Quantification of biomolecules responsible for biomarkers in the surface-enhanced Raman spectra of bacteria using liquid chromatography-mass spectrometry

Abstract

Recently, specific biomarkers in the surface-enhanced Raman scattering (SERS) spectra of bacteria have been successfully exploited for rapid bacterial antibiotic susceptibility testing (AST) - dubbed SERS-AST. The biomolecules responsible for these bacterial SERS biomarkers have been identified as several purine derivative metabolites involved in bacterial purine salvage pathways (W. R. Premasiri, J. C. Lee, A. Sauer-Budge, R. Theberge, C. E. Costello and L. D. Ziegler, Anal. Bioanal. Chem., 2016, 408, 4631). Here we quantified these metabolites in the SERS spectra of Staphylococcus aureus and Escherichia coli using ultra-performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS). The time dependences of the concentrations of these molecules were measured using C- orC-purine derivatives as internal and external standards respectively in UPLC/ESI-MS measurements. Surprisingly, a single S. aureus and an E. coli cell were found to release millions of adenine and hypoxanthine into a water environment in an hour respectively. Furthermore, simulated SERS spectra of bacterial supernatants based on the mixtures of purine derivatives with measured concentrations also show great similarity with those of the corresponding bacterial samples. Our results not only provide a quantitative foundation for the emerging SERS-AST method but also suggest the potential of exploiting SERS for in situ monitoring the changes in bacterial purine salvage processes in response to different physical and chemical challenges.

Rapid antibiotic susceptibility testing of bacteria from patients’ blood via assaying bacterial metabolic response with surface-enhanced Raman spectroscopy

“…These studies thus lay the foundation for the respective origins of the bacterial SERS spectra observed on E. coli and S. aureus. To be noted is that the SERS spectrum of E. coli is dominated by xanthine and hypoxanthine while that of S. aureus by adenine, as observed by Premasiri et al 33 and similarly by our group 34 . The agreement of SERS spectra between reference strains, clinical isolates and blood-culture isolates in our study also implicates the common metabolic profiles of purine derivatives of bacteria from these sources.…”

“…It would be very likely that only very few of all the identified bacterial metabolites (specifically, xanthine and hypoxanthine for E. coli and adenine for S. aureus) are primarily adsorbed on the SERS-active substrate to produce their respective Raman spectra. This preferential adsorption behavior is reflected by the studies of Premasiri et al 33 and our group 34 : the SERS spectra of adenine and xanthine-hypoxanthine mixture are identical to those of S. aureus and E. coli, respectively, though both bacteria secret more types of molecules. Therefore, the ratio between the SERS biomarker signals Techniques from the genomics tool box and studies involving transcriptomics, proteomics and metabolomics have helped reveal the behaviors of E. coli and S. aureus (the prototypical Gram-negative and Gram-positive bacteria, respectively) under differing stressed environments (e.g., nutrient starvation, low/high temperature, high salination, and antibiotic exposure).…”

Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media

“…This spectral modification can be attributed to chemical variations, more specifically, the deamination process, between adenine and hypoxanthine nitrogenous bases. [ 43 ] The molecular structure of both bases (see the inset in Figure 3a) reflects that adenine contains an extra amino group in the purine ring, compared to hypoxanthine.…”

Noble Metal Nanostructured Materials for Chemical and Biosensing Systems

“…Generally, the surface enhancement effect of metal surface on the Raman signal of target is as high as 10 3 -10 5 times, while Au and Ag nanomaterials with special structure can obtain higher enhancement factors, which are enough to detect single molecule or DNA chain. Therefore, SERS has become an important tool for the detection of biomacromolecules or small molecules [166][167][168]. Because organic pollutants, DNA, heavy metal ions and other targets cannot be effectively adsorbed on the surface of noble metal nano-substrate, it is difficult to detect by SERS directly.…”