Molecular Design and Synthesis Discipline, School of Chemistry, Physics and Mechanical Engineering, Science and Engineering Faculty, Queensland University of Technology, Brisbane, QLD, Australia.
SLAS Discov. 2020 Jan;25(1):87-94. doi: 10.1177/2472555219875102. Epub 2019 Sep 19.
The molecular structure of many proteins contains disulfide bonds between their cysteine residues. In this work we demonstrate the utilization of the disulfide bond structure of proteins for their label-free determination by surface-enhanced Raman spectroscopy (SERS). The new approach for label-free SERS detection of proteins is demonstrated for human insulin. The protein was selectively extracted from spiked plasma samples using target-specific functionalized nanomaterial. Enzyme-linked immune assay (ELISA) was used to detect insulin in the blood plasma and cross-validate the SERS method. The disulfide bonds in the molecular structure of the protein were chemically reduced and used for their chemisorption onto the gold-coated copper oxide substrate in a unified orientation at a very short distance from the hotspots. The oriented chemisorption of the protein caused significant enhancement to the signal intensity of its Raman vibration modes. This is attributed to the strong short-range electromagnetic and chemical enhancement effects that are experienced by the immobilized protein. Using this approach, label-free and reproducible SERS detection of insulin, down to 10 zM (relative standard deviation [RSD] = 5.52%), was achieved. Sixty-five percent of proteins contain disulfide bonds in their molecular structure. Therefore, the new label-free SERS detection method has strong potential for the determination of ultralow concentrations of proteins at pathology labs and in biology research.
许多蛋白质的分子结构中含有半胱氨酸残基之间的二硫键。在这项工作中，我们展示了利用蛋白质中二硫键的结构，通过表面增强拉曼光谱（SERS）对其进行无标记测定。我们展示了一种用于无标记 SERS 检测蛋白质的新方法，以人胰岛素为例。该蛋白质使用针对目标的功能化纳米材料从加标的血浆样品中选择性提取。酶联免疫吸附测定（ELISA）用于检测血浆中的胰岛素，并与 SERS 方法交叉验证。蛋白质分子结构中的二硫键被化学还原，并用于在很短的距离内以统一的取向化学吸附到金涂覆的氧化铜基底上，靠近热点。蛋白质的定向化学吸附导致其拉曼振动模式的信号强度显著增强。这归因于固定化蛋白质所经历的强短程电磁和化学增强效应。使用这种方法，可以实现低至 10 zM（相对标准偏差 [RSD] = 5.52%）的无标记和可重复的胰岛素 SERS 检测。65%的蛋白质在其分子结构中含有二硫键。因此，这种新的无标记 SERS 检测方法具有在病理学实验室和生物学研究中确定超低浓度蛋白质的强大潜力。
