## 1 Introduction
As the next generation of SERS substrates, nanoparticle assemblies and three-dimensional nanoscale plasmonic substrates were then emerged, and they contain plasmonic \"hot spots\", where the SERS enhancement factor is much greater [21; 40; 41; 42]. In these nanoparticle assemblies, Raman active molecules are chemically or physically placed at the narrow gap between nanoparticles (\"SERS hot spot\") where they can experience the maximum SERS signal enhancement [14; 29; 43; 44]. The evolution of SERS substrates has resulted in 10-12 orders of maximum Raman signal enhancement, which, under optimum condition, could facilitate even single-molecule detection. The molecular functional interface of a biosensor, which is the interface between the optical transducer (plasmonic nanomaterials in SERS) and the biological environment, is the key element that needs to be carefully designed in order to attain high specificity, and hence, optimize the performance of a biosensor. There are two main factors that need to be considered when designing a functional interface of a nanobiosensor on SERS-based detection; (i) the stability of the SERS substrate in a biological sample matrix and (ii) the molecular recognition moieties (proteins, peptides, nucleic acids, aptamers) that specifically capture biomarkers such as cell membrane proteins, polysaccharides, lipids, or nucleic acids or a matrix that capture and retain the biomarkers or pathogens or infected cells. In addition, the physical distance between the transducer and the analyte should be kept at an optimum, which is <5 nm, to obtain the higher SERS signal enhancement [14; 45; 46]. Therefore, the distance dependence of the SERS signal intensity should also be taken into consideration when designing the molecular recognition moieties and/or spacers. Polymer-derivatives, polyethylene glycol, and surfactants are usually used as capping ligands or surface passivation ligands to ensure the colloidal stability of plasmonic SERS substrates and also to control the non-specific surface adsorption of biomolecules in biological samples [47; 48]. Molecular reignition elements such as antibodies, enzymes, complementary nucleic acid, aptamers, and peptides are either use directly as it is or attached to a passivation ligand or a molecular spacer and then incorporated in the functional interface. Surface functionalization of SERS substrate with molecular reignition elements and surface passivation ligands takes the advantage of inherent high affinity of gold and silver nanomaterials towards moleculesbearing highly electronegative or charged atoms such as nitrogen, sulfur, oxygen, etc., [49]. Therefore, molecular recognition elements are usually modified to contain a functional group such as thiol (SH), primary amine (NH\({}_{2}\)), and carboxylic acid (COOH) that can be attached to SERS substrates through dative covalent bonds to ensure strong and lasting surface attachment [50; 51; 52; 53]. In some cases, molecular recognition elements such as proteins naturally possess primary amines or sulfur, which allows their direct attachment to nanoparticle surfaces. Ligands and molecular recognition elements modified with zwitterionic groups or the charged domains of the molecular reignition elements are also used to electrostatically attach them to plasmonic nanomaterial surfaces. Electrostatic interaction between the charged domains of the biomarkers, such as cell membrane proteins, are often used to directly capture, isolate, and retain the biomarker on plasmonic surfaces. In this case, the overall surface charge of the SERS substrate is modified using charged surface passivation ligands to tailor the functional interface depending on the biomarker of interest. SERS as an analytical tool has been integrated into various disease diagnostic assays, including infectious diseases in two main formats; (1) label-free direct detection and (2) Raman label-enabled indirect detection.