Programmable single-molecule sensing with mobilized receptors

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The COVID19 pandemic has highlighted the need for diagnostic tests that can easily be reconfigured to detect extremely low concentrations of novel targets (such as traces of infectious pathogens). This program establishes a platform for broadly applicable ultrasensitive molecular detection using single-particle tracking to achieve extremely high sensitivity. The anticipated outcome of this program is a set of design principles and corresponding modular building blocks that can be used to construct sensors against any novel chemical and biomolecular target. In doing so, we will resolve multiple longstanding impediments to the commercialization of single-molecule detectors, thereby positioning Canada at the forefront of next-generation sensor technologies. Traditional single-molecule sensors operate by attaching a receptor to a surface and detecting the binding of a target molecule. The problem is that nonspecific absorption of the target molecule onto the surface (background) is difficult to distinguish from the desired receptor-target interaction. Our proposed approach resolves this ambiguity by adding an additional degree of freedom, movement, to distinguish these two cases. By continuously moving receptors at a characteristic velocity, and comparing that rate to the apparent movement of a target molecule, we can fingerprint true positives (where the receptor and target velocities match) and ignore false positives, such as when the target becomes immobilized on a surface. Here we propose to apply this concept and demonstrate the modularity using a bioinspired material that forms the molecular highways in mammalian cells, namely, microtubules. These nanoscale biopolymers have been demonstrated to be highly engineerable, therapeutically interesting, and easily mobilized using their native biological partners, kinesin motor proteins. This will be done in three aims: (1) we will show that correlative movement can be used to confirm binding interactions directly to microtubules; (2) we will encode receptor molecules to pathogenic material onto the microtubules using bioorthogonal chemistry; and (3) demonstrate the parallelizability of the approach for simultaneous detection and characterization of target molecules in complex mixtures. In doing so, this program sets up the long term objective of creating a new class of ultrasensitive molecular sensors that will be explored further. At completion, this program will have trained 2 PhDs 1 Msc and 5 undergraduate students with highly marketable skill sets in quantitative data analysis, instrument development, and problem solving required to take on future global challenges.