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

• 批准号: 10798814
• 负责人: Haiying Liu
• 金额: $ 1.9万
• 依托单位: MICHIGAN TECHNOLOGICAL UNIVERSITY
• 依托单位国家: 美国
• 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/10798814
• 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; biomaterial compatibility; cancer cell; cell injury; computational chemistry; cytotoxicity; design; detection limit; dihydronicotinamide; ethylene glycol; fluorophore; optical spectra; quantum; ratiometric; response; water solubility

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 通过掺入烟酰胺分析活细胞线粒体中NAD（P）H浓度的变化 部分转化为不同的吸电子荧光团作为电子受体， (TPE)二氰基亚甲基-4H-色烯、香豆素、萘二甲酰亚胺、罗丹明、罗丹酚和BODIPY。我们将 开发比率近红外荧光探针，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罗丹酚的荧光探针 接受者，分别。为了显著增强水溶性和生物相容性，我们将引入 支化的寡聚（乙二醇）残基与探针连接。的强吸电子性质， 荧光团如二氰基亚甲基-4H-色烯、香豆素、萘二甲酰亚胺、罗丹明、罗丹酚和 BODIPY染料将促进烟酰胺部分被NAD（P）H快速还原成供电子的 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 探针的性质及其化学结构，例如HOMO和LUMO能级，pKa值， 吸收和发射波长、荧光量子产率和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通过键的能量转移效率， 在NAD（P）H存在下的比率荧光探针。我们将研究底物特异性 葡萄糖和丙酮酸/乳酸不平衡对正常和癌细胞糖酵解途径中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在维持关键的氧化还原平衡中的正常功能， 不同的代谢过程。最后，我们将研究NADH在氧化过程中的独特重要作用。 线粒体磷酸化，并监测线粒体自噬过程中NADH浓度的变化 是由饥饿和药物治疗引起的。