通过切换吡哆醛磷酸盐对金纳米簇荧光的猝灭来实现碱性磷酸酶的灵敏检测。
Sensitive detection of alkaline phosphatase by switching on gold nanoclusters fluorescence quenched by pyridoxal phosphate.
机构信息
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China; Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516 Mansoura, Egypt.
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
出版信息
Biosens Bioelectron. 2017 Sep 15;95:8-14. doi: 10.1016/j.bios.2017.03.073. Epub 2017 Apr 6.
In this work, we designed highly sensitive and selective luminescent detection method for alkaline phosphatase using bovine serum albumin functionalized gold nanoclusters (BSA-AuNCs) as the nanosensor probe and pyridoxal phosphate as the substrate of alkaline phosphatase. We found that pyridoxal phosphate can quench the fluorescence of BSA-AuNCs and pyridoxal has little effect on the fluorescence of BSA-AuNCs. The proposed mechanism of fluorescence quenching by PLP was explored on the basis of data obtained from high-resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS), UV-vis spectrophotometry, fluorescence spectroscopy, fluorescence decay time measurements and circular dichroism (CD) spectroscopy. Alkaline phosphatase catalyzes the hydrolysis of pyridoxal phosphate to generate pyridoxal, restoring the fluorescence of BSA-AuNCs. Therefore, a recovery type approach has been developed for the sensitive detection of alkaline phosphatase in the range of 1.0-200.0U/L (R =0.995) with a detection limit of 0.05U/L. The proposed sensor exhibit excellent selectivity among various enzymes, such as glucose oxidase, lysozyme, trypsin, papain, and pepsin. The present switch-on fluorescence sensing strategy for alkaline phosphatase was successfully applied in human serum plasma with good recoveries (100.60-104.46%), revealing that this nanosensor probe is a promising tool for ALP detection.
在这项工作中，我们设计了一种基于牛血清白蛋白功能化金纳米簇（BSA-AuNCs）作为纳米传感器探针和磷酸吡哆醛作为碱性磷酸酶底物的高灵敏度和选择性的荧光检测方法。我们发现磷酸吡哆醛可以猝灭 BSA-AuNCs 的荧光，而磷酸吡哆醛对 BSA-AuNCs 的荧光几乎没有影响。在高分辨率透射电子显微镜（HRTEM）、动态光散射（DLS）、紫外-可见分光光度法、荧光光谱法、荧光衰减时间测量和圆二色（CD）光谱法等数据的基础上，探讨了 PLP 荧光猝灭的机制。碱性磷酸酶催化磷酸吡哆醛水解生成磷酸吡哆醛，恢复 BSA-AuNCs 的荧光。因此，建立了一种在 1.0-200.0U/L 范围内（R =0.995）用于灵敏检测碱性磷酸酶的恢复型方法，检测限为 0.05U/L。该传感器在葡萄糖氧化酶、溶菌酶、胰蛋白酶、木瓜蛋白酶和胃蛋白酶等各种酶中表现出优异的选择性。该荧光传感策略已成功应用于人血清血浆中的碱性磷酸酶检测，回收率良好（100.60-104.46%），表明该纳米传感器探针是一种有前途的 ALP 检测工具。
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