Stochastic sensing of TNT with a genetically engineered pore

Area of Science:

• Biotechnology
• Nanotechnology
• Analytical Chemistry

Background:

• Engineered protein pores, like alpha-hemolysin (alphaHL), are used for single-molecule sensing.
• Nitroaromatic compounds, common in explosives, have been difficult to detect using this method.

Purpose of the Study:

• To engineer alphaHL pores capable of detecting nitroaromatic analytes.
• To establish a method for distinguishing different nitroaromatics, including TNT, at the single-molecule level.

Main Methods:

• Constructing binding sites within the alphaHL pore lumen using aromatic amino acid side chains (Phe, Tyr, Trp).
• Monitoring ionic current blockades through single engineered pores at a fixed applied potential.
• Analyzing current blockade amplitude and duration to identify and differentiate nitroaromatic analytes.

Main Results:

• Successfully created alphaHL pores with built-in binding sites for nitroaromatics.
• Demonstrated the ability to distinguish various nitroaromatics, including TNT, based on unique current-blocking event signatures.
• Observed that pore structures with fewer than seven aromatic residues exhibit weaker analyte binding, suggesting aromatic-aromatic interactions are key.

Conclusions:

• Engineered alphaHL pores provide a novel platform for single-molecule detection of nitroaromatic explosives.
• This approach offers a sensitive method for identifying and quantifying explosive compounds.
• The findings enhance understanding of noncovalent aromatic-aromatic interactions and could inform the design of future biosensors.