## 3 Immunolabeling Approach  
Although the label-free SERS detection approach represents the simplest method for the identification of biomolecules, there are limitations that can reduce its efficiency, especially when measuring molecules in a complex medium such as blood or serum. In particular, the probability for direct contact between the target molecule and the SERS substrate can be reduced due to absorption of other molecules in the sample. In addition, data analysis can be complicated due to contributions of SERS signals in the SERS spectrum from co-adsorbed molecules. In this respect, the indirect detection approach using antibody-coated SERS substrates and SERS tags increases the degree of specificity and provides specifically strong SERS output signals (Table 2). In this section we review immunolabeling SERS assays that have recently been applied for extraction and identification of exosomes. A sandwich-type immunocomplex containing/based on antibody-labeled magnetic beads and AuNR has been shown to be an effective approach for the detection of tumor-derived exosomes [103]. Two types of antibodies, anti-CD63 and anti-HER2, were used to recognize two corresponding proteins on the surface of exosomes. The sandwich-type immunocomplex was formed by mixing of AuNR, targeted exosomes, and magnetic beads, as shown in Figure 7. The immunocomplex was then precipitated by a magnet and SERS signals detected in the pellet using 5,5'-Dithiobis(2-nitrobenzoic acid)-labeled AuNR. By using this approach, the authors examined the exosomes derived from human breast cancer cells (SKBR3) and compared them to exosomes isolated from normal human embryonic lung fibroblasts (MRC5). The authors tested the different number of exosomes and found that the proposed approach provides a limit of detection of approximately 1200 exosomes. In addition, the presented method is relatively fast with an operation period/analysis time of 2 h and can be used without the purification of exosomes--for example, by using the cell culture media. The selective targeting of exosomes derived from ovarian cancer SKOV3 cells by using an \(\alpha\)381 integrin-specific peptide conjugated to silver nanoparticles (AgNP) for SERS-based detection and analysis was demonstrated by [PERSON]'s group [104]. The peculiarity of this approach is demonstrated in Figure 8. The LXY30 peptide binds first to the \(\alpha\)3\(\beta\)1 integrin on the SKOV3 exosomes followed by the addition of AgNP enabling formation of the exosome-Ag complex via thiol binding. The successful binding of the exosomes to AgNP was verified by the appearance of several distinct SERS peaks that can be assigned to SKOV3 exosomes. The absence of these peaks in the SERS spectra for AgNP-LXY30-Jurkat-derived exosomes confirmed the efficiency/specificity of the proposed method. Figure 7: A scheme of the sandwich-type immunocomplex approach: (**a**) Magnetic nanobead fabrication; (**b**) SERS nanoprobe fabrication; (**c**) sandwich-type structure forming. Reproduced with permission from [103], The Royal Society of Chemistry, 2016. Highly sensitive detection of tumor-derived exosomes using the immunocomplex between SERS-active gold nanostars (AuNS) and magnetic beads was reported in a prior study [105]. SERS nanoprobes comprising of 4-mercaptoethanolacid (4-MBA) as a Raman reporter were functionalized with two cholesterol-modified DNA single strands to form the complex between cholesterol moieties and the exosomal membrane (Figure 9). At the same time, biotin-labeled anti-CD9 was immobilized onto the surfaces of streptavidin-linked magnetic beads for specific capture of CD9-binding exosomes. In the presence of the exosomes, the immunocomplex between magnetic beads, tumor-derived exosomes, and SERS nanoprobes was formed providing strong SERS signals whereas in the absence of the exosomes just poor SERS signals were acquired. The exosomes isolated from liver cancer HepG2 cells were successfully detected with a sensitivity of 27 particles per \(\upmu\)L and analysis time of 50 min.