## 4 Discussion  
The collective physicochemical analyses confirmed the successful surface modification of magnetic beads with EGCG. The SEM and elemental analyses revealed increased surface coverage and carbon contents with higher EGCG concentrations, indicating efficient surface coating. The FT-IR and UV-Vis spectra further supported the presence of EGCG-specific functional groups while preserving the optical characteristics of Fe\({}_{3}\)O\({}_{4}\), suggesting stable composite formation. In addition, the zeta potential and DLS measurements demonstrated enhanced colloidal stability and reduced aggregation with increasing EGCG concentration. These findings are consistent with those of previous studies that reported that polyphenol-based nanoparticle functionalization can improve biomolecular interactions and isolation performance [23]. Building upon these physicochemical properties, we developed a novel EGCG-modified magnetic bead-based method (EGCG@T) for efficient isolation and concentration of exosomes from diverse biofluids. The EGCG@T complex demonstrated superior performance in capturing exosomes compared to conventional methods, with optimization of the composition ratio (10\(\times\) ratio of 0.1 mg EGCG:1 mg T-Fe\({}_{3}\)O\({}_{4}\)) yielding the highest efficiency. Our approach addresses several limitations of existing isolation techniques such as ultracentrifugation, which is time-consuming and equipment-intensive [24, 25], and precipitation methods, which often co-isolate non-specific proteins [26]. The automated protocol developed using the Nextractor NX-Junior platform successfully integrated exosome isolation and protein extraction into a single workflow, significantly reducing the processing time while maintaining or enhancing the isolation efficiency for multiple biofluid types. We demonstrated that biofluid-specific optimization of the isolation protocol is essential, as the physicochemical properties of exosomes vary between sample types. For urine samples, a bead concentration of 12 mg/mL with a 10 min binding and 20 min elution without washing steps proved optimal. In contrast, plasma and serum samples required longer binding times (30 min) and shorter elution times (10 min) to maximize the recovery of various exosomal markers. The combination of RIPA and lysis buffer (RIPA + Lysis) consistently showed a balanced extraction of diverse exosomal markers (CD9, CD63, CD81, TSG101, and ALIX) across sample types, although the efficiency varied by biofluid. These findings align with those of previous studies that reported heterogeneity in exosome populations across different biofluids [27, 28] and the importance of optimizing isolation conditions according to the sample characteristics [29, 30]. One of the key findings of our study is that exosomal marker expression patterns vary significantly depending on the biofluid type. In urine samples, CD9 and CD63 showed high expression in the EGCG@T samples, while TSG101 and ALIX expression was more prominent in plasma and serum samples. These differences likely reflect the heterogeneity of the exosome subpopulations present in each biofluid [31]. [PERSON] et al. reported that extracellular vesicles can be classified into several subpopulations based on the expression of tetraspanins and ESCRT complex components [32]. Our results confirmed that this heterogeneity exists across different biofluids and suggest that isolation methods targeting specific markers should be optimized according to the biofluid type. Notably, the samples obtained using the EGCG@T method on saliva samples showed stable and consistent levels of all exosomal markers. This suggests that salivary exosomes may have more homogeneous physicochemical properties compared to other biofluids. [PERSON] et al. reported that salivary exosomes possess distinct membrane compositions and surface properties that could affect isolation efficiency [33]. Additionally, the lower protein complexity of saliva may facilitate interactions between EGCG and exosome membranes, enhancing isolation efficiency [11].