Electroanalysis at a Single Giant Vesicle Generating Enzymatically a Reactive Oxygen Species

Area of Science:

• Biotechnology
• Electrochemistry
• Synthetic Biology

Background:

• Giant unilamellar vesicles (GUVs) are fundamental structures in artificial cell development, capable of encapsulating molecules and facilitating controlled exchange with their environment.
• Biochemical reactions within GUVs are typically monitored using fluorescence microscopy.
• Developing new methods for in situ analysis of reactions within single vesicles is crucial for advancing synthetic cell research.

Purpose of the Study:

• To demonstrate the suitability of electrochemistry using ultramicroelectrodes for monitoring enzymatic reactions within single giant unilamellar vesicles.
• To establish electroanalysis as a complementary technique to fluorescence microscopy for sensing activities in artificial cells.

Main Methods:

• Microinjection of glucose oxidase (GOx) and glucose into individual GUVs.
• Utilizing a hydrogen peroxide (H2O2)-sensitive ultramicroelectrode (black platinum-modified carbon) placed adjacent to the GUV membrane.
• Employing chronoamperometry for time-resolved, in situ detection of H2O2 flux across the GUV membrane.

Main Results:

• Successful real-time monitoring of glucose oxidase activity within single GUVs over one hour.
• Demonstrated sensitive and selective detection of H2O2 generated by the enzymatic reaction.
• Showcased electroanalysis as a non-invasive method that does not alter the internal GUV medium.

Conclusions:

• Electroanalysis with microsensors is a highly sensitive, selective, and time-resolved method for monitoring enzymatic activities in single GUVs.
• This technique is well-suited and complementary to fluorescence microscopy for sensing reactive oxygen species and other products of enzymatic reactions in artificial cells.
• The findings support the advancement of artificial cell development by providing a robust tool for analyzing biochemical processes at the single-vesicle level.