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Sensitive Fluorescence Probe with Long Analytical Wavelengths for γ-Glutamyl Transpeptidase Detection in Human Serum and Living Cells
Analytical Chemistry ( IF 6.7 ) Pub Date : 2015-07-27 00:00:00 , DOI: 10.1021/acs.analchem.5b01535 Lihong Li 1 , Wen Shi 1 , Zhe Wang 1 , Qiuyu Gong 1 , Huimin Ma 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2015-07-27 00:00:00 , DOI: 10.1021/acs.analchem.5b01535 Lihong Li 1 , Wen Shi 1 , Zhe Wang 1 , Qiuyu Gong 1 , Huimin Ma 1
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A new sensitive fluorescent probe with long analytical wavelengths for γ-glutamyl transpeptidase (GGT) assay has been developed by incorporating the γ-glutamyl group as a recognition unit into the fluorophore of cresyl violet (CV). The detection mechanism is based on the GGT-catalyzed cleavage of the γ-glutamyl group, followed by the release of CV, which leads to a distinct color change from light yellow to pink and a large fluorescence enhancement at 615 nm (λex = 585 nm). Under the optimized conditions, the fluorescence intensity of the probe is directly proportional to the activity of GGT in the range of 1–50 U/L with a detection limit of 5.6 mU/L. By virtue of its high sensitivity and long analytical wavelengths, the probe has been used to directly determine GGT in human serum samples from both healthy people and liver cancer patients, and the obtained results accord well with those acquired by commercial GGT fluorometric assay kit. The probe has also been employed to image endogenous GGT in living cells. Notably, with our probe the expression level of GGT in HepG2 cells under the action of sodium butyrate (an anticancer drug) was studied by fluorescence confocal microscopy, revealing that sodium butyrate can induce the upregulation of GGT in HepG2 cells in a dose- and time-dependent manner. This behavior of sodium butyrate has further been confirmed by lysate assay and inhibitor experiment. The proposed probe is rather simple and may find a wide use in the determination of GGT in clinical and biological samples.
中文翻译：
具有长分析波长的敏感荧光探针，可检测人血清和活细胞中的γ-谷氨酰转肽酶
通过将作为识别单元的γ-谷氨酰基团整合到甲酚紫（CV）的荧光团中，已经开发出了一种用于γ-谷氨酰转肽酶（GGT）分析的长分析波长的新型灵敏荧光探针。检测机制基于GGT催化的γ-谷氨酰胺基团的裂解，然后释放CV，这导致从浅黄色到粉红色的明显颜色变化以及在615 nm处的大荧光增强（λex= 585 nm）。在最佳条件下，探针的荧光强度与GGT的活性直接成正比，在1–50 U / L的范围内，检出限为5.6 mU / L。凭借其高灵敏度和长分析波长，该探针已用于直接测定健康人和肝癌患者的人血清样品中的GGT，所获得的结果与通过商业GGT荧光测定试剂盒获得的结果十分吻合。该探针还已用于在活细胞中对内源GGT成像。值得注意的是，我们的探针通过荧光共聚焦显微镜研究了在丁酸钠（一种抗癌药）作用下HepG2细胞中GGT的表达水平，发现丁酸钠可以诱导HepG2细胞中GGT的上调在剂量和时间上。依赖的方式。丁酸钠的这种行为已通过裂解物测定和抑制剂实验进一步证实。所提出的探针相当简单，可以在临床和生物学样品中GGT的测定中找到广泛的用途。
更新日期：2015-07-27
中文翻译：
具有长分析波长的敏感荧光探针，可检测人血清和活细胞中的γ-谷氨酰转肽酶
通过将作为识别单元的γ-谷氨酰基团整合到甲酚紫（CV）的荧光团中，已经开发出了一种用于γ-谷氨酰转肽酶（GGT）分析的长分析波长的新型灵敏荧光探针。检测机制基于GGT催化的γ-谷氨酰胺基团的裂解，然后释放CV，这导致从浅黄色到粉红色的明显颜色变化以及在615 nm处的大荧光增强（λex= 585 nm）。在最佳条件下，探针的荧光强度与GGT的活性直接成正比，在1–50 U / L的范围内，检出限为5.6 mU / L。凭借其高灵敏度和长分析波长，该探针已用于直接测定健康人和肝癌患者的人血清样品中的GGT，所获得的结果与通过商业GGT荧光测定试剂盒获得的结果十分吻合。该探针还已用于在活细胞中对内源GGT成像。值得注意的是，我们的探针通过荧光共聚焦显微镜研究了在丁酸钠（一种抗癌药）作用下HepG2细胞中GGT的表达水平，发现丁酸钠可以诱导HepG2细胞中GGT的上调在剂量和时间上。依赖的方式。丁酸钠的这种行为已通过裂解物测定和抑制剂实验进一步证实。所提出的探针相当简单，可以在临床和生物学样品中GGT的测定中找到广泛的用途。
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