Tiny Capsules for Disease Detection: Extracellular Vesicles (EVs)

Inside our bodies, cells secrete extremely small, capsule-like substances to communicate with each other. These are called “extracellular vesicles (EVs),” and a specific type, “exosomes,” are known to be released in large quantities from cells affected by diseases like cancer. Therefore, EVs are attracting significant global attention as “biomarkers” for the early detection of diseases and prediction of treatment efficacy.

“Click Chemistry” for Labeling EVs

For EVs to be useful as disease indicators, they must be accurately detected and quantified. One method for this is “labeling,” a technique where fluorescent dyes or other markers are attached to specific molecules. In this study, we utilized “click chemistry,” a chemical reaction where specific molecules efficiently join together, much like snapping LEGO blocks. First, during cell culture, cellular membranes are metabolically functionalized with special “azide groups.” Subsequently, when the fluorescent dye Cy5 is conjugated to these azide groups, the cells internalize them and secrete Cy5-labeled EVs.

Evaluating Labeling Efficiency with Two-color Laser-Induced Fluorometry

Even with “click chemistry” used to label EVs, accurately evaluating how efficiently this reaction proceeds has been a challenging task. To address this, we developed a new measurement system called “two-color laser-induced fluorometry (two-color LIF).” This system involves dual labeling of EVs. One label is Cy5, attached via click chemistry. The other is a “PE-conjugated anti-CD63 antibody,” which binds to “CD63,” a protein found on the surface of EVs (a specific marker for EVs). These two dyes are selectively excited by different colored laser lights (PE by 488 nm, Cy5 by 635 nm). If an EV is labeled with both Cy5 and PE, it will fluoresce in both colors when subjected to the respective laser lights. However, EVs that are only labeled with PE (due to unsuccessful Cy5 attachment) will only fluoresce when excited by the 488 nm laser. By exploiting this difference, our system can precisely distinguish and evaluate the efficiency of click chemistry-based labeling for individual EVs.

Does Cell Culture Condition Affect Labeling Efficiency?

Using this novel system, we investigated how the concentration of “serum” in the cell culture medium affects the click chemistry labeling efficiency of EVs. Our results showed that for both HeLa and A549 cancer cells, the highest labeling efficiency was obtained when the medium was supplemented with “10% fetal bovine serum.” This finding is highly valuable for identifying more efficient and reliable labeling conditions when EVs are used as biomarkers in clinical applications. Our developed two-color LIF system will be a powerful tool for quantitatively evaluating EV labeling efficiency under various cell culture conditions.

The Excitement of Analytical Chemistry

This research demonstrates the fascinating aspect of precisely analyzing extremely small “extracellular vesicles” by combining specific chemical reactions with advanced optical technology. It involves developing techniques to “measure” the invisible microscopic world, aiming for medical applications like early disease detection. That is the essence of “Analytical Chemistry.” If you are interested in contributing to disease diagnosis through chemistry or in precise measurement technologies, we invite you to explore the world of analytical chemistry at university.

Reference Paper

• Arisa Takaya, Takashi Kaneta (2026) “Two-color, laser-induced fluorometry for evaluating the efficiency of click chemistry-based labeling of extracellular vesicles” Analytical Sciences, 2026. DOI: 10.1007/s44211-026-00979-x

Learn more about Prof. Takashi → Analytical Chemistry Laboratory