FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers

Area of Science:

• Biotechnology
• Analytical Chemistry
• Microbiology

Background:

• Staphylococcus aureus (S. aureus) is a foodborne pathogen critical for survival and colonization.
• Early detection of S. aureus is vital for preventing foodborne illnesses.
• Iron-regulated surface determinant protein A (IsdA) is a specific marker for S. aureus, but its detection is underdeveloped.

Purpose of the Study:

• To develop a robust and widely applicable detection method for IsdA.
• To enable sensitive and specific detection of the foodborne pathogen S. aureus.
• To advance aptamer-based sensing for pathogen protein quantification.

Main Methods:

• Computational generation of target-guided aptamers specific to IsdA.
• Utilizing fluorescence resonance energy transfer (FRET)-based single-molecule analysis.
• Verifying aptamer-induced FRET state switching in the presence of IsdA protein.

Main Results:

• Identified three distinct RNA aptamers specific to the IsdA protein.
• Demonstrated picomolar level detection of IsdA (down to ×10⁻¹² M).
• Achieved a dynamic detection range extending to approximately 40 nM.

Conclusions:

• The developed FRET-based single-molecule technique offers high sensitivity and specificity for IsdA detection.
• This method has broad applications in the food industry for pathogen detection.
• Enables quantitative detection of various pathogen proteins using aptamer-based sensing.