Department of Chemistry, Illinois State University, Normal, IL 61790, USA.
Analyst. 2017 Nov 20;142(23):4456-4467. doi: 10.1039/c7an01496a.
Antibody-modified gold nanoparticles (AuNPs) are central to many novel and emerging biosensing technologies due to the specificity provided by antibody-antigen interactions and the unique properties of nanoparticles. These AuNP-enabled assays have the potential to provide significant improvements in sensitivity and multiplexed analysis compared to conventional immunoassays. However, a major challenge for these AuNP platform technologies is the synthesis of stable antibody-AuNP conjugates that resist aggregation in high salt environments and biological matrices. Moreover, synthetic strategies to form stable conjugates often require different solution conditions, e.g., pH, for each unique antibody. Herein we describe our effort to develop an approach to chemically modify lysine residues on antibodies to facilitate the formation of stable antibody-AuNP conjugates over a wide pH range. In this work, we systematically investigated the immobilization of native and chemically modified antibodies to 60 nm citrate-capped AuNPs as a function of pH and evaluated the stability of the antibody-AuNP conjugate in a saline environment. We have developed a method to chemically modify the lysine residues on an antibody prior to conjugation to the AuNP that results in stable conjugates over a wide pH range (6.0-8.5). Amino acid analysis and zeta potential measurements of native and modified antibodies reveal that the requisite modification correlates with the number of lysine residues, and a reduction in positive charge contribution from protonated lysine is required to form stable, pH-independent conjugates. Furthermore, we demonstrate that the chemically modified antibodies maintain antigen-binding capabilities. We apply this novel conjugation strategy to develop a surface-enhanced Raman spectroscopy (SERS)-based assay for the accurate subtyping of avian influenza viruses.
抗体修饰的金纳米粒子（AuNPs）由于抗体-抗原相互作用提供的特异性和纳米粒子的独特性质，是许多新型和新兴生物传感技术的核心。与传统免疫测定相比，这些基于 AuNP 的测定法具有提高灵敏度和进行多重分析的潜力。然而，这些 AuNP 平台技术的一个主要挑战是合成稳定的抗体-AuNP 缀合物，使其能够抵抗高盐环境和生物基质中的聚集。此外，形成稳定缀合物的合成策略通常需要针对每种独特抗体使用不同的溶液条件，例如 pH 值。在此，我们描述了我们开发一种方法的努力，该方法旨在通过化学修饰抗体上的赖氨酸残基来促进在较宽 pH 范围内形成稳定的抗体-AuNP 缀合物。在这项工作中，我们系统地研究了将天然和化学修饰的抗体固定在 60nm 柠檬酸封端的 AuNPs 上作为 pH 函数的稳定性，并评估了在盐环境中抗体-AuNP 缀合物的稳定性。我们开发了一种在与 AuNP 缀合之前对抗体上的赖氨酸残基进行化学修饰的方法，该方法可在较宽的 pH 范围（6.0-8.5）内形成稳定的缀合物。天然和修饰的抗体的氨基酸分析和 zeta 电位测量表明，所需的修饰与赖氨酸残基的数量有关，并且需要减少质子化赖氨酸的正电荷贡献才能形成稳定的、与 pH 无关的缀合物。此外，我们证明了化学修饰的抗体保持抗原结合能力。我们将这种新的缀合策略应用于开发基于表面增强拉曼光谱（SERS）的禽流感病毒的准确亚型测定法。
