Yan Yingchun, Nie Yong, An Liyun, Tang Yue-Qin, Xu Zimu, Wu Xiao-Lei
Institute of New Energy and Low-carbon Technology, Sichuan University, Chengdu, China.
College of Engineering, Peking University, Beijing, China.
Front Bioeng Biotechnol. 2020 Sep 17;8:573777. doi: 10.3389/fbioe.2020.573777. eCollection 2020.
Surface-enhanced Raman scattering (SERS) is a useful tool for label-free analysis of bacteria at the single cell level. However, low reproducibility limits the use of SERS. In this study, for the sake of sensitive and reproducible Raman spectra, we optimized the methods for preparing silver nanoparticles (AgNPs) and depositing AgNPs onto a cell surface. We found that fast dropwise addition of AgNO into the reductant produced smaller and more stable AgNPs, with an average diameter of 45 ± 4 nm. Compared with that observed after simply mixing the bacterial cells with AgNPs, the SERS signal was significantly improved after centrifugation. To optimize the SERS enhancement method, the centrifugal force, method for preparing AgNPs, concentration of AgNPs, ionic strength of the solution used to suspend the cells, and density of the cells were chosen as impact factors and optimized through orthogonal experiments. Finally, the improved method could generate sensitive and reproducible SERS spectra from single cells, and the SERS signals primarily arose from the cell envelope. We further verified that this optimal method was feasible for the detection of low to 25% incorporation of C isotopes by the cells and the discrimination of different bacterial species. Our work provides an improved method for generating sensitive and reproducible SERS spectra.
表面增强拉曼散射（SERS）是一种用于在单细胞水平对细菌进行无标记分析的有用工具。然而，低重现性限制了SERS的应用。在本研究中，为了获得灵敏且可重现的拉曼光谱，我们优化了制备银纳米颗粒（AgNPs）以及将AgNPs沉积到细胞表面的方法。我们发现，将硝酸银快速滴加到还原剂中会产生更小且更稳定的AgNPs，平均直径为45±4纳米。与简单地将细菌细胞与AgNPs混合后观察到的情况相比，离心后SERS信号显著增强。为了优化SERS增强方法，选择离心力、AgNPs的制备方法、AgNPs的浓度、用于悬浮细胞的溶液的离子强度以及细胞密度作为影响因素，并通过正交实验进行优化。最终，改进后的方法能够从单个细胞产生灵敏且可重现的SERS光谱，并且SERS信号主要来自细胞包膜。我们进一步验证了这种优化方法对于检测细胞中低至25%的碳同位素掺入以及区分不同细菌种类是可行的。我们的工作提供了一种用于生成灵敏且可重现的SERS光谱的改进方法。
