College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha, 410082, Hunan, China.
TCM and Ethnomedicine Innovation & Development Laboratory, Sino-Pakistan TCM Research Center, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, Hunan, China.
Anal Bioanal Chem. 2018 Aug;410(21):5219-5228. doi: 10.1007/s00216-018-1174-8. Epub 2018 Jun 22.
As an essential phosphate group hydrolase, alkaline phosphatase (ALP), whose level in serum is correlated with bone disease, liver dysfunction, and cancer, could be used as a biomarker for clinical diagnosis and biomedical studies. Hence, developing a convenient and sensitive method for ALP assay has importance in disease diagnosis, drug treatment, and prognosis assessment. In this work, using a hairpin DNA strand as the substrate, we developed an ultrasensitive and simple fluorescence method for quantitative ALP assay based on the binding difference of reduced graphene oxide (rGO) with different DNA strands coupled with λ exonuclease (λ exo) cleavage. Under the optimal conditions, the limit of detection (LOD) of ALP is estimated to be 0.01 U/L with the linear region from 0.5 U/L to 70 U/L. Furthermore, the proposed assay was used to detect ALP in complicated cell-free extracts and evaluate the inhibitory effects of two well-known inhibitors of ALP activity. Finally, the method was used to investigate the effect of natural compounds on ALP activity and five compounds with different inhibitory capability were screened. In summary, we propose that the new method for ALP assay can be applied for therapeutic drug monitoring (TDM) and high-throughput compound screening in combination with multiwell plate technology.
作为一种重要的磷酸基团水解酶，碱性磷酸酶（ALP）的血清水平与骨骼疾病、肝功能障碍和癌症相关，可作为临床诊断和生物医学研究的生物标志物。因此，开发一种方便、灵敏的 ALP 测定方法对于疾病诊断、药物治疗和预后评估具有重要意义。在这项工作中，我们使用发夹 DNA 链作为底物，基于与不同 DNA 链结合的还原氧化石墨烯（rGO）的结合差异，结合 λ 外切酶（λ exo）切割，开发了一种用于定量 ALP 测定的超灵敏和简单的荧光方法。在最佳条件下，ALP 的检测限（LOD）估计为 0.01 U/L，线性范围为 0.5 U/L 至 70 U/L。此外，该测定法用于检测复杂的无细胞提取物中的 ALP，并评估两种 ALP 活性的著名抑制剂的抑制效果。最后，该方法用于研究天然化合物对 ALP 活性的影响，并筛选出五种具有不同抑制能力的化合物。总之，我们提出，新的 ALP 测定方法可与微孔板技术结合，用于治疗药物监测（TDM）和高通量化合物筛选。
