Continuous Protein Sensing Using Fast-Dissociating Antibody Fragments in Competition-Based Biosensing by Particle...

Sensing technologies for the continuous monitoring of protein concentrations are important for understanding time-dependent behaviors of biological systems and for controlling bioprocesses. We present a continuous sensing methodology based on tethered particle motion (t-BPM) that utilizes fast-dissociating antibody fragments (Fabs) for continuous protein monitoring. A competition-based t-BPM sensor was developed and characterized utilizing custom-made Fabs. The sensing concept was demonstrated for lactoferrin, an 80 kDa iron-binding glycoprotein that is part of the innate immune response. Thirteen Fabs were compared using free particle motion sensing as well as surface plasmon resonance, of which six Fabs showed rapid association and dissociation. The integration of the Fabs into the t-BPM sensor enabled nanomolar lactoferrin detection in both buffer solutions and milk matrices over tens of hours. This work demonstrates how continuous protein sensing can be realized using fast-dissociating antibodies in a competitive sensor format.