Krishna Thej Pammi Guru

It is important to isolate exosomes (8 exosomes/mL were isolated from serum. With the limit of detection of 10 pg, the designed cell showed a linear detection of HER2 from 0.1 ng to 1 µg. It was observed from the results that the electric field lysis of exosomes not only plays a significant role in releasing the cargo protein but also improves the sensing of surface proteins associated with exosomes.

Exosomes, whose mean diameter ranges from 20 nm to 200 nm, are cell-secreted vesicles and are abundant in most biological fluids, such as blood, urine, tears, sweat, breast milk, etc. Exosomal size variations and their composition can be attributed to several factors, such as age, gender and disease conditions of the individual. Existing techniques, such as ultracentrifugation and density gradient ultracentrifugation, for exosome isolation are instrument-dependent, time-consuming and lack specificity. In the present work, a gold-nanoparticle (GNP)-coated silicon (Si) wafer, functionalized with polyethylene glycol (PEG) was used for conjugation with anti-CD63 antibody via EDC NHS chemistry and incubated with serum to immobilize the exosomes on the Si surface. The surface-immobilized exosomes were eluted and quantified by a nanoparticle tracking analyzer (NTA). It was observed that an increase in GNP density on the Si wafer increases the size range and total number of exosomes that are being isolated. Western blotting performed for proteins such as HSP 70 and calnexin confirmed the immobilization and elution of exosomes. The proposed technique can be used as an alternative to existing techniques, as it has several benefits such as reusability of the Si surface for several isolations, minimal instrumental requirement, isolation of exosomes in two hours and compatibility with the microfluidic platform, making the technique suitable for real-time application. The proposed method could be useful in isolating a specific subrange of exosomes by altering the size of the GNP used for coating the Si wafer.

Exosomes are cell-secreted vesicles secreted by a majority of cells and, hence, populating most of the biological fluids, namely blood, tears, sweat, swab, urine, breast milk, etc. They vary vastly in size and density and are influenced by age, gender and diseases. The composition of exosomes includes lipids, DNA, proteins, and coding and noncoding RNA. There is a significant interest in selectively isolating small exosomes (≤50 nm) from human serum to investigate their role in different diseases and regeneration. However, current techniques for small exosome isolation/purification are time-consuming and highly instrument-dependent, with limited specificity and recovery. Thus, rapid and efficient methods to isolate them from bio fluids are strongly needed for both basic research and clinical applications. In the present work, we explored the application of a bench-top centrifuge for isolating mostly the small exosomes (≤50 nm). This can be achieved at low g-force by adding additional weight to the exosomes by conjugating them with citrate-capped gold nanoparticles (CGNP). CGNPs were functionalized with polyethylene glycol (PEG) to form PEGylated GNP (PGNP). EDC/SNHS chemistry is used to activate the –COOH group of the PEG to make it suitable for conjugation with antibodies corresponding to exosomal surface proteins. These antibody-conjugated PGNPs were incubated with the serum to form PGNP-exosome complexes which were separated directly by centrifugation at a low g-force of 7000× g. This makes this technique efficient compared to that of standard ultracentrifugation exosome isolation (which uses approximately 100,000× g). Using the technique, the exosome isolation from serum was achieved successfully in less than two hours. The purification of small exosomes, characterized by the presence of CD63, CD9 and CD81, and sized between 20 nm to 50 nm, was confirmed by western blot, dynamic light scattering (DLS), transmission electron microscopy (TEM) and nanoparticle tracking analyser (NTA).