Shu Chang, Li Duo, Li Tengfei, Ji Shunli, Ding Li
Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education Nanjing 210009 China.
Department of Pharmaceutical Analysis, School of Pharmacy, China Pharmaceutical University 24 Tongjiaxiang Nanjing 210009 P. R. China
RSC Adv. 2018 Oct 29;8(64):36527-36533. doi: 10.1039/c8ra06973e. eCollection 2018 Oct 26.
Alkaline phosphatase (ALP) is an important biomarker for many diseases. Therefore, the sensitive and accurate detection of ALP activity is essential for fundamental biochemical processes and clinic diagnosis. Herein, we design a fluorescent on-off-on switch for sensitive and visual detection of ALP activity. Meanwhile, mass barcode-modified quantum dots (QDs) amplified the LC-MS/MS detection signal in complex biological samples. Firstly, the QDs were modified with phosphorylated Gly-Gly-Phe-Phe-Tyr (OPOH) peptide (GGFFYp) and the mass barcode. The fluorescence of QDs-SS-Yp was quenched by fluorescence resonance energy transfer (FRET) between QDs-SS-Yp and dansyl chloride (DNS). ALP can hydrolyze the phosphorylated peptide to form peptide self-assemblies on the QDs-SS-Yp surfaces. The effective separation distance between the QDs-SS-Yp donor and DNS acceptor becomes larger, restricting FRET between the QDs-SS-Yp and DNS. At this point, the obvious QDs-SS-Yp fluorescence signal can be restored. However, the absence of ALP results in no peptide self-assembly on the QDs-SS-Yp surface and no obvious QDs-SS-Yp fluorescence signal was detected. Therefore, the ALP activity can be analyzed according to the degree of fluorescence restoration by the fluorescence on-off-on switch. Finally, the small tag molecules obtained by cleaving the disulfide bond of the QDs-SS-Yp as a mass barcode were used to amplify the LC-MS/MS detection signal. The proposed approach shows a good linear relationship (from 0.01 to 2.4 U L) and has the significant advantage of a low detection limit of 0.001 U L.
碱性磷酸酶（ALP）是许多疾病的重要生物标志物。因此，灵敏且准确地检测ALP活性对于基础生化过程和临床诊断至关重要。在此，我们设计了一种用于灵敏且可视化检测ALP活性的荧光开-关-开开关。同时，质量条形码修饰的量子点（QDs）放大了复杂生物样品中的液相色谱-串联质谱（LC-MS/MS）检测信号。首先，用磷酸化的甘氨酰-甘氨酰-苯丙氨酰-苯丙氨酰-酪氨酸（OPOH）肽（GGFFYp）和质量条形码修饰量子点。QDs-SS-Yp的荧光通过QDs-SS-Yp与丹磺酰氯（DNS）之间的荧光共振能量转移（FRET）而猝灭。ALP可水解磷酸化肽，在QDs-SS-Yp表面形成肽自组装体。QDs-SS-Yp供体与DNS受体之间的有效分离距离变大，限制了QDs-SS-Yp与DNS之间的FRET。此时，可恢复明显的QDs-SS-Yp荧光信号。然而，缺乏ALP会导致QDs-SS-Yp表面无肽自组装体，未检测到明显的QDs-SS-Yp荧光信号。因此，可根据荧光开-关-开开关的荧光恢复程度分析ALP活性。最后，将通过切割QDs-SS-Yp的二硫键获得的小标签分子作为质量条形码用于放大LC-MS/MS检测信号。所提出的方法显示出良好的线性关系（从0.01至2.4 U/L），且具有0.001 U/L的低检测限这一显著优势。
