## Bacterial Infections
### Nucleic Acid Biomarkers
The complete hybridization of these three sequences is exhibited by the unique SERS signal of the Raman tag, indicating the presence of target DNA and resulting in a low LOD. Recently, [PERSON]'s group also developed a new assay format for multiplexed SERS-based indirect detection and quantification of three meningitis pathogens using a sandwich assay [5]. They used two modified complementary DNA sequences to the target DNA sequence, a 3'-biotinylated modified sequence (capture probe) and a 5'-Raman tag-modified sequence (reporter probe). The newly formed duplex is retained on a streptavidin functionalized bead. Then, they used l-exonuclease to digest the double-stranded DNA. The DNA sequences are designed in such a way that the digestion product contains the Raman tag. The digestion products were then added to a solution containing silver nanoparticles for the SERS detection of the digestion product. The complete hybridization of these three sequences provides the unique SERS signal of the Raman tag, indicating the presence of the target DNA and resulting the LOD in the picomolar range. This approach provides higher specificity due to the hybridization between the target and two complementary sequences. Direct, label-free SERS detection of nucleic acids has also been used despite common indirect detection methods [73; 74]. [PERSON] et al. introduced a new class of label- free (direct SERS) SERS-based assay to detect _Neisseria meningitidis_, _Streptococcus pneumoniae_, and _Haemophilus influenzae_ in cerebrospinal fluid (CSF) using their unique cell membrane biomarkers such as lipids and polysaccharides [74]. Their assay is based on the Au-Ag coated polycarbonate membranes that allow simultaneous filtration of CSF and immobilization of CSF components to enhance their intrinsic  
Figure 1: (**a**) An indirect surface-enhanced Raman spectroscopy (SERS)-based method for PCR-amplified pathogenic DNA detection. This method utilizes a sandwich hybridization event between a capture DNA sequence on gold nanowires and Raman reporter-labeled DNA sequence on with a target pathogenic DNA. (**b**) SERS spectra from each of the four nanowire systems in the presence of only the target DNAs of _Enterococcus facetium_ and _Stenotrophomonas maltolilia_. SERS spectra of the Raman reporter (Cy5) detected only for _E. facetium_ and _S. maltolophilia_ sensors. (**c**) SERS-based identification of amplified pathogenic DNA in clinical samples. (**d**) SERS-based multiplexed detection when all four target DNAs are present at \(10^{-8}\) M each. Reproduced with permission from Reference [66] American Chemical Society (2010). SERS signature. This approach facilitates the direct detection of nitrogenous bases of target DNA and neopterin at the single-bacteria cell level. Direct detection of nucleic acids can be challenging because of the background noise from the biological matrix. SERS-based diagnostic methods using clinical samples have also been conducted to determine the applicability towards POC applications [78; 79; 80; 87; 88; 89; 90]. [PERSON]'s group reported a novel sandwich SERS immunoassay for an antigenic biomarker for tuberculosis found in bodily fluids using a handheld Raman spectrometer [90]. They used serum samples to detect phospho-myo-inositol-capped lipoarabinomannan (PILAM), a stimulant of an antigenic biomarker found in tuberculosis infected patients. Their method requires sample pretreatment in order to eliminate any possible non-specific interaction between the PILAM and the other constituents found naturally in the serum sample. It is important to eliminate unnecessary biological interactions to ensure high detection specificity and, more importantly, to remove the biological matrix interferences to achieve high signal-to-noise ratio in SERS signal for high analytical sensitivity. The sample was acidified in order to denature proteins that could bind to PILAM. These denatured proteins and insoluble components were separated from the PILAM by centrifugation.