## 4 SERS-Based Diagnostic Devices and Assay Platforms
They investigated the effect of the morphological features of gold nanoparticles (spherical vs. star-shaped and the varying tip curvature of gold nanostars) as SERS substrates on the sensitivity of SERS-based LFA [70]. The SERS enhancement of the Raman reporter molecules 1,2-bis(4-pyridyl)ethylene and 4-mercaptoethanol acid were optimized and compared using different types of gold nanoparticles for multiplexed detection of zika and dengue non-structural protein 1 (NS1). They concluded that the nanoparticles with sharp tips such as nanostars provide higher enhancement factors, hence enabling higher sensitivities in SERS immunoassay. In addition, they also observed that the SERS intensity distribution within the test zone is greater in the area where the fluid first encounters the immobilized antibody, and such factors can be used to strategically design LFA to decrease the overall assay cost. In 2018, the same group focused on selecting Raman reporters for quantitative multiplexed SERS detection of protein (IgG) biomarkers in a dipstick immunoassay [125]. In this study, gold nanostars conjugated to polyclonal anti-human IgG antibodies were tagged with 15 different Raman reporters. They used machine learning to evaluate the spectral overlap and quantitative contribution of Raman reporters. Using the information gathered from the comprehensive correlation matrix analysis, they were able to select multiple reporters that provide sufficient spectral resolution and higher quantitative accuracy in multiplexed SERS-based igG biomarker detection in LFAs. Microfluidic SERS immunoassays using antigen-antibody interactions are also a popular device architecture for POC infectious disease detection in clinical samples [7; 73; 124]. [PERSON] et al. developed a conceptually new SERS diagnostic assay integrated into a microfluidic devices for duplex detection of two antigen biomarkers of the _Entamoeba histolytica_ pathogen [7]. The bioassay platform was designed using a glass chip-coated with Raman tags-encoded gold nanoparticles (SERS substrate) which was then then fixed into a microfluidic device (Figure 8). The glass chip (SERS substrate) is comprised of multiple channels that carry different Raman tags-encoded gold nanoparticles for multiplexed detection of antigens. In order to reduce the cost associated with the device fabrication, this platform used inexpensive nanoyeast single-chain variable fragments (NYscFv) reagents instead of antibodies as the recognition moieties of antigen biomarkers. Using this new SERS-based microfluid bioassays, they achieved highly specific and highly sensitive detection of individual _E. histolytica_ antigen EHI_15350 and EHI_182030 at the LOD of 1 pg/mL and 10 pg/mL, respectively. Overall this microfluid device exhibits greater potential as a SERS-based analytical platform for high throughput multiplexed detection of pathogen antigens. [PERSON] et al. introduced a microfluidic system coupled to a \"lab-on-a-chip\" system that enables label-free SERS-based rapid detection and differentiation of _Staphylococcus aureus_ (MRSA) and methicillin-sensitive _S. aureus_ (MSSA) in clinical isolates [73]. This integrated device is based on the working principle of an optofluidic system where the SERS detection is carried under controlled flow conditions as opposed to traditional stationary conditions. A microfluidic SERS-based assay with an optofluidic design provides many advantages such as highly reliable handling of small sample volumes and high sample throughput. By coupling the novel  
Figure 8: SERS-based diagnostic devices and assay platforms. Lateral flow assay (LFA) designs: (**a**) Representation of a traditional LFA and a SERS-based LFA that utilized Raman reporter-labeled hollow gold nanospheres to detect the presence of _staphylococcal enterotoxin_ B (SEB). (**b**) Average SERS spectra at the mapping area of the test zone for varying concentrations of SEB. (**c**) SERS maps acquired using a peak intensity at 1650 cm\({}^{-1}\) for varying concentrations of SEB concentrations.