Wei Hui, Chen Chaogui, Han Bingyan, Wang Erkang
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China.
Anal Chem. 2008 Sep 15;80(18):7051-5. doi: 10.1021/ac801144t. Epub 2008 Jul 29.
Colorimetric assay based on the unique surface plasmon resonance properties of metallic nanoparticles has received considerable attention in bioassay due to its simplicity, high sensitivity, and low cost. Most of colorimetric methods previously reported employed gold nanoparticles (GNPs) as sensing elements. In this work, we develop a sensitive, selective, simple, and label-free colorimetric assay using unmodified silver nanoparticle (AgNP) probes to detect enzymatic reactions. Enzymatic reactions concerning adenosine triphosphate (ATP) dephosphorylation by calf intestine alkaline phosphatase (CIAP) and peptide phosphorylation by protein kinase A (PKA) were studied. In the absence of the enzymes, unreacted ATP could protect AgNPs from salt-induced aggregation, whereas in the presence of the enzymes, the reaction product of ATP (i.e., adenosine for CIAP and ADP for PKA) could not. Via our method, dephosphorylation and phosphorylation could be readily detected by the color change of AgNPs, with a detection limit of 1 unit/mL for CIAP and a detection limit of 0.022 unit/mL for PKA. More importantly, the enzymatic inhibition by inhibitors and enzymatic activity in complex biological fluids could also be realized. This work is an important step toward a colorimetric assay using AgNPs and might provide a promise for enzyme assay in realistically complex systems and for screening of different enzyme inhibitors in future.
基于金属纳米颗粒独特的表面等离子体共振特性的比色测定法，因其简单性、高灵敏度和低成本，在生物测定中受到了广泛关注。此前报道的大多数比色法都使用金纳米颗粒（GNPs）作为传感元件。在这项工作中，我们开发了一种灵敏、选择性好、简单且无需标记的比色测定法，使用未修饰的银纳米颗粒（AgNP）探针来检测酶促反应。研究了小牛肠碱性磷酸酶（CIAP）催化三磷酸腺苷（ATP）去磷酸化以及蛋白激酶A（PKA）催化肽磷酸化的酶促反应。在没有酶的情况下，未反应的ATP可以保护AgNPs不发生盐诱导的聚集，而在有酶的情况下，ATP的反应产物（即CIAP反应生成的腺苷和PKA反应生成的二磷酸腺苷）则不能。通过我们的方法，通过AgNPs的颜色变化可以很容易地检测到去磷酸化和磷酸化反应，CIAP的检测限为1单位/毫升，PKA的检测限为0.022单位/毫升。更重要的是，还可以实现复杂生物流体中抑制剂对酶的抑制作用以及酶活性的检测。这项工作是朝着使用AgNPs进行比色测定迈出的重要一步，可能为实际复杂系统中的酶测定以及未来不同酶抑制剂的筛选提供希望。
