# Enhancing Disease Diagnosis: Biomedical Applications of Surface-Enhanced Raman Scattering
## 6 Conclusions and Future Outlook  
In this review, we have discussed recent applications of SERS and shown that this method is a versatile physicochemical tool that can be used to extract diagnostic and quantitative information within cancers and microbial infections, as well as in respiratory disease. Both direct and indirect SERS play vital roles in targeted detection of specific biomarker in cells or human biofluids and their use have been extended to several diseases in need of urgent attention. However, to harness full analytical capabilities and to accelerate translation of SERS to the clinic, there are pending technical and methodological issues that need urgent improvements. We know very well that SERS peak intensity or area is linearly proportional to the concentration of sample analytes which facilitates accurate quantitative detection of diseases. However, due to saturation of NP surfaces in label-free SERS, whereby some molecules reside outside the LSPR, or instability of a radiation source and inconsistent cumulative aggregation, reproducibility and linearity are lost. To overcome this problem in quantitative analysis, a suitable internal standard (IS) can be integrated so that characteristic biomarker bands are normalised to distinct IS peak. This reduces spectral signal fluctuations since analyte and internal standard spectral bands are affected in exactly the same way, especially when isotopologues are used as illustrated recently [154; 155]. Alternatively the standard addition method can be used as this also accounts for any sample background as shown for the quantification of uric acid in the urine of pregnant individuals [156]. This way quantitative detection and prediction of disease/prognostic markers in a clinical environment would be more accurate and reliable. One other area which perhaps needs more attention to simplify SERS interpretation is the use of functionalised NPs (label-based SERS) for selective detection of specific markers within complex backgrounds. It is worth noting that reporters and capture species prevent undesirable aggregation of NPs and resolve known multiple biomarkers simultaneously without any prior separation or enrichment procedures. The use of SERS in parallel with multiparameter tools is also showing promising trends; it is clear that SERS has improved time between diagnostics and cure, specificity and quantitative analysis of biomarkers identified by chromatography and spectrometry. Moreover, several problematic issues, due to nonspecific binding or cross-reactivity among untargeted species, NPs and capture elements, especially polyclonal antibodies, which hampered detection of desirable targets, have been addressed in recent years. Generally, a series of washing steps and biocompatible coating materials such as polymers and silica as protective layers or core/encapsulation-shells have been applied successfully. The main objective here is to prevent leaching of Raman reporter and capture probes, enhance specificity and to shield interfering contaminants from accessing NP surfaces and paratopes of capture elements. Another fundamental area of focus for reliable interpretation of biomedical SERS in future is spectral band assignment. For bacterial SERS, metabolomics and isotopic labelling experiments have played an indispensable role; we now understand significant contributions of purines and pyrimidines to characteristic SERS spectral bands [157; 158]. Nonetheless, there is still a pressing need to formulate standard operating protocols and a unified library of reference biomaterials for SERS to allow for repeatability, unambiguous and detailed understanding of molecular dynamics and metabolic pathways. In light of this, it should be emphasised that only when pending problems are effectively overcome will SERS be convincingly relevant and acceptable to clinicians.