Single-molecule readout of reversible nanoswitches enables continuous monitoring of low biomarker concentrations.

Continuous sensing technologies are expected to change how dynamic bioprocesses will be monitored and controlled in the future. However, a fundamental challenge in the field of biomolecular sensing is that low concentrations invariably cause slow sensor responses. In this paper, we explain how continuous sensors can be developed for quantifying very low biomarker concentrations with fast sensor response times. The measurement concept is based on combining reversible affinity-based nanoswitches with single-molecule readout. A generalizable rate-based simulation model was developed and validated on experimental data of a sandwich-based nanoswitch. The results show how the nanoswitch design and the sensing acquisition parameters control the amplitude and stochastic variations of the sensor response, and how these affect the precision of the concentration determination. We show that the measurement precision is limited by the counting statistics of molecular sandwich events. Using realistic design parameters, we predict limits-of-quantification in the low picomolar range within measurement timescales of minutes. These results pave the way for the development of intrinsically reversible nanoswitch sensors that can access unexplored concentration-time spaces of dynamic biosystems.