## Bacterial Infections
### Nucleic Acid Biomarkers  
Nucleic acids are a well-established group of biomarkers for detecting bacterial infections [5; 66; 71; 72; 73; 74]. Knowing the pathogenic DNA and RNA sequences facilitates the design of complementary strand(s) to specifically capture pathogenic RNA or single-stranded DNA, and hence, to identify them either directly or indirectly using SERS [75; 76; 72]. The nature of DNA and RNA, however, poses challenges in preparing sample to develop SERS-based diagnostic assays. The presence of smaller copy numbers of pathogenic nucleic acid biomarkers at the early stages of disease onset and their extraction and isolation from sample matrices make nucleic acid biomarker detection with no amplification very challenging. Therefore, as a common practice, synthetic nucleic acid sequences and also polymerase chain reaction (PCR)-amplified nucleic acid biomarkers from clinical samples are usually used in the proof-of-concept assays to optimize the assay parameters, and hence, the assay performance. Once the assay is optimized for analytical sensitivities and specificities, analysis in real biological/clinical samples with no nucleic acid amplification is carried out for field-transferable adaptation and optimization of the SERS-based diagnostic assays. Most of the studies have used cultured bacterial suspensions to test SERS-based assays as a preliminary step for proof-of-concept testing [77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 7]. The extraction of the DNA/RNA from the cultured bacterial cells of interest is crucial in order to remove background noise from other biomarkers, like cell membrane and metabolite biomarkers. The extracted nucleic acid is often amplified using PCR in order to reach the analytical sensitivities of the instrumentation as the concentrations of nucleic acid biomarkers are very low at early disease stages. For instance, [PERSON]'s group developed a combinatorial approach using PCR and SERS to detect epizootic pathogen DNA from cultured cells [72]. However, the sample preparation can be extensive when using PCR as it requires culturing of bacterial cells to extract DNA, followed by extensive purification and isolation steps. Clinical/biological samples have high background noise in SERS assays, which originates from the biological matrix in which the target DNA resides, therefore it requires more sample preparation. While sample preparation for nucleic acid detection has its inherent challenges, SERS-based high sensitivities at the picomolar to attomolar levels can still be achieved. For instance, in 2010, [PERSON] et al. used bacterial genomic DNA as the biomarker for four different pathogenic strains of bacteria and reported a limit of detection (LOD) of each pathogen ranging from 10 pM-10 nM using indirect SERS [66]. In their method, the target genomic DNA from four bacterial pathogens that causes various infections were first extracted and then amplified by PCR. The target DNA biomarkers for this study originated from reference bacteria and as well as clinically isolated samples such as cerebrospinal fluid, stool, pus, and sputum. Among SERS-based nucleic acid detection methods, direct and indirect sandwich assays are the most common approach. It involves hybridization of a target nucleic acid sequence (target probe) with a capture sequence (capture probe) and reporter sequence linked to a Raman tag (reporter probe) and a plasmonic nanoparticle. For instance, [PERSON] et al. presented an indirect SERS detection of PCR-amplified pathogenic DNA using a sandwich assay (Figure 1) [66]. They used two modified complementary DNA sequences, a 3'-thiol modified sequence attached to a gold nanowire (capture probe) and a 5'-Raman tag-modified sequence attached to gold nanoparticles through the 3'-thiol functional group (reporter probe), that hybridize specifically to two different sequence positions of the target DNA sequence.