P)H, and assess the through-bond energy transfer efficiency from the donors to the acceptors of ratiometric fluorescent probes in the presence of NAD(P)H. We will investigate the effects of substrate-specific glucose and pyruvate/lactate imbalance on NADH levels in the glycolysis pathway in normal and cancer cells. We will also investigate the co-relationship between GSH/GSSG (glutathione/glutathione disulfide) and the cellular redox regulator NAD+/NADH in maintaining a critical redox balance for proper functioning in the different metabolic processes. Finally, we will investigate the unique significant role of NADH in oxidative phosphorylation in mitochondria, and monitor NADH concentration changes during the mitophagy process caused by starvation and drug treatment.

该项目旨在开发水溶性近红外荧光探针，用于精确定量掺入烟酰胺分析活细胞线粒体中 NAD(P)H 浓度变化部分进入不同的吸电子荧光团作为电子受体，例如四苯乙烯 (TPE)、二氰基亚甲基-4H-色烯、香豆素、萘二甲酰亚胺、罗丹明、对甲酚胺和 BODIPY。我们将开发比例近红外荧光探针以克服基于强度的系统误差通过将近红外 BODIPY 供体引入近红外罗丹明和对甲氨基酚来制备荧光探针分别是接受者。为了显着增强水溶性和生物相容性，我们将引入将支链寡聚（乙二醇）残基连接到探针上。其强吸电子特性荧光团，例如二氰基亚甲基-4H-色烯、香豆素、萘二甲酰亚胺、罗丹明、对甲酚等 BODIPY 染料将促进 NAD(P)H 快速将烟酰胺部分还原为供电子体二氢烟酰胺部分并显着打开探针荧光。我们将使用计算化学指导如何设计有效的荧光探针并研究光学之间的关系探针的特性及其化学结构，例如 HOMO 和 LUMO 能级、pKa 值、吸收和发射波长、荧光量子产率以及探针之间的氧化还原反应和NAD(P)H，并评估从供体到受体的键通能量转移效率 NAD(P)H 存在下的比率荧光探针。我们将研究特定底物的影响正常细胞和癌细胞中糖酵解途径中的葡萄糖和丙酮酸/乳酸失衡对 NADH 水平的影响。我们还将研究 GSH/GSSG（谷胱甘肽/谷胱甘肽二硫化物）与细胞氧化还原调节剂 NAD+/NADH 维持关键的氧化还原平衡，以保证细胞的正常功能不同的代谢过程。最后，我们将研究 NADH 在氧化中的独特重要作用。线粒体磷酸化，并监测线粒体自噬过程中 NADH 浓度的变化由饥饿和药物治疗引起。