Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

Peptide-based hydrogels were shown to serve as good matrices for 3D cell culture and to be applied in the field of regenerative medicine. The study of the cell-matrix interaction is important for the understanding of cell attachment, proliferation, and migration, as well as for the improvement of the matrix. Here, we used scanning ion conductance microscopy (SICM) to study the growth of cells on self-assembled peptide-based hydrogels. The hydrogel surface topography, which changes during its formation in an aqueous solution, were studied at nanoscale resolution and compared with fluorescence lifetime imaging microscopy (FLIM). Moreover, SICM demonstrated the ability to map living cells inside the hydrogel. A zwitterionic label-free pH nanoprobe with a sensitivity > 0.01 units was applied for the investigation of pH mapping in the hydrogel to estimate the hydrogel applicability for cell growth. The SICM technique that was applied here to evaluate the cell growth on the peptide-based hydrogel can be used as a tool to study functional living cells.

The growing interest in biomimetic hydrogels is due to their successful applications in tissue engineering, 3D cell culturing and drug delivery. The major characteristics of hydrogels include swelling, porosity, degradation rate, biocompatibility, and mechanical properties. Poor mechanical properties can be regarded as the main limitation for the use of hydrogels in tissue engineering, and advanced techniques for its precise evaluation are of interest. The current research aims to demonstrate the suitability of scanning ion conductance microscopy (SICM) for assessing the stiffness of various hydrogels – Fmoc-FF peptide hydrogel, polyacrylamide and gelatin, – which differ by two orders of magnitude in Young's modulus (E). We provide a direct comparison between SICM measurements and atomic force microscopy (AFM) data, the latter being a widely used method for assessing the mechanical properties of scaffolds. The results of these methods showed good agreement, however, for materials with various stiffness two SICM-based approaches – application of hydrostatic pressure and application of intrinsic force – should be used. For hydrogels with Young's modulus of more than 2.5 kPa the application of SICM using hydrostatic pressure is recommended, whereas for soft materials with E ∼ 200–400 Pa the technique using intrinsic force can also be applied. We have shown that SICM and AFM methods can be used for the evaluation of the mechanical properties of soft hydrogels with nanometer resolution, while SICM is a completely non-invasive method, which requires a minimum influence on the sample structure.