Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme
Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme
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DOI：
10.1021/acs.analchem.5b02728
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发表时间：
2015-10-20
影响因子：
7.4
通讯作者：
Wang, Guang-Li
中科院分区：
文献类型：
--
作者：
Jin, Lu-Yi;Dong, Yu-Ming;Wang, Guang-Li
碱性磷酸酶（ALP）生物催化，随后原位酶促生成可见光响应纳米酶，通过酶级联反应阐明用于多功能生物传感的新型扩增策略。由于烯二醇配体对 Ti(IV) 的特异性和高亲和力，ALP 酶促水解邻膦氧基苯酚 (OPP) 生成儿茶酚 (CA) 可以在 TiO2 纳米粒子 (NPs) 的表面上配位。在 ALP 产生的 CA 刺激下，惰性 TiO2 NP 被激活，表现出高效的氧化酶模拟活性，可利用溶解氧作为电子受体，在可见光 (lambda >= 400 nm) 照射下催化 3,3',5,5'-四甲基联苯胺 (TMB) 典型底物的氧化。基于ALP和CA配位TiO2（TiO2-CA）纳米颗粒的级联反应，我们设计了精美的比色生物传感器，用于探测ALP活性及其2,4-二氯苯氧基乙酸（2,4-DA）抑制剂。实现了在 0.01 至 150 U/L 的宽线性范围内定量检测 ALP 活性，检测限为 0.002 U/L，这使得该方法具有足够高的灵敏度，可用于人血清实际样本（成人 ALP 水平为 40-190 U/L）的潜在实际应用。此外，利用ALP/纳米酶级联放大反应作为信号传感器，验证了以小鼠IgG为例的新型免疫分析方案。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) 试剂盒的检测限低 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可以推广到其他 ELISA 系统。该方法为放大和多功能生物传感开辟了新视野，包括探测 ALP 活性和后续基于 ALP 的 ELISA 免疫测定。
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 (lambda >= 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.