Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education and Fujian Province), Department of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350108, PR China.
Anal Chim Acta. 2013 May 7;776:79-86. doi: 10.1016/j.aca.2013.03.034. Epub 2013 Mar 21.
Enzyme-linked immunosorbent assay (ELISA) methods based on natural enzyme-labeled probes have been applied in the immunoassays, but most have some inevitable limitations (e.g. harsh preparation, purification and storage) and are unsuitable for routine use. Herein we synthesized a new class of irregular-shaped platinum nanoparticles (ISPtNP) with a mean length of 7.0 nm and a narrowing width from 2.0 to 5.0 nm along the longitudinal axes, which were utilized as peroxidase-like mimics for the development of colorimetric immunoassays. Compared with bioactive horseradish peroxidase (HRP), the synthesized ISPtNP exhibited a low Km value (0.12 mM) and a high Kcat value (2.27×10(4)s(-1)) for 3,3',5,5'-tetramethylbenzidine (TMB) with strong thermal stability and pH tolerance. The catalytic mechanism of the ISPtNP toward TMB/H2O2 was for the first time discussed and deliberated in this work. Based on a sandwich-type assay format, two types of colorimetric immunoassay protocols were designed and developed for the detection of rabbit IgG (RIgG, as a model) by using the synthesized ISPtNP and conventional HRP as the labeling of detection antibodies, respectively. Similar detection limits (LODs) of 2.5 ng mL(-1) vs. 1.0 ng mL(-1) were obtained toward RIgG with the ISPtNP labeling compared to HRP format. Intra- and inter-assay coefficients of variation were less than 13%. Importantly, the ISPtNP-based assay system could be suitable for use in a mass production of miniaturized lab-on-a-chip devices and open new opportunities for protein diagnostics and biosecurity.
基于天然酶标记探针的酶联免疫吸附测定（ELISA）方法已应用于免疫分析中，但大多数方法都存在一些不可避免的局限性（例如苛刻的制备、纯化和储存条件），不适合常规使用。在此，我们合成了一类新型的不规则形状的铂纳米粒子（ISPtNP），其平均长度为 7.0nm，沿纵轴的宽度从 2.0nm 变窄至 5.0nm，可用作过氧化物酶模拟物，开发比色免疫分析。与生物活性辣根过氧化物酶（HRP）相比，合成的 ISPtNP 对 3,3',5,5'-四甲基联苯胺（TMB）表现出较低的 Km 值（0.12mM）和较高的 Kcat 值（2.27×10(4)s(-1)），具有较强的热稳定性和 pH 耐受性。首次讨论和审议了 ISPtNP 对 TMB/H2O2 的催化机制。基于夹心型测定模式，设计并开发了两种比色免疫分析方案，分别使用合成的 ISPtNP 和传统 HRP 作为检测抗体的标记，用于检测兔 IgG（作为模型的 RIgG）。与 HRP 格式相比，使用 ISPtNP 标记检测 RIgG 时，检测限（LOD）相似，均为 2.5ngmL(-1)vs.1.0ngmL(-1)。内和间分析的变异系数均小于 13%。重要的是，ISPtNP 为基础的检测系统可适用于大规模生产微型化的芯片实验室设备，并为蛋白质诊断和生物安全开辟新的机会。
