Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou, 450046, China.
Pharmacy College, Henan University of Chinese Medicine, Zhengzhou, 450046, China.
Anal Bioanal Chem. 2021 Mar;413(7):1827-1836. doi: 10.1007/s00216-020-03150-1. Epub 2021 Jan 22.
As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".
作为一种非特异性磷酸单酯酶，碱性磷酸酶（ALP）在组织矿化和成骨中起着关键作用，是骨和肝胆疾病临床诊断的重要生物标志物。在此，我们描述了一种新的电化学方法，该方法使用氨基二茂铁（AFC）作为电活性探针来检测 ALP 活性。在咪唑和 N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐（EDC）的条件下，AFC 探针可以通过一步偶联直接标记在单链 DNA（ssDNA）上。具体而言，巯基化的 ssDNA 修饰到电极表面的 3'末端是通过 Au-S 键。在没有 ALP 的条件下，AFC 可以通过与磷酸基团结合而标记在 ssDNA 上。在存在 ALP 的情况下，磷酸基团被催化从 ssDNA 的 5'末端去除。AFC 探针不能标记在 ssDNA 上。因此，实现了对 ALP 活性的电化学检测。在最佳条件下，该策略在 20 至 100 mU/mL 的范围内呈现出与电流强度和 ALP 浓度之间的良好线性关系，检测限（LOD）为 1.48 mU/mL。更重要的是，该方法具有高选择性和令人满意的适用性，可用于 ALP 活性检测。此外，该方法具有操作简单、成本低、环保等优点。因此，该策略在实际应用中具有很大的潜力用于 ALP 活性检测。通过电化学“信号关闭”，开发了一种用于检测碱性磷酸酶活性的简单、灵敏和可靠的策略。
