Generalizable Molecular Switch Designs for In Vivo Continuous Biosensing

Continuous biosensors have the potential to transform medicine, enabling healthcare to be more preventative and personalized as compared to the current standard of reactive diagnostics. Realizing this transformative potential requires biosensors that can function continuously in vivo without sample preparation and deliver molecular specificity, sensitivity, and high temporal resolution. Molecular switches stand out as a promising solution for creating such sensors for the continuous detection of many different types of molecules. Molecular switches are target-binding receptors designed such that binding causes a conformational change in the switch's structure. This structure switching induces a measurable signal change via reporters incorporated into the molecular switch, enabling highly specific, label-free sensing. However, there remains an outstanding need for generalizable switch designs that can be adapted for the detection of a wide range of molecular targets. In this Account, we chronicle the work our lab has done to develop generalizable molecular switch designs that allow more rapid development of high-performance biosensors across a broad range of biomarkers. Pioneering efforts toward molecular switch-based biosensing have employed aptamers─nucleic acid-based receptors with sequence-specific target affinity. However, most of these early demonstrations relied upon aptamers with intrinsic structure-switching capabilities. To accelerate aptamer switch design for more targets, we have applied rational design and knowledge of an aptamer's structure to engineer switching functionality into pre-existing aptamers. Our designs contained several structural parameters that enabled us to easily tune the sensitivity and binding kinetics of the resulting switches. Using such rationally designed aptamer switches, we demonstrated continuous optical detection of cortisol and dopamine at physiologically relevant concentrations in complex media. In an effort to move beyond aptamers with well-characterized structural properties, we developed a high-throughput screening method that allowed us to simultaneously screen millions of candidates derived from a single aptamer to find sensitive switches without any prior structural knowledge of the parent aptamer. In subsequent work, we reasoned that we could enhance our ability to design a broader range of biosensors by leveraging other classes of receptors besides aptamers. Antibodies offer excellent affinity and specificity for a wide range of targets, but lack the capacity for intrinsic structure switching. We therefore developed a set of strategies to augment antibodies with the capacity to act as molecular switches with a diverse range of target-binding properties. We combined both the high binding affinity of an antibody with the structure-switching capabilities of an aptamer to develop a chimeric switch with 100-fold enhanced sensitivity for a protein target and improved function in interferent-rich samples. In a second design, we developed a competitive immunoassay-inspired scheme to engineer switching behavior into an antibody for minutes-scale temporal resolution with nanomolar sensitivity. We used this competitive antibody-switch to demonstrate the first continuous detection of cortisol directly in whole blood. Together, these advances in molecular switch development have expanded our capability to rapidly engineer new continuous biosensors, thereby increasing opportunities to track health via a wide range of biomarkers to deliver more personalized and preventative medicine.