N.M. Emanuel Institute of Biochemical Physics RAS, Kosygina Str. 4, 119334 Moscow, Russia.
Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia.
Molecules. 2024 Feb 8;29(4):793. doi: 10.3390/molecules29040793.
The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an -phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.
基于辣根过氧化物酶（HRP）作为酶标记物、邻苯二胺（oPD）作为底物和 2,3-二氨基吩嗪（DAP）作为酶产物的基于表面增强拉曼散射（SERS）的酶联免疫吸附测定（ELISA）是分析化学中一个日益增长的领域，因为它具有潜在的高灵敏度。然而，这种众所周知的方法的全部功能尚未被揭示。本研究解决了 SERS 检测阶段性能的一个以前未被认识的问题。使用银纳米粒子和 oPD 和 DAP 的模型混合物，广泛评估了 pH 值、聚集剂浓度和颗粒表面氯离子稳定剂的影响。在最佳的微酸性 pH 值 3 下，使用 0.93 至 1 M 柠檬酸盐缓冲液和用 20 mM 氯离子稳定的 AgNPs，与比色法相比，DAP 和 HRP 的 SERS 检测限（LOD）提高了两个数量级。对于 HRP 的检测限为 0.067 pmol/L（每个测定 1.3 个毫摩尔），这表明所开发的方法是一种高灵敏度的技术。我们假设这种改进的检测系统可以成为开发基于 SERS 的 ELISA 协议的有用工具。
