Protein detection by nanopores equipped with aptamers

Area of Science:

• Biotechnology
• Nanotechnology
• Biochemistry

Background:

• Protein nanopores serve as stochastic sensors for detecting various analytes by monitoring ionic current modulation.
• Existing methods for analyte detection can be limited in sensitivity and scope.

Purpose of the Study:

• To develop a new stochastic sensor utilizing an alpha-hemolysin (αHL) pore modified with a DNA aptamer.
• To demonstrate the sensor's capability for detecting and quantifying thrombin.

Main Methods:

• An oligonucleotide-linked DNA aptamer was covalently attached to a modified αHL pore via a disulfide bond.
• The binding of thrombin to the aptamer was detected by observing changes in ionic current through the single nanopore.

Main Results:

• The aptamer-modified nanopore sensor successfully detected thrombin binding, altering the ionic current.
• The sensor enabled quantification of thrombin in nanomolar concentrations.
• Key kinetic parameters, including association and dissociation rate constants and equilibrium dissociation constants, were determined for thrombin-aptamer interactions.

Conclusions:

• Aptamer-modified protein nanopores represent a promising new class of stochastic sensors.
• This approach facilitates the sensitive detection and kinetic analysis of analytes like thrombin.
• The technology holds potential for integration into arrays for parallel, multi-analyte sensing.