| Popis: |
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. |
