Pammi Guru Krishna Thej, Praween Nusrat, Basu Palash Kumar
Department of Avionics, Indian Institute of Space Science and Technology, Thiruvanathapuram 695547, India.
Nanomaterials (Basel). 2023 Jan 18;13(3):387. doi: 10.3390/nano13030387.
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.
外泌体是细胞分泌的囊泡，其平均直径范围为20纳米至200纳米，在大多数生物流体中都很丰富，如血液、尿液、眼泪、汗液、母乳等。外泌体的大小变化及其组成可归因于几个因素，如个体的年龄、性别和疾病状况。现有的外泌体分离技术，如超速离心和密度梯度超速离心，依赖仪器、耗时且缺乏特异性。在本研究中，用聚乙二醇（PEG）功能化的金纳米颗粒（GNP）包被的硅（Si）晶片，通过EDC-NHS化学方法与抗CD63抗体结合，并与血清孵育，将外泌体固定在Si表面。通过纳米颗粒跟踪分析仪（NTA）对外泌体进行洗脱和定量。观察到Si晶片上GNP密度的增加会扩大所分离外泌体的大小范围并增加其总数。对热休克蛋白70（HSP 70）和钙连接蛋白等蛋白质进行的蛋白质印迹证实了外泌体的固定和洗脱。所提出的技术可作为现有技术的替代方法，因为它具有几个优点，如Si表面可重复用于多次分离、仪器需求最少、两小时内可分离外泌体以及与微流控平台兼容，使得该技术适用于实时应用。通过改变用于包被Si晶片的GNP的大小，所提出的方法可能有助于分离特定大小范围的外泌体。
