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  • Advancement in diagnostic capabilities is an important step

    2018-11-05

    Advancement in diagnostic capabilities is an important step to improving outcomes of trauma-related infections. Rapid diagnosis, treatment, and control of bacterial infections are necessary to reduce morbidity and mortality. Raman spectroscopy combined with nanotechnology is a potential platform upon which an effective novel diagnostic system can be developed. Devices based on spectroscopic techniques are fast, efficient, and have the potential to reduce the overall time of diagnosis while requiring little to no technical expertise and additional equipment. Raman spectroscopy is a vibrational spectroscopic technique for ‘molecular fingerprinting’ of tissues, cells, proteins, nucleic acids, and other small organic and inorganic compounds. Raman spectroscopy has been investigated for use in prediction of preterm birth [35] and for the diagnosis of several pre-cancerous and malignant pathologies including basal cell carcinoma, leukemia, prostate cancer, and dysplastic growth in Barrett\'s dpp-iv inhibitors [6,10,30,34]. The addition of nanotechnology further improves the capabilities of Raman spectroscopy. Surface enhanced Raman scattering (SERS) was developed by utilizing nanoparticles as enhancing substrates to improve overall Raman signal intensity, resolution, and limits of detection down to single molecules. Silver nanorod (AgNR) substrates have been developed for detection and identification of viral and bacterial pathogens [11] such as E. coli[43], pathogenic mycoplasmas [23], and the human immunodeficiency virus [18]. Enhancement factors of up to 5 x 108 have been reported with the length of nanorods directly affecting the measured SERS signal intensity [11,18]. Identification and discrimination of bacteria at the species and strain level using AgNR substrates have been demonstrated [14]. The rapid measurement time, ease of operation, and field deployable potential make this technology a promising alternative infection diagnostic tool for military and civilian caregivers alike. Benchtop Raman microscopes have been the gold standard for characterization of bacteria with SERS. Benchtop SERS systems are capable of label free detection in situ[12], measuring multiple species and strains of bacteria simultaneously [37], discerning between species in mixed bacterial cultures [22], and integrating with microfluidic devices [42]. Raman microscopes are powerful with respect to their resolution and sensitivity, but size and cost restrict their use to the laboratory setting. Raman systems such as the Enwave Raman HRC-10HT [45] and the handheld FirstDefender RM [7] have been developed to address concerns of device footprint and portability. Recently the use of handheld Raman spectrometers for use in SERS identification of bacteria filtered from bean sprout samples have been demonstrated [43]. The objective of this research was to expand the number of bacterial species examined with a field deployable SERS system. The SERS diagnostic system consisting of a handheld Raman spectrometer and multi-well AgNR substrates was characterized with multiple pathogenic bacterial species and strains. Furthermore, the effects of pooled human serum lysis filtration on handheld SERS spectra of pathogenic bacteria were determined in order to characterize the separation of bacteria from increasingly complex biological media.
    Materials and methods
    Results
    Discussion The purpose of the present study was to determine if a handheld SERS diagnostic system could accurately detect and identify pathogenic bacteria separated from human serum. The handheld SERS system with AgNR substrates measures the extracellular, whole cell spectra of bacteria. If no changes occur to the bacteria, the SERS spectra are a characterization of the cell envelope with intensity correlating to the contents of the bacterial cell wall and plasma membrane. The SERS spectra of preloaded samples are representative of the intact, living cell. The pattern of major SERS intensity bands observed between 650 and 800cm are highly similar to SERS spectra of E. coli from mung bean sprout samples with high intensity peaks at 654cm, 730cm, and 795cm[43]. Bacterial cell wall constituents normally characterized by SERS include peptidoglycans, phospholipids, glycoproteins, and lipopolysaccharides [19,40]. The relative SERS peak intensity changed for all bacteria to varying extents after separation from serum. All recovered samples of E. coli and K. pneumoniae were unclassifiable by PLSDA when utilizing a pure culture reference library, which was due to the additional peaks of the recovered samples that were not accounted for by the model data set, Table 1. The change in position of bacterial groupings observed using PCA, Fig. 2, reflects the changes to the bacterial cell surface and biomolecular composition due to lysis filtration.