How Highly Heterogeneous Sensors with Single‐Molecule Resolution can Result in Robust Continuous Monitoring Over Long Time Spans

Abstract

Biomolecular sensors with single‐molecule resolution are composed of multitudes of transducers that measure state changes related to single‐molecular binding and unbinding events. Conventionally, signals are aggregated from many individual transducers in order to achieve sufficient statistics. However, by aggregating signals, transducer‐to‐transducer differences are lost and heterogeneities cannot be studied. Here, transducers with single‐molecule resolution over long time spans are studied, enabling the collection of sufficient statistics from independent transducers. This allows comparisons between transducers that reveal fundamental heterogeneities in their molecular assemblies related to stochastic variations. The study is performed with biosensing by particle motion, a sensing methodology with thousands of particles that dynamically interact with a sensing surface. The signals of individual particles are studied for series of modulations of analyte concentration over 25 h. The results show large differences in individual concentration‐dependent responses. Monte Carlo simulations clarify that heterogeneities can be attributed to stochastic fluctuations in the numbers of binder molecules, and that gradual changes of the response characteristics can be related to losses of molecules in the single‐particle transducers. The results give insights into molecular and temporal heterogeneities of continuous transducers with single‐molecule resolution and explain how sensors can be engineered to achieve robust, precise, and stable biomolecular monitoring.