## 4 Discussion
In contrast, plasma and serum samples may require longer binding times for optimal isolation due to interference from abundant proteins such as albumin that interact with EGCG. [PERSON] et al. reported that protein-EGCG interactions in plasma could affect exosome isolation efficiency [34]. The optimized protocols developed in this study have been adjusted for these biofluid-specific differences, providing an important methodological advancement for exosome research and diagnostic applications using various biofluids. In this study, we systematically evaluated the impact of various extraction buffer compositions on the recovery of exosomal marker proteins. Testing RIPA buffer, lysis buffer, and various combinations of the two revealed that the buffer composition critically influences the levels of specific exosomal markers. Notably, we found that a 2:1 ratio mixture of RIPA and lysis buffers provided a balanced recovery of diverse exosomal markers from most biofluids. This aligns with the findings reported by [PERSON] et al. who demonstrated that composite buffer systems may be more effective for extracting various membrane and cytosolic proteins than single lysis buffers [1]. Interestingly, ESCRT (Endosomal Sorting Complex Required for Transport) complex-associated proteins such as TSG101 and ALIX were more sensitive to the lysis buffer components, while tetraspanin proteins including CD9, CD63, and CD81 were more effectively extracted with RIPA buffer. This suggests differences in protein composition and membrane characteristics among exosome subpopulations. [PERSON] et al. reported that tetraspanins are predominantly located in lipid rafts, and these membrane microdomains may be more effectively solubilized by the detergents included in RIPA buffer [35]. In contrast, [PERSON] et al. demonstrated that ESCRT-related proteins could be more effectively extracted using buffers containing denaturants such as urea [36]. The RIPA + Lysis mixed buffer system developed in this study leverages these properties to efficiently and simultaneously extract a broad range of marker proteins from various exosome subpopulations. This could be an important advantage for future clinical applications such as disease diagnosis through liquid biopsy. [PERSON] et al. showed that analyzing diverse exosomal markers simultaneously could significantly improve diagnostic accuracy compared to relying on single markers [37]. In this study, we established an integrated workflow for efficient exosome isolation from various biofluids using a combination of the Nextractor NX-Junior automated system and EGCG-modified magnetic beads. One of the major advantages of this automated platform is the integration of exosome isolation and protein extraction steps, which have traditionally been performed as separate processes, into a single procedure. This not only significantly reduces the sample processing time but also minimizes the sample loss that can occur during multi-step isolation processes. [PERSON] et al. reported that additional exosome isolation steps can substantially reduce the final recovery rates [5]. The quantitative assessment using ELISA confirmed that the automated system using the EGCG@T method isolated CD63-positive exosomes from urine samples more than twice as efficiently as the conventional PEG precipitation method. This suggests that the synergistic effect of EGCG's specific binding to exosome membrane phospholipids [38] and the efficient magnetic separation characteristics of magnetic beads enables high-efficiency exosome isolation. Moreover, the system can be used for various biofluids and can isolate exosomes from small sample volumes (200-1000 \(\mu\)L), indicating its high potential for application in clinical diagnostic settings. [PERSON] et al. emphasized that effective exosomeisolation from small volumes of biofluids could make liquid biopsy using exosomes an innovative tool for early diagnosis and monitoring of various diseases including cancer [39].