Reversible Sandwich‐Based Particle Nanoswitch for Continuous Protein Monitoring at Picomolar Concentrations with Automated Calibration

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.