Department of Chemistry, Jinan University, Guangzhou 510632, People's Republic of China.
Department of Chemistry, Jinan University, Guangzhou 510632, People's Republic of China.
J Chromatogr A. 2018 Nov 23;1577:47-58. doi: 10.1016/j.chroma.2018.09.045. Epub 2018 Sep 27.
This study combines a high-performance liquid chromatography-fluorescent detection method (HPLC-FLD) with in-situ cell imaging for the sensitive analysis of glutathione (GSH), cysteine (Cys) and homocysteine (Hcys), using BODIPY®507/545 IA as a labeling reagent. The analytical potential of BODIPY®507/545 IA in cell imaging was deeply explored, concerning fluorescent response, selectivity, cell-permeability, biotoxicity and so on. It is demonstrated that BODIPY®507/545 IA has good biocompatibility and the fluorescence intensity is enhanced remarkably after reacting with thiols. The best derivative condition was obtained in boric acid buffer (0.05 mmol/L, pH 9.5) at 45 °C for 15 min. For chromatographic method, two sensitive methods, HPLC-FLD and capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) were both developed, validated, and compared. The detection limits for the thiols ranged from 5 to 10 nmol/L with HPLC-FLD and 0.5 nmol/L for the CE-LIF method. Finally, HPLC-FLD is adopted to quantify the thiols in HepG2 cell samples after cell imaging.
本研究结合高效液相色谱-荧光检测法（HPLC-FLD）和原位细胞成像，使用 BODIPY®507/545 IA 作为标记试剂，对谷胱甘肽（GSH）、半胱氨酸（Cys）和同型半胱氨酸（Hcys）进行灵敏分析。深入探讨了 BODIPY®507/545 IA 在细胞成像中的分析潜力，包括荧光响应、选择性、细胞通透性、生物毒性等。结果表明，BODIPY®507/545 IA 具有良好的生物相容性，与巯基反应后荧光强度显著增强。在硼酸缓冲液（0.05mmol/L，pH9.5）中于 45°C 反应 15min 可获得最佳衍生条件。对于色谱方法，建立并比较了两种灵敏方法，高效液相色谱-荧光检测法（HPLC-FLD）和毛细管电泳-激光诱导荧光检测法（CE-LIF）。HPLC-FLD 法检测巯基的检出限范围为 5-10nmol/L，CE-LIF 法为 0.5nmol/L。最后，采用 HPLC-FLD 对细胞成像后的 HepG2 细胞样品中的巯基进行定量。
