TDP#3 Amplified Force Sensors

Harnessing cellular force measurements for applications such as diagnosis of disease and drug screening requires ultrasensitive and highly specific tension probe signal. Under TDP#3, we will leverage the programmability of DNA to generate tension probes with enhanced sensitivity and amplified response to mechanical input. The common theme in this TDP is not a specific driving biomedical question, rather the uniting theme is overcoming the common challenge of detecting highly transient biophysical events that are sparse and distributed across the cell surface.

Hybridization Chain Reaction (HCR)-based Force Sensing and in-situ Signal Amplification

Molecular tension probe signal is quantified using high resolution fluorescence microscopes equipped with single photon sensitivity EMCCDs. Total internal reflection fluorescence (TIRF) imaging is typically required to enhance the S/N. The need for such dedicated microscopy instrumentation hinders widespread adoption and application of tension probes. Ideally, one would use conventional epifluorescence imaging or other bulk fluorescence methods to readout cell force levels. To address this need, we have developed an in situ hybridization chain reaction (HCR) to amplify the mechanical signal using DNA strand displacement reactions. HCR is akin to PCR and ELISA- type assays, in that this reaction generates an amplified fluorescent signal in response to an input. In this case the input is a specific magnitude mechanical event generated by cells.

Mechano-Cas12a Assisted Tension Sensor (MCATS) for Massively Amplified Cell Traction Force Measurements

Cells transmit piconewton forces to mediate essential biological processes such as coagulation. One challenge is that cell-generated forces are infrequent, transient, and difficult to detect. Here, we report the development of Mechano-Cas12a Assisted Tension Sensor (MCATS) that utilizes CRISPR-Cas12a to transduce and amplify the
molecular forces generated by cells. We demonstrate the power of MCATS by detecting the forces generated by as few as ~10^3 human platelets in a high-throughput manner. Platelet forces are significantly inhibited when blood samples are treated with FDA-approved drugs such as aspirin, eptifibatide(integrilin®), 7E3(Reopro®), and ticagrelor (Brelinta®). Because MCATS requires <5uL of blood/measurement, a single blood draw can generate a personalized dose-response curve and IC50 for this panel of drugs. Platelet activity and force-generation are tightly associated, and hence MCATS was used to quantify platelet dysfunction following cardiopulmonary bypass (CPB) in a pilot study of 7 cardiac patients. We found that MCATS detected platelet dysfunction which strongly correlated with the need for platelet transfusion to limit bleeding. These results indicate MCATS may be a useful assay for clinical applications.

Adapted from Duan et al. Nat. BME 2023

We are developing other types of probes that are currently manuscripts in preparation and we will be sharing the details of this new technology when these developments are published.