The handheld SERS biosensor successfully identified

The handheld SERS biosensor successfully identified bacteria at the species level from pure culture and serum samples. Strain level detection was not observed, Fig. 3. Discrimination between bacterial strains is advantageous because many strains of a single species have differences with respect to properties such as pathogenesis, antibiotic resistance, biofilm production, and generation of toxins. Nanoparticle substrates are capable of generating sufficiently large enhancement factors to discern between bacterial strains, which necessitate spectrometer instrumentation to be on an equal level with respect to measurement sensitivity. Microfluidic SERS systems have achieved strain level resolution with support vector machine accuracy of 92% [42]. Hardware improvements to the handheld SERS biosensor are necessary to reach strain level resolution. Poly-microbial detection is another major concern as more than one species will likely be present in a chronic infection. The separate PCA score grouping of the combined pure culture sample of A. baumannii and S. aureus, Fig. 4, demonstrates the potential to identify the presence of a mixed species of Gram positive and Gram negative bacteria. Additional studies will need to be pursued to determine SERS spectra of mixed cultures as a function of more species and varying cell concentration. The effects of serum processing on SERS spectra of poly-microbial populations also need to be addressed.
Conclusions The effective recovery of live bacteria is important for consistent whole cell SERS characterization. The SERS biosensor utilized in this study was capable of detection, identification, and classification of a majority of the bacteria of military interest sampled from human serum. Bacteria were identifiable at the species level, and the potential for detecting poly-microbial cultures by the unique spectra that they generate was demonstrated. Raman signal for recovered bacteria is likely generated from a combination of inactive cells, active viable cells, and altered cell membranes, but it should be noted that SERS measurement is sensitive to even the smallest changes in biomolecular composition. Lysis ubiquitin conjugating enzyme successfully purified hydrophilic bacteria without significantly affecting Raman spectra. However, shifts in relative peak intensities of SERS spectra for hydrophobic bacteria due to lysis filtration were seen and must be overcome. Generation of a reference library using pure culture samples can be effective for identifying hydrophilic bacteria such as A. baumannii and S. aureus. Bacteria sensitive to lysis filtration will require a more complex reference library for SERS identification or milder conditions for separation.
Acknowledgment The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. The authors are employees of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person\'s official duties. This work was funded by BUMED Advanced Medical Development, supported by work unit number G1211.
Introduction Currently, the most widespread technique to detect antigens is enzyme-linked immunosorbent assay (ELISA). This method uses a cascade of interactions between antibodies and antigens leading to a visual response that indicates the presence or absence of a given antigen or substance [1]. There are several different ELISA methods, such as direct and sandwich assays [2–4], but the underlying feature is the requirement for antibody–antigen binding to a surface followed by a secondary reaction to produce a response. This multi-step process always starts by binding the target antigen, either directly or via a capture antibody, to the surface of the assay chamber. The next step is blocking unbound sites with an unrelated protein-based solution. The third step is detection, where an antibody, conjugated with an enzyme such as horseradish peroxidase (HPR), binds the target antigen. The addition of a chromogenic substrate for the enzyme then creates a quantifiable signal. Washing processes separate each of these steps. Therefore, ELISA is a relatively lengthy process typically performed in a laboratory environment. Examples for other developing techniques for biosensors include surface plasmon resonance (SPR) [5] and microfluidics [6].