Tetraspanins, including
CD9, CD63, and CD81, are transmembrane
biomarkers that play a crucial role in regulating cancer cell proliferation,
invasion, and metastasis, as well as plasma membrane dynamics and
protein trafficking. In this study, we developed simple, fast, and
sensitive immunosensors to determine the concentration of extracellular
vesicles (EVs) isolated from human lung cancer cells using tetraspanins
as biomarkers. We employed surface plasmon resonance (SPR) and quartz
crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal
antibodies targeting CD9, CD63, and CD81 were oriented vertically
in the receptor layer using either a protein A sensor chip (SPR) or
a cysteamine layer that modified the gold crystal (QCM-D) without
the use of amplifiers. The SPR studies demonstrated that the interaction
of EVs with antibodies could be described by the two-state reaction
model. Furthermore, the EVs’ affinity to monoclonal antibodies
against tetraspanins decreased in the following order: CD9, CD63,
and CD81, as confirmed by the QCM-D studies. The results indicated
that the developed immunosensors were characterized by high stability,
a wide analytical range from 6.1 × 104particles·mL–1to 6.1 × 107particles·mL–1, and a low detection limit (0.6–1.8) ×
104particles·mL–1. A very good
agreement between the results obtained using the SPR and QCM-D detectors
and nanoparticle tracking analysis demonstrated that the developed
immunosensors could be successfully applied to clinical samples.