## 3 Mosquito-Borne Infections
### Metabolites as Biomarkers  
Changes in the red blood cells (RBC) have been traditionally used to detect mosquito-borne infection, and it has also been found that some of the molecular changes in RBC can also be detected using SERS to potentially achieve high diagnostic sensitivities. [PERSON]'s group at the University of Georgia used the SERS spectral features apparent in RBC infected with _Plasmodium falciparum_ (iRBC) to differentiate varying stages of the parasite in the blood as iRBC at different post-invasion times exhibit different cell membrane modifications [104]. In this study, they analyzed and compared the intrinsic spectral signatures of healthy RBC and iRBC on silver nanorod array substrates. For instance, a unique SERS peak at \(\Delta\)v = 1599 cm\({}^{-1}\) for ring stage iRBCs, and a SERS speak characteristic to trophozoite, and schizoid stages were at \(\Delta\)v = 723 cm\({}^{-1}\) identified while none of them were present in healthy RBC (Figure 7). This intrinsic SERS-based assay reported 1.5 \(\times\) 10\({}^{7}\)/mL LOD for both RBCs and iRBCs. In addition, there are several other reports that uses either Raman or resonance Raman to identify the erythrocyte membrane alterations at iRBC at different post-invasion times [105; 106; 107; 108].  
Hemozoin, a metabolite found in malaria-infected blood has also been used as a biomarker for early malaria diagnosis [109; 110; 111; 112]. In 2012, [PERSON] et al. used SERS-active magnetic nanoparticles composed of iron oxide core and silver shell to detect hemozoin in iRBC based on its intrinsic SERS signature [113]. Hemozoin, which is paramagnetic in nature, was enriched using an external magnetic field on the iron oxide core and then the amplified SERS signal on silver shell was measured. They observed that the SERS signal without the use of the magnetic field is about two orders of magnitude lower than signals reported without the magnetic enrichment (Figure 7). The LOD for \(\beta\)-hematin was determined to be 5 nM, which is approximately equivalent to 30 parasites/\(\mu\)L present at the early stages of malaria infection [113]. The same group later developed a method for SERS-based hemozoin detection in iRBC using only plasmonic nanoparticle, but with no need for an external magnetic field [109]. This improvement was done in order to eliminate any additional variation in SERS readings that could have originated from the magnetic enrichment and hence improve the SERS-based quantification capability. They used two approaches; (i) synthesized silver nanoparticles in the blood or (ii) pre-synthesized silver nanoparticles added to a blood sample. The first approach has exhibited lower variation in the SERS measurement, which could be more suitable for quantification of the parasitemia level. On the other hand, the second method has resulted in a higher sensitivity, with the LOD as low as 0.00005% parasitemia level in the ring stage, which is approximately 2.5 parasites/\(\mu\)L of blood. The reported LOD is comparable to the most sensitive detection techniques currently available, thus this approach could be more effective in early malaria diagnosis. [PERSON] et al. also developed a novel SERS platform, which is composed of gold-coated butterfly wings for SERS-based direct detection of malarial hemozoin in lysed blood samples [114]. Using this new SERS platform, they identified key characteristic spectral features of the hemozoin in the iRBC lysate (e.g., strong pyrrole in-phase breathing vibration peak at 1375 cm\({}^{-1}\)) that indicate the presence of early-ring stage parasitemia levels and provided the LOD between 0.0005% and 0.005%.