Driskell Jeremy D, Kwarta Karen M, Lipert Robert J, Porter Marc D, Neill John D, Ridpath Julia F
Institute for Combinatorial Discovery, Department of Chemistry, Ames Laboratory-U.S. DOE, Iowa State University, Ames, Iowa 50011-3020, USA.
Anal Chem. 2005 Oct 1;77(19):6147-54. doi: 10.1021/ac0504159.
The need for rapid, highly sensitive, and versatile diagnostic tests for viral pathogens spans from human and veterinary medicine to bioterrorism prevention. As an approach to meet these demands, a diagnostic test employing monoclonal antibodies (mAbs) for the selective extraction of viral pathogens from a sample in a chip-scale, sandwich immunoassay format has been developed using surface-enhanced Raman scattering (SERS) as a readout method. The strengths of SERS-based detection include its inherent high sensitivity and facility for multiplexing. The capability of this approach is demonstrated by the capture of feline calicivirus (FCV) from cell culture media that is exposed to a gold substrate modified with a covalently immobilized layer of anti-FCV mAbs. The surface-bound FCVs are subsequently coupled with an extrinsic Raman label (ERL) for identification and quantification. The ERLs consist of 60-nm gold nanoparticles coated first with a layer of Raman reporter molecules and then a layer of mAbs. The Raman reporter molecule is strategically designed to chemisorb as a thiolate adlayer on the gold nanoparticle, to provide a strong and unique spectral signature, and to covalently link a layer of mAbs to the gold nanoparticle. The last feature provides a means to selectively tag substrate-bound FCV. This paper describes the development of the assay, which uses cell culture media as a sample matrix and has a linear dynamic range of 1 x 10(6)-2.5 x 10(8) viruses/mL and a limit of detection of 1 x 10(6) viruses/mL. These results reflect the findings from a detailed series of investigations on the effects of several experimental parameters (e.g., salt concentration, ERL binding buffer, and sample agitation), all of which were aimed at minimizing nonspecific binding and maximizing FCV binding efficiency. The performance of the assay is correlated with the number of captured FCV, determined by atomic force microscopy, as a means of method validation.
对病毒病原体进行快速、高灵敏度且通用的诊断测试的需求，涵盖了从人类医学、兽医学到生物恐怖主义预防等多个领域。作为满足这些需求的一种方法，已开发出一种诊断测试，该测试采用单克隆抗体（mAb），以芯片规模的夹心免疫分析形式从样品中选择性提取病毒病原体，并使用表面增强拉曼散射（SERS）作为读出方法。基于SERS的检测优势包括其固有的高灵敏度和多重检测能力。通过从暴露于用抗猫杯状病毒（FCV）mAb共价固定层修饰的金基底的细胞培养基中捕获FCV，证明了该方法的能力。随后，表面结合的FCV与外部拉曼标记（ERL）偶联，用于鉴定和定量。ERL由60纳米的金纳米颗粒组成，首先涂覆一层拉曼报告分子，然后涂覆一层mAb。拉曼报告分子经过精心设计，可作为硫醇盐吸附层化学吸附在金纳米颗粒上，以提供强烈而独特的光谱特征，并将一层mAb共价连接到金纳米颗粒上。最后一个特征提供了一种选择性标记基底结合的FCV的方法。本文描述了该检测方法的开发过程，该方法使用细胞培养基作为样品基质，线性动态范围为1×10⁶ - 2.5×10⁸病毒/毫升，检测限为1×10⁶病毒/毫升。这些结果反映了一系列详细研究的结果，这些研究涉及几个实验参数（如盐浓度、ERL结合缓冲液和样品搅拌）的影响，所有这些研究旨在最小化非特异性结合并最大化FCV结合效率。该检测方法的性能与通过原子力显微镜测定的捕获FCV数量相关，以此作为方法验证的手段。
