## 3 Immunolabeling Approach
[108] by using a sandwich structure formed by a polydopamine (PDA)-modified immunocapture substrate, exosomes, and polydopamine-encapsulated antibody-reporter-Ag(shell)-Au(core) multilayer (PEARL) nanotags, as illustrated in Figure 12. Due to a self-polymerizing porous hydrophilic PDA layer, specific antibodies are simultaneously encapsulated and captured by the surface. PDA encapsulation provides higher antibody capture efficiency and exosome detection sensitivity compared with other antibody capture methods. A PEARL tag consists of AuNP as a core, a silver shell, 4-aminobenzenzenethiol (pATP) as a Raman reporter, BSA, PDA, and antibodies. The intensity of the pATP Raman peak at 1072 cm\({}^{-1}\) was chosen as a quantitative signal, because it is one of the strongest peaks in immunoassays' SERS spectra and is not influenced by any impure surrounding peaks. An optimal low PDA layer thickness in PEARL provides stability and high Raman intensity of the nanotag. Comparing the intensities of anti-CD9, anti-CD63, anti-GPC1, and anti-MIF groups for exosomes derived from cancerous and healthy pancreatic cells revealed that anti-MIF and anti-GPC1 platforms can distinguish between them. The developed immunoassay provides an extra-low detection limit of one exosome in a 2 \(\mu\)L sample. Validation of the immunosensor in diluted clinical serum samples from pancreatic cancer patients proved its capability to discriminate between cancer patients and healthy controls as well as between different cancer stages (the latter can be provided merely by anti-MIF). Figure 11: A miniaturized antibody array-based device for exosome analysis in a target-specific manner: (**a**) A schematic design of the Raman exosome assay using a 3D-printed array template. AuNR binding to the exosome’s lipid membrane: (**b**) Side view; (**c**) top view. Reproduced with permission from [107], Ivyspring International Publisher Pty Ltd. Licensed under CC BY-NC 4.0., 2018. A microfluidic bioch, which is potentially appropriate for exosome analysis, was implemented by [PERSON] et al. [89]. They used SERS for a rapid parallel analysis of several cancer protein biomarkers in a microfluidic chip based on magnetic nanochains (Magchains), and cellular and subcellular analysis is considered among its potential applications. The bioconjugated Magchains are driven by an external magnetic field and act as both nanoscale stir bars for rapid liquid mixing and capturing agents for bioseparation. The developed magnetic nanochain integrated microfluidic biochip (MiChip) has a simple planar design shown in Figure 13a,b, also it can be easily transformed to a multichannel array for a multiple parallel analysis (Figure 13c). The Magchains are made as superparamagnetic Fe\({}_{3}\)O\({}_{4}\) nanoparticles coated with polydopamine that is self-polymerized under weak basic conditions, providing strong adhesion and forming nanochains, which are then antibody(Ab-I)-functionalized (Figure 13d). The Raman nanotags are AuNR with different Raman reporter molecules, coated with thiolated PEG for surface passivation and functionalized by detection antibodies (Ab-II). The assay process is the following: A mixture of Magchains and SERS probes is injected via the first inlet while a liquid specimen is injected via the second one. Then, they are both transferred and confined in the mixing chamber by two pneumatic valves and their mixing is operated by a spinning magnetic field generated by electromagnetic coils. Hereby sandwich-type immunocomplexes are formed between Magchains, targets of interest, and SERS probes. Finally, valve 2 opens, and the Magchains are magnetically driven to the detection chamber, where Raman spectroscopic detection is provided. By this route, the authors simultaneously detected cancer protein biomarkers for prostate, colorectal, and hepatocellular cancers--prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and \(\alpha\)-fetoprotein (AFP). They also examined clinical serum samples and successfully distinguished patients with these types of cancer.