Jin Lu-Yi, Dong Yu-Ming, Wu Xiu-Ming, Cao Gen-Xia, Wang Guang-Li
The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University , Wuxi 214122, Jiangsu, China.
State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University , Nanjing 210093, Jiangsu, China.
Anal Chem. 2015 Oct 20;87(20):10429-36. doi: 10.1021/acs.analchem.5b02728. Epub 2015 Oct 6.
The alkaline phosphatase (ALP) biocatalysis followed by the in situ enzymatic generation of a visible light responsive nanozyme is coupled to elucidate a novel amplification strategy by enzymatic cascade reaction for versatile biosensing. The enzymatic hydrolysis of o-phosphonoxyphenol (OPP) to catechol (CA) by ALP is allowed to coordinate on the surface of TiO2 nanoparticles (NPs) due to the specificity and high affinity of enediol ligands to Ti(IV). Upon the stimuli by CA generated from ALP, the inert TiO2 NPs is activated, which demonstrates highly efficient oxidase mimicking activity for catalyzing the oxidation of the typical substrate of 3,3',5,5'-tetramethylbenzidine (TMB) under visible light (λ ≥ 400 nm) irradiation utilizing dissolved oxygen as an electron acceptor. On the basis of the cascade reaction of ALP and the nanozyme of CA coordinated TiO2 (TiO2-CA) NPs, we design exquisitely colorimetric biosensors for probing ALP activity and its inhibitor of 2, 4-dichlorophenoxyacetic acid (2,4-DA). Quantitative probing of ALP activity in a wide linear range from 0.01 to 150 U/L with the detection limit of 0.002 U/L is realized, which endows the methodology with sufficiently high sensitivity for potentially practical applications in real samples of human serum (ALP level of 40-190 U/L for adults). In addition, a novel immunoassay protocol by taking mouse IgG as an example is validated using the ALP/nanozyme cascade amplification reaction as the signal transducer. A low detection limit of 2.0 pg/mL is attained for mouse IgG, which is 4500-fold lower than that of the standard enzyme-linked immuno-sorbent assay (ELISA) kit. Although only mouse IgG is used as a proof-of-concept in our experiment, we believe that this approach is generalizable to be readily extended to other ELISA systems. This methodology opens a new horizon for amplified and versatile biosensing including probing ALP activity and following ALP-based ELISA immunoassays.
碱性磷酸酶（ALP）生物催化与原位酶促生成可见光响应纳米酶相结合，以阐明一种通过酶级联反应实现多功能生物传感的新型放大策略。由于烯二醇配体对Ti(IV)的特异性和高亲和力，碱性磷酸酶将邻膦酰基苯酚（OPP）酶水解为儿茶酚（CA）的反应得以在二氧化钛纳米颗粒（NPs）表面进行。在碱性磷酸酶产生的CA刺激下，惰性的二氧化钛纳米颗粒被激活，在可见光（λ≥400 nm）照射下，利用溶解氧作为电子受体，它表现出高效的氧化酶模拟活性，可催化典型底物3,3',5,5'-四甲基联苯胺（TMB）的氧化。基于碱性磷酸酶与CA配位的二氧化钛（TiO2-CA）纳米颗粒的纳米酶的级联反应，我们精心设计了用于检测碱性磷酸酶活性及其抑制剂2,4-二氯苯氧乙酸（2,4-DA）的比色生物传感器。实现了在0.01至150 U/L的宽线性范围内对碱性磷酸酶活性的定量检测，检测限为0.002 U/L，这赋予了该方法足够高的灵敏度，可在人血清实际样品（成人碱性磷酸酶水平为40 - 190 U/L）中进行潜在的实际应用。此外，以小鼠IgG为例，使用碱性磷酸酶/纳米酶级联放大反应作为信号转导器，验证了一种新型免疫分析方案。小鼠IgG的检测限低至2.0 pg/mL，比标准酶联免疫吸附测定（ELISA）试剂盒低4500倍。尽管在我们的实验中仅使用小鼠IgG作为概念验证，但我们相信这种方法具有通用性，可轻松扩展到其他ELISA系统。该方法为放大和多功能生物传感开辟了新视野，包括检测碱性磷酸酶活性和基于碱性磷酸酶的ELISA免疫分析。
