Institute of Nanomedicine and Nanobiosensing, MOE Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University , Fuzhou 350108, China.
ACS Appl Mater Interfaces. 2014 Oct 22;6(20):18243-50. doi: 10.1021/am505342r. Epub 2014 Oct 2.
Nanostructure-based visual assay has been developed for determination of enzymatic activity, but most involve in poor visible color resolution and are not suitable for routine utilization. Herein, we designed a high-resolution colorimetric protocol based on gold/silver core/shell nanorod for visual readout of alkaline phosphatase (ALP) activity by using bare-eyes. The method relied on enzymatic reaction-assisted silver deposition on gold nanorod to generate significant color change, which was strongly dependent on ALP activity. Upon target ALP introduction into the substrate, the ascorbic acid 2-phosphate was hydrolyzed to form ascorbic acid, and then, the generated ascorbic acid reduced silver ion to metal silver and coated on the gold nanorod, thereby resulting in the blue shift of longitudinal localized surface plasmon resonance peak of gold nanorod accompanying a perceptible color change from red to orange to yellow to green to cyan to blue and to violet. Under optimal conditions, the designed method exhibited the wide linear range 5-100 mU mL(-1) ALP with a detection limit of 3.3 mU mL(-1). Moreover, it could be used for the semiquantitative detection of ALP from 20 to 500 mU mL(-1) by using the bare-eyes. The coefficients of variation for intra- and interassay were below 3.5% and 6.2%, respectively. Finally, this method was validated for the analysis of real-life serum samples, giving results matched well with those from the 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP)-based standard method. In addition, the system could even be utilized in the enzyme-linked immunosorbent assay (ELISA) to detect IgG at picomol concentration. With the merits of simplification, low cost, user-friendliness, and sensitive readout, the gold nanorod-based colorimetric assay has the potential to be utilized by the public and opens a new horizon for bioassays.
基于纳米结构的比色法已被开发用于测定酶活性，但大多数方法的可见颜色分辨率较差，不适合常规使用。在此，我们设计了一种基于金/银核/壳纳米棒的高分辨率比色方案，通过肉眼对碱性磷酸酶（ALP）活性进行可视化读出。该方法依赖于酶反应辅助金纳米棒上的银沉积来产生显著的颜色变化，该颜色变化强烈依赖于 ALP 活性。在引入目标 ALP 进入基底后，抗坏血酸 2-磷酸被水解形成抗坏血酸，然后，生成的抗坏血酸将银离子还原为金属银并覆盖在金纳米棒上，从而导致金纳米棒的纵向局域表面等离激元共振峰蓝移，同时颜色从红色变为橙色，再变为黄色，再变为绿色，再变为青色，再变为蓝色，最后变为紫色。在最佳条件下，所设计的方法表现出 5-100 mU mL(-1) ALP 的宽线性范围，检测限为 3.3 mU mL(-1)。此外，通过肉眼可以对半定量检测 20-500 mU mL(-1) 的 ALP。内和间试验的变异系数分别低于 3.5%和 6.2%。最后，该方法用于真实血清样本的分析，结果与 4-硝基苯磷酸二钠盐六水合物（pNPP）标准方法吻合。此外，该系统甚至可以用于酶联免疫吸附测定（ELISA）中检测皮摩尔浓度的 IgG。基于金纳米棒的比色测定具有简化、低成本、友好用户和灵敏读出等优点，有可能被公众使用，并为生物测定开辟新的视野。
