A Reversible ATR-FTIR Biosensor with Single-Residue Sensitivity

Infrared spectroscopy provides label-free access to protein structure information with high sensitivity. Challenges in infrared spectroscopy include artifacts arising from temperature changes, baseline drifts, and the strong water absorption, especially in difference spectroscopy, where signals are orders of magnitude smaller than the total absorbance. Previously, we introduced an ATR-based immuno-infrared sensor (iRS) based on sophisticated surface chemistry. This provides a stable platform for covalent protein immobilization and allows its application with body fluids such as cerebrospinal fluid or blood without unspecific background. It detects misfolding of small amounts of antigen (Aβ or α-synuclein) by binding to an antibody or antibody fragment, which allows for the diagnosis of various neurodegenerative diseases. Here, we extend this platform by introducing reversible binding via the NColE7/Im7 pair. Im7-tagged proteins are captured with subpicomolar affinity and quantitatively released under mild conditions (high salt or low pH), allowing repeated capture-regeneration cycles with continuous spectral monitoring of the Im7 fusion protein. Using an Im7-antibody Fab fusion protein, we present a reusable immuno-IR sensor that enables multiple measurements on the same surface. For example, different antibodies can be used for differential screening. Using alternating Im7 protein attachments to the internal reflection element, we further compare Im7 single point mutants to wild-type Im7 to resolve infrared signatures of specific amino acid substitutions. Difference spectra reveal the distinct tyrosine absorption associated with either the introduction or removal of a single tyrosine residue, providing a proof-of-concept for single-residue sensitivity in infrared difference spectroscopy enabled by complete protein exchange instead of induced perturbations on the same protein.