Decoding aptamer-protein binding kinetics for continuous biosensing using single-molecule techniques

Area of Science:

• Biomolecular Engineering
• Biosensor Technology
• Biophysics

Background:

• Continuous biosensing offers real-time biochemical process monitoring for health applications.
• Aptamers are increasingly used as biorecognition elements in biosensors, but their binding interactions are not fully understood.
• Understanding aptamer-target binding kinetics is essential for optimizing biosensor performance.

Purpose of the Study:

• To present a novel single-molecule technique for decoding aptamer-protein binding interactions.
• To elucidate the binding kinetics of structurally similar aptamers.
• To enable the rational design of high-performance aptamer-based biosensors.

Main Methods:

• Development and application of a single-molecule assay to analyze aptamer-protein binding.
• Utilizing computational simulations to guide the interpretation of binding data.
• Validation of the technique using quartz crystal microbalance experiments.

Main Results:

• The single-molecule assay successfully decoded binding kinetics of aptamers with similar affinities.
• Quantitative insights into binding interactions enabled rational understanding of biosensor sensitivity and limit of detection.
• Demonstrated capability using thrombin and structurally similar aptamers (HD1, RE31, NU172).

Conclusions:

• The developed single-molecule technique provides high temporal resolution for studying aptamer-protein interactions.
• This approach facilitates a deeper understanding of biosensor mechanisms.
• Paves the way for rational design and optimization of aptamer-based biosensors.