Discrimination of nanovesicles using two-color laser-induced fluorescence detection

Area of Science:

• Biotechnology
• Analytical Chemistry
• Nanotechnology

Background:

• Nanovesicles, including extracellular vesicles (EVs), play crucial roles in cellular communication.
• Differentiating specific EV subtypes, such as CD63-positive EVs, is essential for understanding their biological functions.
• Accurate quantification and characterization of EVs are vital for diagnostic and therapeutic applications.

Purpose of the Study:

• To develop and validate a two-color fluorescence detection system for distinguishing and quantifying different types of nanovesicles.
• To apply the system for differentiating CD63-positive extracellular vesicles (EVs) from other EVs in cell culture media.
• To investigate the impact of doxorubicin on EV secretion from cancer cells.

Main Methods:

• Utilized a two-color fluorescence detection system with two lasers (488 and 635 nm) emitting at distinct wavelengths.
• Reshaped laser beams into sheets and focused them at separate positions within a square capillary for vesicle interrogation.
• Employed fluorescence emission position to identify specific fluorophores and count vesicles, validating with labeled liposomes.

Main Results:

• Successfully differentiated and counted nanovesicles labeled with distinct fluorescent dyes using the developed system.
• Applied the system to distinguish CD63-positive EVs from other EVs in media from HeLa and A549 cells.
• Observed and analyzed the effect of the anticancer drug doxorubicin on EV secretion in the studied cancer cell lines.

Conclusions:

• The developed two-color fluorescence system provides a robust method for nanovesicle discrimination and quantification.
• The system is effective in identifying specific EV populations, such as CD63-positive EVs, in complex biological samples.
• This technology offers a valuable tool for studying EV dynamics and the effects of therapeutic agents on EV secretion in cancer research.