Reversible Sandwich-Based Particle Nanoswitch for Continuous Protein Monitoring at Picomolar Concentrations with...

Continuous monitoring of specific proteins is essential for understanding the dynamics of biological systems and for enabling real-time measurement-and-control strategies in bioprocesses. Ideally, sensors for continuous monitoring should be intrinsically reversible and able to perform accurate measurements over long time spans. Here, we present a particle nanoswitch sensor containing two different antibody fragments that bind reversibly to a protein of interest and thus form transient sandwich complexes. The antibody fragments are incorporated into the sensor using site-specific conjugation strategies to achieve optimal antibody orientation. Short-lived sandwich complexes are detected with single-molecule resolution, by tracking the motion of tethered particles. The sensing concept is demonstrated for lactoferrin, an iron-binding and immune-modulating protein. We show continuous measurements of picomolar concentrations, with a delay time of less than 12 min, over a monitoring period of more than 12 h. Automated calibration strategies are described that result in a mean absolute relative difference below 10% compared to reference measurements. These results demonstrate how rapid continuous protein sensing at picomolar concentrations can be achieved using reversible sandwich-based particle nanoswitches, enabling long-term monitoring of dynamic bioprocesses.