College of Chemistry, Jilin University, Qianjin Street 2699, Changchun, 130012, China.
College of Life Sciences, Jilin University, Qianjin Street 2699, Changchun, 130012, China.
Anal Chim Acta. 2021 Dec 15;1188:339163. doi: 10.1016/j.aca.2021.339163. Epub 2021 Oct 11.
Alkaline phosphatase (ALP) is a commonly used marker in clinical practice, and this enzyme is a key indicator for diagnosing various diseases. In this study, we describe the development of a reliable and novel fluorescent assay for ALP detection based on chitosan carbon dots (C-CDs, peak emission, 412 nm) and calcein (peak emission, 512 nm). In the presence of Eu (which binds calcein), the fluorescence intensity of calcein is quenched. Utilizing the ALP-triggered generation of phosphate ions (PO) from the substrate p-nitrophenyl phosphate (pNPP), the Eu ions bind PO (which shows a higher affinity toward Eu than calcein), and the fluorescence of calcein is recovered. As a consequence, C-CDs fluorescence is decreased by inner filter effect (IFE). Exploiting these changes in the fluorescence intensity ratio of C-CDs and calcein, we developed a high sensitivity, accurate, and easily synthesized ratiometric fluorescence probe. Our novel fluorescent bioassay demonstrates good linear relationship in the 0.09-0.8 mU mL range, with a low detection limit of 0.013 mU mL. The excellent applicability of this novel assay in HepG2 cells and human serum samples demonstrates that our novel method has excellent biomedical research and disease diagnosis prospects.
碱性磷酸酶（ALP）是临床实践中常用的标志物，该酶是诊断各种疾病的关键指标。在本研究中，我们描述了一种基于壳聚糖碳点（C-CDs，峰值发射，412nm）和钙黄绿素（峰值发射，512nm）的新型荧光 ALP 检测方法。在 Eu（与钙黄绿素结合）存在的情况下，钙黄绿素的荧光强度被猝灭。利用 ALP 从底物对硝基苯磷酸酯（pNPP）中产生的磷酸根离子（PO），Eu 离子与 PO 结合（Eu 与 PO 的亲和力高于钙黄绿素），钙黄绿素的荧光恢复。因此，C-CDs 的荧光通过内滤效应（IFE）降低。利用 C-CDs 和钙黄绿素荧光强度比值的这些变化，我们开发了一种高灵敏度、准确且易于合成的比率荧光探针。我们的新型荧光生物测定法在 0.09-0.8 mU mL 范围内表现出良好的线性关系，检测限低至 0.013 mU mL。该新型测定法在 HepG2 细胞和人血清样本中的优异适用性表明，我们的新方法具有优异的生物医学研究和疾病诊断前景。
