## 3 Results
### Automated Exosome Isolation: Evaluation of Buffer Systems Using the NX-Jr Platform  
To effectively isolate exosomes from various types of biological samples, the buffer conditions were optimized using the automated Nextractor NX-Junior platform and the EGCG-modified magnetic beads. A Western blot analysis was used to compare the exosomal marker profiles obtained using the different extraction conditions against those obtained using the conventional PEG precipitation method (Figure 8). Figure 7: Comparison of exosomal marker profiles under different extraction buffer conditions. For urine samples, the buffers evaluated included RIPA buffer, lysis buffer, and a mixed buffer (RIPA + Lysis). THP (\(\sim\)100 kDa) levels were high using the beads and all the NX-Jr conditions compared to PEG. TSG101 (\(\sim\)45 kDa) showed the highest levels using PEG and was detectable in the lysis and mixed buffer samples, but not in the RIPA buffer alone sample. Tetraspanins CD81, CD63, and CD9 (\(\sim\)20-60 kDa) showed high levels in the PEG, RIPA buffer, and mixed buffer condition samples but lower levels with the lysis buffer alone. Thus, the mixed buffer (RIPA + Lysis) provided the optimal extraction of urine-derived exosomes. For plasma samples, ALIX (\(\sim\)75 kDa) levels were highest with the lysis buffer and moderate with the mixed buffer, but it had weak levels with PEG and RIPA buffer alone. CD63 (\(\sim\)45 kDa) showed high levels with RIPA buffer and the mixed buffer and moderate levels with PEG. CD9 (\(\sim\)20-25 kDa) levels were highest with lysis buffer and lowest with RIPA buffer alone. TSG101 (\(\sim\)45 kDa) had high levels with PEG and RIPA buffer and moderate levels with the mixed buffer. Therefore, the mixed buffer condition offered balanced extraction; however, specific buffer choices can be used to selectively concentrate specific markers. Figure 8: Evaluation of extraction buffer systems for automated exosome isolation. Western blot analysis of exosomal markers isolated from (**A**) urine, (**B**) plasma, (**C**) serum, and (**D**) saliva samples using conventional PEG precipitation (PEG) and automated extraction by NX-Jr instrument with different buffers: RIPA, LYS, and mixed RIPA/LYS. Molecular weights (kDa) indicated on the left. Optimal buffer conditions varied by sample type and target marker. In serum samples, ALIX (\(\sim\)75 kDa) and TSG101 (\(\sim\)45 kDa) exhibited the highest levels in the PEG samples, with moderate levels in the lysis and mixed buffer samples, but weak levels with RIPA buffer alone. CD63 (\(\sim\)50 kDa) had the highest levels with PEG and lower levels in all the bead samples. CD9 (\(\sim\)22 kDa) showed high levels with PEG and the lysis buffer, but minimal levels with RIPA buffer and the mixed buffer. CD81 was undetectable in all the samples. Overall, PEG yielded the highest levels of most markers, with the lysis and mixed buffers performing best among the bead samples. For saliva samples, TSG101 (\(\sim\)50 kDa) levels were significantly higher in all the bead samples compared to the PEG samples, indicating their effectiveness. CD81 (\(\sim\)25 kDa) showed the highest levels with RIPA buffer, moderate levels with the mixed buffer, and weak levels with the lysis buffer. CD63 (\(\sim\)50 kDa) and CD9 (\(\sim\)22 kDa) displayed consistently high levels across all the samples, with slightly better results with RIPA buffer and the mixed buffer. Thus, saliva-derived exosomes exhibited stable extraction patterns across the different buffer conditions, with the mixed buffer being most practical. In conclusion, the RIPA + Lysis mixed buffer provided balanced and effective extraction from urine, plasma, serum, and saliva samples, with selective buffer adjustments recommended for targeted marker enrichment. A Western blot analysis of exosomal markers isolated from (A) urine, (B) plasma, (C) serum, and (D) saliva samples using conventional PEG precipitation (PEG) and automated extraction with an NX-Jr instrument with different buffers (RIPA, LYS, and mixed RIPA/LYS) was performed.