A universal and enzyme-free immunoassay platform for biomarker detection based on gold nanoparticle enumeration with a dark-field microscope
A universal and enzyme-free immunoassay platform for biomarker detection based on gold nanoparticle enumeration with a dark-field microscope
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一种通用的无酶免疫分析平台，用于基于金纳米粒子计数和暗视野显微镜的生物标志物检测。
作者：
Wu Xi;Li Tian;Tao Guangyu;Lin Ruoyun;Pei Xiaojing;Liu Feng;Li Na
开发一种无酶、非扩增的生物标志物检测策略，具有普遍性和易于实施，在临床诊断和治疗监测中具有核心重要性。在此，我们首次报道了一种通用的、无酶的、基于磁珠的生物标记物检测平台，该平台通过将二抗功能化的AuNP和自动AuNP计数读出相结合来进行生物标记物检测。对于前列腺特异性抗原（PSA），检测限为1 ng mL−1，10%胎牛血清的加标回收率（n = 3）为113.5%（2 ng mL−1）和107.7%（10 ng mL−1）。该试验还具有合理的重复性，如60天内5次测量的变异系数为13.1%所示。该方法已成功应用于癌胚抗原（CEA）和甲胎蛋白（AFP）的测定，证明了该方法的普适性。我们提出的非扩增平台具有与酶联免疫吸附测定（ELISA）相当的灵敏度，具有更好的重复性;更重要的是，我们的方法比大多数基于扩增的方法具有更好的简单性，因此更适合常规分析。我们工作的亮点表明，这是一种很有前途的方法，并且可能成为实验室中密集进行常规分析的ELISA的替代方案。
Developing an enzyme-free, non-amplification strategy for biomarker detection with universality and easy implementation is of central importance in clinical diagnosis and therapeutic monitoring. Herein, we report for the first time a universal and enzyme-free magnetic bead-based sandwich-format immunoassay platform for biomarker detection by combining secondary antibody functionalized AuNPs and automatic AuNP counting readout. For the prostate specific antigen (PSA), the detection limit is found to be 1 ng mL−1, and the spike recoveries (n = 3) with 10% fetal bovine serum are 113.5% for 2 ng mL−1 and 107.7% for 10 ng mL−1. The assay also presents reasonable repeatability as indicated by the coefficient of variance of 13.1% with 5 measurements in 60 days. This strategy has been successfully applied to the determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), demonstrating the universality of this strategy. Our proposed non-amplification platform presents sensitivity comparable to that of the enzyme-linked immunosorbent assay (ELISA) with better repeatability; and more importantly, our method has better simplicity than most of the amplification-based methods, and thus is more suitable for routine analysis. The highlights of our work suggest that it is a promising method and would be potentially an alternative for ELISA in laboratories where routine analyses are intensively performed.