Nanoliter Plasma Sample for Disease Diagnosis by Using a Visualization Platform of Counting Extracellular Vesicle Particles

Extracellular vesicles (EVs) are an excellent candidate for noninvasive diagnostic and prognostic assessments for liquid biopsy applications owing to reflecting the dynamic changes of pathological and physiological states in the body and exceptional stability in vivo. Herein, we developed a single-extracellular vesicle fluorescence visualization and counting platform containing three parts of the nanoliter-liquid operating platform for aspirating the sample, antibody-functionalized coverslip for capturing targeted EVs, and the single-molecule fluorescence labeling and imaging system for the visualization and counting of EVs. Compared to previously reported studies, our developed platform not only minimized operational complexity but also successfully detected single EVs using a minimal volume of 25 nL of plasma. Moreover, the results of fluorescence spot counts demonstrated that the developed platform exhibits a wide dynamic range and an excellent linear correlation between fluorescence intensity and particle numbers for the detection of CD9⁺ EVs from standard samples. Remarkably, our developed single EVs capture and analysis platform was successfully applied not only in the quantitative analysis of EpCAM⁺ EVs to CD9⁺ EVs for the diagnosis of breast cancer (BC) but also in the quantitative analysis of CD14⁺ EVs to CD9⁺ EVs for the diagnosis of type 2 diabetes (T2DM). Collectively, our work provides new ideas for the diagnosis of breast cancer and type 2 diabetes while developing a versatile method for distinguishing other diseases by simultaneous detection of multibiomarkers of Exos subtypes in trace samples due to the substitutability of antibodies.