Functionalized screen-printed electrodes for non-invasive detection of vascular-endothelial cadherin in extracellular vesicles

Abstract

In this study, we developed a biosensor using a gold screen-printed electrode (Au-SPE) functionalized with mercaptoundecanoic acid (MUA) and an antibody for detecting the vascular-endothelial cadherin (CD144) as a endothelial biomarker protein on extracellular vesicles (EVs) isolated from saliva. The MUA functionalization provides a stable platform for immobilizing the CD144 antibody, ensuring the detection of the target protein. This biosensor combines Au-SPE technology with an immunoassay, offering a rapid, sensitive, and non-invasive method for detection of CD144 carried by EVs. Characterization of saliva-derived EVs using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) confirmed their morphology and size, which fell within the expected range of 80-180 nm. NTA indicated a lower concentration of particles in saliva-EVs than in serum-EVs (controls), highlighting the need for sensitive detection of EV cargos in this type of EV. Immunodetection confirmed the presence of CD144 in both saliva and serum-derived EVs, with higher concentrations in serum. Functionalization of Au-SPEs with MUA and CD144 antibodies was confirmed by significant resistance changes, and atomic force microscopy (AFM) was used to verify the preservation of EV morphology and their capturing post-immune adsorption. A calibration curve demonstrated the high sensitivity of the biosensor prototype for detecting CD144-positive EVs, with a limit of detection (LOD) of 0.111 ng mL^-1 and a limit of quantification (LOQ) of 0.37 ng mL^-1, requiring only 3 μL of EV-sample. This biosensor shows potential as a novel method for detecting and studying endothelial biomarkers associated with cardiovascular disease in EVs isolated from saliva, a capability not currently available with existing tools. Furthermore, it provides a key platform for expanding research to other biomarkers and diseases by monitoring protein cargos in the EVs, enhancing its utility across diverse clinical applications.

Fig. 1. Overview.

Fig. 2. (A and B) Transmission electron microscopy image of EV-Sal. The scale bar represents 100 nm. (C–E) Nanoparticle tracking analysis of EV-Sal, EV-Serum and EV-GFP.

Fig. 3. (A) Protein quantification by BSA for EV-GFP, EV-Sal, and EV-Serum, n = 3. (B–D) Dot Blot tests of CD144, CD81, and CD9 proteins for EV-GFP, EV-Sal, and EV-Serum. (E) Calibration curve of ELISA test for CD144, and (F) Quantification of CD144 for EV-Sal and EV-Serum, n = 3.

Fig. 4. (A) EIS Nyquist plot of functionalization of the Au-SPE with MUA and Cd-144 antibody. (B) A model of CPE with diffusion was used in the simulation to calculate the electron-transfer resistance.

Fig. 5. AFM topography images: (A and B) purified EVs from human saliva. (C and D) Height measurement analysis of purified EVs from human saliva on AFM topography image. (E and F) Distance measurement analysis of purified EVs from human saliva in AFM topography image.

Fig. 6. Image of 3D atomic force microscopy (AFM) topography of the biosensor surface after the capturing of EV-Sal.

Fig. 7. The calibration curve of the human CD144 biosensor with purified EVs from saliva, n = 3.

Scheme 1. (A) Schematic diagram of the IgG-modified electrode. (B) Schematic diagram of the IgG-modified electrode with CD144-positive EV-Sal.