Extracellular Vesicle (EV) Dot Blotting for Multiplexed EV Protein Detection in Complex Biofluids.

Extracellular vesicles (EVs) are nanoscale vesicles secreted from cells, carrying biomolecular cargos similar to their cells of origin. Measuring the protein content of EVs in biofluids can offer a crucial insight into human health and disease. For example, detecting tumor-derived EVs' protein markers can aid in early diagnosis of cancer, which is life-saving. In order to use these EV proteins for diagnosis, sensitive and multiplexed methods are required. The current methods for EV protein detection typically require large sample consumption due to challenges with sensitivity and often need an EV isolation step for complex biofluid samples such as blood plasma. In this work, we have developed a simple and sensitive method for multiplexed detection of protein markers on EV membrane surfaces, which we call "EV dot blotting", inspired by conventional dot blotting techniques. After optimization of multiple factors such as antibody concentration, blocking reagent, type of 3D membranes, and use of gold nanoparticles for signal enhancement, cancer-cell-derived EVs were spiked in pooled normal human plasma for conducting a multiplexed assay in a microarray format. Without the need of isolating EVs from blood plasma, a limit of detection of 3.1 × 10 EVs/mL or 1863 EVs/sample was achieved for CD9 protein, 4.7 × 10 EVs/mL or 281 EVs/sample for CD24, and 9.0 × 10 EVs/mL or 538 EVs/sample for EpCAM, up to 4 orders of magnitude lower than those of conventional ELISA. This platform offers sensitive, multiplexed, simple, and low-cost EV protein detection directly from complex biofluids with minimal sample consumption, providing a useful tool for multiplexed EV protein quantification for a variety of applications.