Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells

Tetraspanins, includingCD9, CD63, and CD81, are transmembranebiomarkers that play a crucial role in regulating cancer cell proliferation,invasion, and metastasis, as well as plasma membrane dynamics andprotein trafficking. In this study, we developed simple, fast, andsensitive immunosensors to determine the concentration of extracellularvesicles (EVs) isolated from human lung cancer cells using tetraspaninsas biomarkers. We employed surface plasmon resonance (SPR) and quartzcrystal microbalance with dissipation (QCM-D) as detectors. The monoclonalantibodies targeting CD9, CD63, and CD81 were oriented verticallyin the receptor layer using either a protein A sensor chip (SPR) ora cysteamine layer that modified the gold crystal (QCM-D) withoutthe use of amplifiers. The SPR studies demonstrated that the interactionof EVs with antibodies could be described by the two-state reactionmodel. Furthermore, the EVs' affinity to monoclonal antibodiesagainst tetraspanins decreased in the following order: CD9, CD63,and CD81, as confirmed by the QCM-D studies. The results indicatedthat the developed immunosensors were characterized by high stability,a wide analytical range from 6.1 x 10(4) particles center dot mL(-1) to 6.1 x 10(7) particles center dot mL(-1), and a low detection limit (0.6-1.8) x10(4) particles center dot mL(-1). A very goodagreement between the results obtained using the SPR and QCM-D detectorsand nanoparticle tracking analysis demonstrated that the developedimmunosensors could be successfully applied to clinical samples.