## 3 Mosquito-Borne Infections
### Proteins  
Antibodies are used in immunoassays to detect protein biomarkers associated with infectious diseases. [PERSON] et al. used SERS nanoprobes in an immunoassay to detect Zika virus [97]. They used gold shell-isolated nanoparticles as the SERS substrate, where a Raman reporter (Nile Blue) is embedded in a silica shell to prevent leaching and the silica surface is functionalized with monoclonal anti-Zika NS1 antigens. This method was able to ensure high specificity that they observed no cross-reactivity with DENV NS1 antigens and to provide 10 ng/mL LOD for ZIKV NS1. In 2010,  
Figure 4: (**a**) Direct detection of _plasmodium falciparum_, malaria parasite, using an unamplified DNA and RNA biomarker. RNA biomarker sequence hybridized to the capture probe sequence on a magnetic bead and the reporter probe sequence on an ultra-bright SERS nanorattle with in a lab-in-a-stick device detection scheme. (**b**) SERS spectra displaying malaria synthetic DNA versus blank and (**c**) Malaria synthetic DNA with a concentration range of 10 fm to 1 nM. (**d**) Quantification of target DNA using the SERS intensity at the reporter signature peak at 930 cm\({}^{-1}\), (**e**). Malaria _P. falciparum_ 18s rRNA detection in blood lysate. Reproduced with permission from Reference [96] Scientific Reports (2018). [PERSON] et al. focused on designing a SERS immunoassay to detect the WNV. They used WNV antigen-coated gold nanostars and matchite green Raman reporter-conjugated proteins. When the antigen functionalized gold nanostars captures the target WNV proteins, it can be detected using the Raman spectral signature of matchite green. The LOD of this SERS immunoassay was determined to be 2 ng/mL while resulting sensitivity of 50 pg/mL [98]. Later, they designed another type of SERS immunoassay to detect the surface envelope and capsid antigens of WNV and RVF. Raman reporter coated-gold nanoparticles and paramagnetic nanoparticles were conjugated with disease specific polyclonal antibodies, which resulted LOD near 5 fg/mL [99]. Then, in 2018, they reported multiplexed immunoassay SERS for the detection of viral pathogenic antigens associated with WNV, RVF, and Yersinia pestis [100]. Both magnetic nanoparticles and SERS reporter-embedded silica-coated gold nanoparticles were functionalized with the polyclonal antibodies in order to capture the disease specific antigens. An external magnetic field was used to aggregate the antigen-, magnetic nanoparticle-, and silica-coated gold nanoparticle complex. The unique SERS spectrum of the Raman tags embedded in the silica-coated gold nanoparticles was then used to indirectly detect each captured antigen, which has enabled the multiplexed SERS-based protein biomarker detection with the LOD at 10 pg/mL. Protein-induced plasmonic nanoparticle aggregation has also been used to detect protein biomarkers using their intrinsic SERS signatures (direct SERS). [PERSON] et al. used gold nanostars (GNS) to design an approach for label-fee direct SERS detection of Enterovirus 71 (EV71) [101]. This approach is very attractive for POC diagnosis given the fact that it is based on the intrinsic SERS of proteins (Figure 5). In this study, GNS were conjugated to a recombinant scavenger receptor class B, member 2 (SCARB2) protein, which has an established affinity for EV71. When the GNS-SCARB2 is introduced to the infected sample, they conjugate to EV71 hence facilitating the direct detection of EV71 while preventing GNS aggregation. This method has yield LOD comparable to other approaches at 10\({}^{6}\) to 10\({}^{8}\) pfu/mL. In addition, the high sensitivity (10\({}^{7}\) pfu/ mL), low amount of sample preparation, and only 15-minute analysis time are favorable characteristics, which make this assay more appealing for POC applications. Recently, [PERSON]'s group developed a multiplexed SERS-based dipstick immunoassay to distinguish between Zika and dengue [102].