Identification of Isomerically Diverse Ginsenosides Using Engineered Aerolysin Nanopore via Non-Translocation...

Area of Science:

• Biophysics
• Analytical Chemistry
• Glycoscience

Background:

• Traditional nanopore sensing relies on analyte translocation, which often leads to low signal precision and resolution.
• Distinguishing structurally similar molecules, like diverse ginsenosides, remains a significant challenge in current sensing technologies.

Purpose of the Study:

• To develop a non-translocation blockade sensing strategy for the precise identification of isomerically diverse ginsenosides.
• To engineer aerolysin protein for enhanced molecular trapping and characterization.

Main Methods:

• Engineered aerolysin S278K with a positively charged interior to create electro-osmotic flow and steric/enthalpic barriers.
• Utilized non-translocation blockade sensing to trap ginsenosides, prolonging residence time.
• Integrated deep learning for quantitative analysis of complex ginsenosides in real samples.

Main Results:

• Achieved a 43-fold increase in ginsenoside residence time within the nanopore.
• Successfully identified 30 distinct ginsenosides based on variations in glycosyl composition, isomerism, modification, and aglycone.
• Demonstrated quantitative analysis of complex ginsenoside mixtures from real samples.

Conclusions:

• Non-translocation nanopore sensing offers a promising alternative for analyzing structurally complex and diverse small molecules.
• Engineered aerolysin provides enhanced capabilities for detailed molecular characterization, advancing glycoscience research.
• This method significantly improves the detection and differentiation of challenging analyte pools.