High-Throughput Microrheology for the Assessment of Protein Gelation Kinetics

Abstract

A high-throughput microrheological assay is employed to assess the gelation kinetics of a coiled-coil protein, Q, across a compositional space with varying ionic strengths and pH values. Two methods of passive microrheologymultiple particle tracking (MPT) and differential dynamic microscopy (DDM)are used to determine mean-squared displacements of tracer beads embedded in protein solutions with respect to lag time over a fixed period. MPT data was analyzed to determine gelation kinetics in a high-throughput, automatable manner by fitting relaxation exponents to sigmoidal curves and verifying with the more traditionally used time-cure superposition. DDM-determined gelation time was assessed as the last resolvable time, which we found to be on a similar scale to gelation times given by MPT. Both methods show distinct advantages with regard to being used in a highthroughput, automatable setup; DDM can serve as an effective initial screen for rapid gelation kinetics due to it requiring less user intervention and inputs, with MPT giving a more complete understanding of the entire gelation process. Using these methods, a clear optimum for rapid gelation was observed near the isoelectric point of Q and at higher ionic strengths over the compositional space studied.

Particle-Based Microrheology As a Tool for Characterizing Protein-Based Materials

“…Recently, the authors of this review compared the effectiveness of DDM and MPT in tracking gelation, using the previously described coiled-coil protein, Q , , as a model system . Using an imaging setup similar to that of Schultz et al, 25 combinations of environmental conditions were screened for their effect on the gelation kinetics of Q .…”

“…Recently, the authors of this review compared the effectiveness of DDM and MPT in tracking gelation, using the previously described coiled-coil protein, Q , , as a model system . Using an imaging setup similar to that of Schultz et al, 25 combinations of environmental conditions were screened for their effect on the gelation kinetics of Q . Notably, this allowed for the rapid determination of an optimum at a pH near the isoelectric point and at the highest ionic strength within the range of conditions studied where gelation was observed on the shortest time scale .…”

“…This was used as a model case to compare the effectiveness of MPT and DDM in tracking gelation in high throughput. For MPT, gelation kinetics were analyzed using two methods; while the traditionally done time–cure superposition is a time-intensive, user-heavy process and thus not amenable for high-throughput implementation, relaxation exponents were also fit to sigmoidal curves such that analysis became automatable and user-bias was largely removed . For DDM, the resolution limit previously described by Martineau et al was used as an estimate for the gelation time .…”

“…A key finding from this study, is that the methods largely agree with each other in determining gelation times of the protein at different environmental conditions (Figure ). Although DDM is more amenable to high-throughput use due to its robust analysis and lack of user intervention, MPT also remains a key method because of its ability to track particle movement through the entire gelation process . Thus, a possible combination of the two methods in assessing protein gelation kinetics may consist of using DDM to identify promising gelation conditions or variants within a library and then using MPT to fully elucidate the gelation kinetics and mechanism.…”

“…(b) Gelation times for pH 10–12 plotted on a smaller y -axis for better visualization of data trends. Reproduced with permission from ref . Copyright 2022 American Chemical Society …”