## 3 Mosquito-Borne Infections
### Nucleic Acid Biomarker Detection  
Nucleic acids, both DNAs and RNAs (coding and non-coding), are valuable biomarkers for infectious disease detection. In 2011, [PERSON] et al. designed a proof-of-concept SERS assay to detect DNA oligonucleotides obtained from the West Nile virus (WNV) genome. They used the classical sandwich assay where hybridization between the complementary oligonucleotide probes covalently linked to paramagnetic nanoparticle (capture probe) and Raman-reporter conjugated-gold nanoparticles (reporter probe) and the target DNA sequence to design an indirect SERS assay for WNV detection [92]. They later simplified this method to do multiplexed detection of viral DNA of WNV and  
Figure 3: (Top Left) Sample preparation steps for urine samples spiked with bacterial strains of urinary tract infection (UTI) clinical isolates for SERS-based direct detection of nucleic acid metabolites. (Right) SERS spectra of 12 different UTI bacterial strains exhibiting unique biomarker signatures that were produced from sample on an aggregated gold nanoparticle-covered silica substrate. (Bottom Left) Intrinsic SERS spectra of seven key metabolite biomarkers used in the differentiation of the 12 UTI bacterial strains. Reproduced with permission from Reference [87] [PERSON] (2017). Rift Valley virus (RVFV) and also intensify the SERS response by magnetic enrichment [93]. They used a capture probe attached to a gold-coated paramagnetic nanoparticle that have both magnetic and SERS properties, and a reporter probe with a Raman tag as the key components. Each of these probes has specific complementary oligonucleotide sequences to the opposing ends of the target WNV DNA sequence. In the presence of the target DNA, a hybridized complex between capture-target-reporter probe is formed. It is then magnetically enriched using Au-coated paramagnetic nanoparticle and the SERS signal is recorded. Herein, the capture of target DNA is indirectly detected by monitoring the SERS spectrum of the Raman tag associated with the reporter probe. The LOD was determined to be in the low nanomolar range (20-100 nM) requiring only 1 h for the assay. Lab-on-a-chip SERS substrates are another common approach for DNA biomarker detection. The [PERSON] group at Duke University developed a bioassay-on-chip using plasmonic bimetallic nanowire to detect dengue viral DNA [94]. They used gold and silver nanowires to develop a highly sensitive SERS substrate, which was subsequently functionalized with reporter probes carrying a Raman tag for dengue viral DNA. They designed a new type of reporter probe with a placeholder, which is covalently linked to the plasmonic nanowaves. In the absence of the target sequence, the reporter probe and the placeholder maintain a partially duplex complex. It keeps the Raman tag at the end of the reporter probe away from the SERS substrate at a distance where the SERS effect is negligible and therefore no significant SERS signal is detected--SERS \"off\" state. In the presence of target viral DNA, placeholder hybridizes with the target DNA, leaving the reporter probe to form a hair loop. This hair loop structure brings the Raman tag back into very close proximity to the SERS substrate (SERS \"ON\") hence producing a measurable SERS signal. The LOD of this approach was determined to be 6 attomoles. The same group later developed another simpler detection strategy based on sandwich DNA hybridization to detect malaria DNA biomarkers [95]. The target DNA was identified using single nucleotide polymorphism discrimination via ultrabright SERS nanoparticles known as nanoratlles and then enriched using magnetic beads. The ultrabright nanoratlles are functionalized with SERS reporter DNA probes and the magnetic beads are coated with the capture DNA probes. Once the target DNA is introduced, complexation of magnetic beads and nanoratlles takes place due to the sandwich DNA hybridization between target-capture and reporter DNA probes.