## 3 Results
### Automated Exosome Isolation: Evaluation of Buffer Systems Using the NX-Jr Platform
The molecular weights (kDa) are indicated on the left. The optimal buffer conditions varied depending on the sample type and target marker. Comparative Analysis of CD63 Expression in Exosomes Isolated from Urine, Saliva, Plasma, and Serum Samples Using Different Methods  
To evaluate the differences in CD63 levels based on the exosome isolation method, both Western blot and ELISA analyses were performed for four types of biological samples: urine, saliva, plasma, and serum (Figure 9). For urine samples, exosomes were isolated using PEG precipitation, an EGCG solution (EGCG Sol. ), the EGCG-modified magnetic beads (EGCG@T), and the magnetic bead core (T core). The Western blot analysis showed detectable CD63 (\(\sim\)45-60 kDa) levels using all the methods, with particularly strong bands observed in the PEG, EGCG@T, and T core samples. The EGCG Sol. group showed relatively weak levels. The EGCG@T method yielded the most intense and distinct CD63 bands. ELISA quantification confirmed this trend, revealing the highest CD63 levels with EGCG@T (0.29 ng/mL), over twice that with PEG (0.13 ng/mL), EGCG Sol. (0.12 ng/mL), and T core (0.13 ng/mL). These results suggest a synergistic effect of EGCG and magnetic beads in enhancing CD63-positive exosome isolation from urine. Building on these findings, plasma samples were tested using PEG and EGCG@T. The Western blot analysis demonstrated stronger CD63 bands with the EGCG@T method than with PEG. The ELISA quantification supported this result, with the EGCG@T method yielding a CD63 level of approximately 5.7 ng/mL, compared to 3.4 ng/mL for PEG. This indicates that EGCG@T has significantly better isolation efficiency for CD63-positive exosomes from plasma. In serum samples, both the PEG and EGCG@T methods showed strong CD63 bands in the Western blot, with similar intensities. The ELISA results showed comparable CD63 levels using both methods, with 5.6 ng/mL for PEG and 5.8 ng/mL for EGCG@T. These findings suggest that while both methods are effective for serum-derived exosome isolation, EGCG@T provides a slightly better performance. Saliva samples were also analyzed using the PEG and EGCG@T methods. The Western blot results showed broad and strong CD63 bands (\(\sim\)37-75 kDa) using both methods, suggesting the presence of abundant CD63-positive exosomes in saliva. The ELISA analysis indicated a higher CD63 level with the EGCG@T method (0.13 ng/mL) compared to PEG (0.10 ng/mL), which is consistent with the trend observed in urine, though with a smaller difference. This may reflect matrix effects or intrinsic differences in salivary exosome characteristics. In summary, across all four types of biological samples, the EGCG@T method consistently demonstrated better or comparable isolation efficiency for CD63-positive exosomes compared to the PEG method. The difference was most pronounced for urine and plasma samples, indicating that EGCG-modified magnetic beads are a promising tool for high-efficiency exosome isolation from diverse biological fluids. Western blot and ELISA analyses were performed to compare CD63 expression in exosomes isolated from urine, saliva, plasma, and serum samples. Four different isolation methods were evaluated for urine samples: PEG precipitation (PEG), an EGCG solution (EGCG sol), the EGCG-modified magnetic beads (EGCG@T), and the magnetic bead core (T core). EGCG@T demonstrated the highest CD63 expression in both the Western blot and ELISA results (0.29 ng/mL). For saliva, plasma, and serum samples, exosomes were isolated using the PEG and EGCG@T methods. The Western blot results showed comparable or stronger CD63 expression with EGCG@T, and the ELISA confirmed higher CD63 levels in  
Figure 9: Comparative analysis of CD63 expression in exosomes isolated from various types of biological samples using Western blot and ELISA: (**A**) urine, (**B**) plasma, (**C**) serum, and (**D**) saliva. EGCG@T-treated samples compared to PEG samples, particularly for plasma.