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

Abstract

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 × 10 4 particles•mL −1 to 6.1 × 10 7 particles•mL −1 , and a low detection limit (0.6−1.8) × 10 4 particles•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.

Immunoassays for Extracellular Vesicle Detection via Transmembrane Proteins Using Surface Plasmon Resonance Biosensors

“…The LOD was estimated according to 3σ/ S , where σ is the standard deviation of the blank signal (Figure S5), and S is the slope of the calibration curve. The LOD of our EV detection was estimated to be 5.9 × 10 6 EVs/mL, which is about 2 orders of magnitude larger than a recently reported value . Even though CD81 on the EV surface was used as a target tetraspanin in the earlier work, the EVs were produced from human lung cancer cells and CD81 mAbs were immobilized on the surface via protein A, which may offer a better antibody orientation to improve the LOD.…”

“…SPR biosensors have been extensively applied in the detection of EV samples using antibodies (Abs) − with focus on the targeting of tetraspanin markers like CD81, CD63, CD9, and disease-specific TMPs. Biotin–avidin chemistries for antibody (Ab) immobilization are most often used, followed by EV detection targeting TMPs.…”

“…Similarly, HER-2 Abs were oriented onto SPR chips using biotin–streptavidin chemistry to detect EVs in serum from BT474 cells, which resulted in a linear range and LOD of (0.8–3.31) × 10 7 EVs/mL and 8.28 × 10 6 EVs/mL, respectively . Recently, Kowalczyk et al used SPR biosensors to detect EVs from human lung cancer cells . Their study reports equilibrium dissociation constants of 4.77 μM, 44.1 nM, and 0.09 nM when EVs were respectively targeted via CD81, CD63, and CD9 using Abs immobilized onto protein A-functionalized SPR chips.…”

Probing the Interaction Mechanism of Sodium Oleate and Dodecyl Amine with Quartz Surfaces in the Presence of Ca2+ Ions by AFM Force Measurement

“…Several substrates have been utilized to track the adsorption kinetics of polymers, , DNA, − and proteins , using QCM-D. In this study, we installed four clean silica QCM-D sensors in the QCM-D measurement chamber.…”

Methylene Blue-Stained Single-Stranded DNA Aptamers as a Highly Efficient Electronic Switch for Quasi-Reagentless Exosomes Detection: An Old Dog with New Tricks

“…Unfortunately, the practical application of these methods in clinical diagnosis is limited by several shortcomings such as tedious operation, the need for expensive and complex instruments, low sensitivity, or the demand for large volumes of samples. To overcome these problems, several sensing methods including fluorescence, electrochemistry, surface plasmon resonance (SPR), colorimetry, surface-enhanced Raman scattering (SERS), etc. have been established for sensitive detection of exosomes.…”