# Enhancing Disease Diagnosis: Biomedical Applications of Surface-Enhanced Raman Scattering
## 5 Applications of SERS in Disease Diagnostics
### Cancer
In subsequent studies, attention shifted towards ex vivo analysis of human biofluids to test novel biosensors in real biological environments. Within this framework, [PERSON] et al. analysed diagnostic and prognostic markers of pancreatic cancer, mucin (MUC4) protein and serum carbohydrate (CA-19-9) antigen, and compared SERS results to ELISA and RIA [90]. In addition to quick readout time, SERS demonstrated much better sensitivity (LOD 33 ng/mL) than ELISA (LOD 30 ug/mL) for MUC4, and an LOD of 0.8 U/mL compared to RIA's 1.0 U/mL for CA-19-9. Interestingly, SERS quantified trace levels of MUC4 in human serum for pancreatic cancer patients whereas ELISA and RIA failed to register signals under same conditions. Many other articles have appeared showing that SERS is also versatile as it can detect various cancers both in vitro and in vivo [91; 92; 93; 94]. The last decade has seen substantial progress directed towards the application of SERS for detecting multiple disease markers simultaneously aimed to reduce risks of false positives associated with singular biomarker detection and to strengthen differential diagnostics. One of the earliest multiplexed assays was reported by [PERSON], who identified five DNA sequences (5-plex) quantitatively at 1 pM detection limit within a single assay [95]. Quite recently, a similar sensitivity regime was reported for prostate-free prostate specific antigens (f-PSA) and complexed prostate specific antigens (c-PSA) [96] and pancreatic CA19-9, matrix metalloproteinase-7 (MMP7) and MUC4 [97], cancers in 2-plex and 3-plex in human serum, respectively. The multiplex SERS method shown in Figure 2 of the former study, proposed a very sensitive and selective sandwich immunocomplex (SIC) nanotags, similar to those reported in recent years [98; 99]. The robust and specific SIC assay showed negligible nonspecific binding and cross reactivity between f-PSA and c-PSA [96]. This represents a remarkable leap toward reduction of false positives and assay non-reactivity driven by random binding to interferents which hamper the vast majority of diagnostic immunoassay. More important, the computed values of free to total PSA ratio for clinical samples clearly indicated that optimal dual-binding SERS assay can match the sensitivity of chemiluminescence standard used in clinics. Figure 2: **(I)** Schematic illustration of SERS immunoassay approach for multiplex detection. i. analytical mixture of free-prostate specific antigens (f-PSA) and complexed prostate specific antigens (c-PSA) with antibody-conjugated magnetic beads, ii. formation of sandwich immunocomplex (SIC) upon addition of SERS nanotags, iii. magnetic separation of immunocomplexes and iv. and v. simultaneous detection of f-PSA and c-PSA in serum, respectively. **(II)** Quantitative analysis (a) and (b). Characteristics SERS spectra at different concentrations for f-PSA and c-PSA. (c) Corresponding calibration curves. Reprinted with permission from [PERSON] et al. [96]. Copyright © 2017, American Chemical Society. Huge efforts have also been made to deploy automated digital microfluidics-SERS for on-site biosensing of multiple cancer protein markers in minute sample volumes. Recently, a simple prototype microfluidics-SERS, with minimal sample processing, detected subinfective dose of prostate cancer [100]. [PERSON] et al. and [PERSON] et al. quantified breast cancer biomarkers at 6.5 fM in serum [92] and 0.1 ppm in plasma [101], respectively. The latter study demonstrated a novel sensor for rapid sorting and quantitative detection of peptides which may play a vital role where multiplexed analysis tends to be complicated by intermolecular interactions between analyte biomarkers. Other researchers have gone so far as to suggest multiplex PCR-SERS to study melanoma DNA mutations associated with various cancers [102]. By combining the biochemical superiority of PCR, and the sensitivity and specificity of SERS, mutant alleles as low as 0.1% could be identified in serum.