Improvement of Surface-Enhanced Raman Scattering Method for Single Bacterial Cell Analysis.

Yingchun Yan,Liyun An,Yue-Qin Tang,Yong Nie,Zimu Xu,Xiao-Lei Wu

doi:10.3389/fbioe.2020.573777

Abstract

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 AgNO3 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 Escherichia coli 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 13C isotopes by the cells and the discrimination of different bacterial species. Our work provides an improved method for generating sensitive and reproducible SERS spectra.

Highlights

Microbes are the most diverse and abundant organisms on Earth and play crucial roles in biogeochemical carbon and nitrogen cycling
The average diameter of AgNPs-4, which were prepared by fast dropwise addition of hydroxylamine-hydrochloride into AgNO3 solution, was significantly smaller than that of AgNPs-3 prepared by slow dropwise addition
Single cell Surface-enhanced Raman scattering (SERS) has been reported for diverse applications in discrimination of bacterial species (Jarvis and Goodacre, 2004; Berus et al, 2020) and detection of pathogens (Boardman et al, 2016), how to improve the reproducibility and sensitivity is still an important issue

Summary

Introduction

Microbes are the most diverse and abundant organisms on Earth and play crucial roles in biogeochemical carbon and nitrogen cycling. Certain tools, including nanometerscale secondary ion mass spectrometry (NanoSIMS) (Orphan et al, 2001) and a combination of microautoradiography and fluorescence in situ hybridization (MAR-FISH) (Kindaichi et al, 2004), have been used to detect the functions and activities of single microbial cells in situ within complex communities Among these tools, Raman spectroscopy combined with microscopy (Raman microspectroscopy) has received increasing attention for label-free analysis of bacteria at the single cell level (Wagner, 2009; Huang et al, 2010; Li et al, 2012b). This method is often used to detect and screen carotenoidcontaining photosynthetic microorganisms (Li et al, 2012a) because it depends on the resonance Raman-active compounds, such as carotenoids

Methods

Results

Conclusion