Ultrasensitive Quantification of Single Extracellular Vesicles in Unprocessed Plasma using Droplet Digital Nanoplasmonic Assay

Abstract

Cancer is the top five cause of death worldwide, and cancer diagnosis and treatment costs increase yearly. The most common method for cancer detection is CT, MRI, endoscopy, and tissue biopsy. However, these methods are expensive, invasive, and time-consuming. Because of these traits, a cancer diagnosis is limited to underprivileged people. Also, one of the critical factors for a high survival rate is the early detection of cancer. So, researchers have been searching for quick and cheap methods for cancer detection. The most promising method for this is a liquid biopsy. Liquid biopsy is a cancer detection method using biological fluids such as blood, urine, sweat, semen, and more. Many biomarkers, such as cell-free DNA, circulating tumor cells, RNA, and extracellular vesicles, are used. Among different biomarkers, extracellular vesicles have advantages such as stability and rich information internally and externally. So, for cancer detection, tumor-derived extracellular vesicles (tEV) can be used for liquid biopsy-based cancer detection. ELISA and western blot were used for cancer detection. Although these two methods are considered a gold standard, the proportion of EVs containing cancerous markers must be improved, usually less than 0.1% of the EV population. In addition, EV heterogeneity brings challenges to the quantitative detection of cancer. Here, we report a wash-free single EV detection platform using a nanoplasmonic biosensor with droplet microfluidics and dark-field microscopy. Using 10nm antibody-coated gold nanoparticle and multiple ANP conjugation on the surface of EV, plasmonic resonance signal changes, and these changes are detectable under dark-field microscopy. Furthermore, cell-free DNA, circulating tumor cells, RNA, and extracellular vesicles are used and digital absolute quantification. Sample and ANP are flown together in a droplet microfluidics chip and encapsulated in a 20µm droplet. Before analysis, we proved why wash-free detection is possible by explaining plasmonic resonance signal, different scattering cross sections, and scattering intensity with simulation and UV spectrometry. We revealed the difference in plasmonic signal between individual ANP and EV-ANP conjugation. We developed quick and straightforward wash-free detection of tumor-derived EVs. In addition, we validated whether our platform can be used for different types of cell line-derived EVs and with different target proteins. From our results, our method could detect cancer with 10 µL of patient plasma, with a total assay time of less than an hour. Also, our method could be used for the detection of prostate cancer and lung cancer with 95% accuracy.