Plasmonic and Electrostatic Interactions Enable Uniformly Enhanced Liquid Bacterial Surface-Enhanced Raman Scattering (SERS)

Abstract

Surface-enhanced Raman spectroscopy (SERS) is a promising cellular identification and drug susceptibility testing platform, provided it can be performed in a controlled liquid environment that maintains cell viability. We investigate bacterial liquid-SERS, studying plasmonic and electrostatic interactions between gold nanorods and bacteria that enable uniformly enhanced SERS. We synthesize five nanorod sizes with longitudinal plasmon resonances ranging from 670 to 860 nm and characterize SERS signatures of Gram-negative Escherichia coli and Serratia marcescens and Gram-positive Staphylococcus aureus and Staphylococcus epidermidis bacteria in water. Varying the concentration of bacteria and nanorods, we achieve large-area SERS enhancement that is independent of nanorod resonance and bacteria type; however, bacteria with higher surface charge density exhibit significantly higher SERS signal. Using cryo-electron microscopy and zeta potential measurements, we show that the higher signal results from attraction between positively charged nanorods and negatively charged bacteria. Our robust liquid-SERS measurements provide a foundation for bacterial identification and drug testing in biological fluids.

Keywords: bacteria; cryo-electron microscopy; gold nanorods; infectious disease; surface-enhanced Raman spectroscopy (SERS).

Figure 1

Overview of liquid-SERS chamber and plasmonic nanoparticles employed. (A) Liquid well imaging setup with bacteria (green) and nanorods (gold) both suspended in water and mixed together in the well. The well is fabricated using a Si wafer base and a double-sided adhesive spacing layer and sealed with a coverslip (see also Figure S15). (B) Absorption spectra of nanorods in the liquid well with longitudinal plasmon resonances ranging from 670–860 nm. Inset shows aspect ratios plotted against peak resonance wavelength. The dashed line indicates the 785 nm incident laser used for measurement. The region from ∼835–915 nm (annotated with redline) represents the bacterial Raman shift region. (C) TEM (left) and SEM (right) of synthesized gold nanorod particles used for liquid SERS measurement, showing monodispersity in nanorod size and shape. Additional images are included in Figure S1.

Figure 2

Spectral enhancement consistency across a large surface area of the well and among multiple liquid-SERS chambers. (A) Comparison of E. coli Raman spectra with and without the 670 nm nanorods, acquired at 60 s with 5 accumulation, showing no significant spectral signature from liquid E. coli samples without nanorods. (B) Liquid-SERS spectra of the four bacterial species mixed with the 670 nm nanorods with the average and standard deviation of 15 measurements at 10 s acquisition across 100 × 200 μm² dimension on the well (inset in C). (C) Heatmap plot of the S. epidermidis spectra shown in B highlighting the consistency of enhancements across the large area. Each row is a spectrum recorded from unique locations on the well. The inset is a schematic showing the xy map region scanned on the liquid well.

Figure 3

SERS spectra comparison across nanorods and bacteria. (A) S. epidermidis spectra combined with the five NRs, color coded and arranged such that the most blue-shifted is on top and most red-shifted at the bottom. (B) S. aureus, (C) E. coli, and (D) S. marcescens SERS spectra when mixed with the five nanorods tested. (E) Area under the peak values for signature bacterial peaks near 1000, 1300, and 1500 cm^–1 showing higher values for S. epidermidis and S. aureus, which have higher negative surface charge compared to E. coli and S. marcescens. The bar plots shown are for the 670 and 863 nm resonant nanorods (see Figure S9.

Figure 4

Interaction between bacterial cells and nanoparticles and its effect on Raman signal enhancement. Cryo-EM images of S. epidermidis (left column) mixed with three NRs with longitudinal plasmon resonance peaks and color coding: (A) 670 nm, purple; (B) 717 nm, green; and (C) 860 nm, red, respectively. Similarly, E. coli (right column) mixed with three NRs with longitudinal plasmon resonance peaks and color coding: (D) 670 nm, purple; (E) 717 nm, green; and (F) 860 nm, red, respectively. The nanoparticles show tight binding to S. epidermidis cell surface compared to E. coli cell surfaces, as the former has a higher negative surface charge. Scale bar indicates 500 nm.