A fluorometric sensing method for sensitive detection of trypsin and its inhibitor based on gold nanoclusters and gold nanoparticles
A fluorometric sensing method for sensitive detection of trypsin and its inhibitor based on gold nanoclusters and gold nanoparticles
复制标题
基于金纳米团簇和金纳米粒子的灵敏检测胰蛋白酶及其抑制剂的荧光传感方法
DOI：
10.1007/s00216-018-1292-3
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发表时间：
2018
影响因子：
4.3
通讯作者：
Su Xingguang
中科院分区：
文献类型：
--
作者：
Wang Mengke;Su D;an;Wang Guannan;Su Xingguang
基于巯基十一烷酸修饰的金纳米团簇（AuNCs）和金纳米颗粒（AuNPs）之间通过鱼精蛋白桥的荧光共振能量转移（FRET），建立了一种简便、无标记、灵敏的荧光检测胰蛋白酶及其抑制剂的方法。鱼精蛋白可以触发AuNP的聚集并通过静电相互作用将AuNC与聚集的AuNP连接。与单分散金纳米粒子相比，聚集金纳米粒子的紫外-可见吸收光谱与金纳米粒子的发射光谱有很大的重叠。因此，通过FRET，聚集的AuNPs明显淬灭AuNCs的荧光。在胰蛋白酶的作用下，鱼精蛋白水解成小片段，导致金纳米粒子的解聚，打破了金纳米粒子与金纳米粒子之间的短距离，从而抑制了FRET过程，使金纳米粒子的荧光恢复。AuNCs荧光强度的增加与胰蛋白酶的量直接相关。因此，胰蛋白酶可以测定的基础上的荧光强度的变化，与线性范围为5-5000 ng mL-1和1.9 ng mL-1的检测限。以大豆中分离的胰蛋白酶抑制剂为模型，将该体系用于胰蛋白酶抑制剂的检测。将该方法应用于人尿和市售多酶片样品中胰蛋白酶的检测，结果令人满意。
In this work, a facile, label-free, and sensitive fluorometric strategy for detection of trypsin and its inhibitor was established on the basis of the fluorescence resonance energy transfer (FRET) between mercaptoundecanoic acid functionalized gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) via protamine as a bridge. Protamine can trigger the aggregation of AuNPs and link AuNCs with aggregated AuNPs through electrostatic interaction. Compared with monodisperse AuNPs, the UV–vis absorption band of aggregated AuNPs overlapped considerably with the emission spectrum of AuNCs. Thus, the fluorescence of AuNCs was obviously quenched by the aggregated AuNPs through FRET. In the presence of trypsin, protamine was hydrolyzed into small fragments, leading to the deaggregation of AuNPs and breaking of the short distance between AuNPs and AuNCs, so the FRET process was inhibited, and the fluorescence of AuNCs was recovered. The increase in the fluorescence intensity of AuNCs was directly related to the amount of trypsin. Hence trypsin can be determined on the basis of the variation of fluorescence intensity, with a linear range of 5–5000 ng mL-1 and a detection limit of 1.9 ng mL-1. In addition, this system was used for the detection of trypsin inhibitor by application of the inhibitor isolated from soybean as a model. The sensing method was applied for trypsin detection in human urine and commercial multienzyme tablet samples with satisfactory results.