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，以进行精确和定量 通过掺入烟酰胺，分析活细胞线粒体的NAD（P）H浓度变化 作为电子受体（例如四苯基乙二烯），将一部分作为不同的电子吸引荧光团绘制的荧光团 （TPE），二氰基甲基-4H-CHROMENE，香豆素，萘酰亚胺，若丹明，犀牛和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 荧光探针通过将近红外卫星供体引入近红外杜鹃胺和杜尔 受体分别。为了显着提高水的溶解度和生物相容性，我们将引入 探针的分支寡醇（乙二醇）残基。强的电子吸引特性 荧光团，例如二氰基甲基-4H-Chromene，香豆素，萘酰亚胺，若丹明，犀牛和 Bodipy染料将促进NAD（P）h快速减少烟酰胺部分 二氢氨基氨酸酰胺部分，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的关系 探针及其化学结构的特性，例如同性和Lumo水平，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，并评估从捐助者到受体的底键能量转移效率 在NAD（P）h存在下的比率荧光探针。我们将研究底物特异性的影响 正常和癌细胞中糖酵解途径中NADH水平的葡萄糖和丙酮酸/乳酸不平衡。 我们还将研究GSH/GSSG（谷胱甘肽/谷胱甘肽二硫键）与 细胞氧化还原调节剂NAD+/NADH保持关键的氧化还原平衡，以在 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。 线粒体中的磷酸化，并在线粒体过程中监测NADH浓度变化 由饥饿和药物治疗引起。