Luminescent quantum dots fluorescence resonance energy transfer-based probes for enzymatic activity and enzyme inhibitors
Luminescent quantum dots fluorescence resonance energy transfer-based probes for enzymatic activity and enzyme inhibitors
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DOI：
10.1021/ac0614644
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发表时间：
2007-01-01
影响因子：
7.4
通讯作者：
Rosenzweig, Zeev
中科院分区：
文献类型：
--
作者：
Shi, Lifang;Rosenzweig, Nitsa;Rosenzweig, Zeev
本文介绍了基于量子点的探针的开发和表征的分析性能的酶活性和筛选酶抑制剂。发光探针基于作为供体的发光量子点和通过肽接头固定到量子点表面的罗丹明受体之间的荧光共振能量转移（FRET）。采用一步配体交换法制备了肽包覆的CdSe/ZnS量子点，其中RGDC肽分子取代三辛基氧化膦（TOPO）分子作为量子点的封端配体。肽分子通过肽半胱氨酸残基的巯基结合到CdSe/ZnS量子点的表面。用罗丹明标记肽包被的量子点以形成FRET探针。量子点FRET探针的发射量子产率比TOPO封端的量子点的发射量子产率低4倍。然而，量子点FRET探针足够明亮，能够进行定量酶和酶抑制测定。首先使用探针来测试溶液中的胰蛋白酶的酶活性，基于在蛋白水解酶存在下基于量子点的酶探针的FRET信号变化。例如，量子点FRET探针暴露于500 μ g/mL胰蛋白酶15分钟导致量子点的光致发光增加60%，罗丹明分子的发射相应减少。这些变化是由于肽分子的酶促裂解导致罗丹明分子从量子点表面释放而引起的。基于量子点FRET的探针被用来监测胰蛋白酶的酶活性，并筛选胰蛋白酶抑制剂的抑制效率。
The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trioctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thiol group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FRET signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 mu g/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRET-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency.