A Reversible ATR-FTIR Biosensor with Single-Residue Sensitivity.

Area of Science:

• Biophysical Chemistry
• Spectroscopy
• Biosensing

Background:

• Infrared spectroscopy offers label-free protein structure insights but faces challenges like water absorption and artifacts.
• Previous ATR-based immuno-IR sensors (iRS) enabled protein immobilization for disease diagnosis but lacked reusability.
• Detecting small amounts of misfolded proteins (Aβ, α-synuclein) is crucial for neurodegenerative disease diagnosis.

Purpose of the Study:

• To develop a reusable immuno-IR sensor platform using reversible protein binding.
• To demonstrate continuous spectral monitoring and repeated capture-regeneration cycles.
• To achieve single-residue sensitivity in infrared difference spectroscopy.

Main Methods:

• Implemented reversible protein capture and release using the NColE7/Im7 protein pair.
• Engineered an Im7-antibody Fab fusion protein for the reusable sensor.
• Utilized alternating protein attachments and spectral monitoring for analysis.

Main Results:

• Achieved subpicomolar affinity capture and quantitative release of Im7-tagged proteins.
• Demonstrated a reusable immuno-IR sensor enabling multiple measurements on the same surface.
• Resolved infrared signatures of single amino acid substitutions, showing tyrosine absorption differences.

Conclusions:

• The NColE7/Im7 system enables reusable immuno-IR sensors with continuous monitoring.
• This platform facilitates differential screening and protein analysis with high sensitivity.
• The study proves the potential for single-residue sensitivity in infrared difference spectroscopy.