Department of Chemical Engineering, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China.
School of Chemistry and Chemical Engineering, Nantong University, Nantong, Jiangsu, 226019, China.
Analyst. 2022 May 30;147(11):2494-2499. doi: 10.1039/d1an02159a.
Alkaline phosphatase (ALP) is a widely used indicator in the diagnosis of various diseases. Thus, it is also urgent to develop simple and efficient methods, which can meet the accurate determination of ALP activity in physiological environments. In this study, enzyme-triggered click chemistry combined with the surface-enhanced Raman spectroscopy (SERS) technique was developed for the highly sensitive detection of the ALP activity in complex biological samples. ALP was able to catalyze the ascorbic acid-phosphate (AAP) to generate ascorbic acid (AA). Then, AA could reduce Cu(II) to produce Cu(I), which plays the role of a catalyst to promote the click reaction of azide terephthalic acid (ATA) and 4-acetylene biphenyl (4-AB), resulting in the SERS signal intensity of free 4-AB in the solution was clearly reduced along with the click reaction carried on, and showed a quantitative relationship with the concentration of ALP. The proposed method has the advantages of high sensitivity, selectivity and excellent repeatability. As a proof of concept, the new developed SERS-click strategy was applied to the specific determination of the ALP activity in clinical serum samples, cellular lysate samples and the ALP inhibitor assessment successfully, indicating that the proposed ALP-triggered click chemistry assay has a significant potential application in medical diagnosis.
碱性磷酸酶（ALP）是诊断各种疾病的常用指标。因此，开发简单、高效的方法来满足生理环境中 ALP 活性的准确测定也是当务之急。在这项研究中，开发了酶触发点击化学与表面增强拉曼光谱（SERS）技术相结合的方法，用于在复杂生物样品中高灵敏度检测 ALP 活性。ALP 能够催化抗坏血酸-磷酸（AAP）生成抗坏血酸（AA）。然后，AA 可以将 Cu(II)还原为 Cu(I)，Cu(I)起到催化剂的作用，促进叠氮苯二甲酸（ATA）和 4-乙炔联苯（4-AB）的点击反应，导致溶液中游离 4-AB 的 SERS 信号强度随着点击反应的进行而明显降低，并与 ALP 的浓度呈定量关系。该方法具有灵敏度高、选择性好、重现性好等优点。作为概念验证，新开发的 SERS-点击策略成功应用于临床血清样品、细胞裂解物样品中 ALP 活性的特异性测定和 ALP 抑制剂的评估，表明所提出的基于 ALP 触发的点击化学分析具有在医学诊断中有重要的潜在应用价值。
