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ConfocAl Microscopy and real-time Rheology of dynamIc hyroGels

Periodic Reporting for period 4 - CAM-RIG (ConfocAl Microscopy and real-time Rheology of dynamIc hyroGels)

Reporting period: 2021-11-01 to 2023-04-30

Supramolecular hydrogels have become an important research area in the last decade due to their unique properties including easy preparation, self-healing and stimuli-responsiveness for applications such as sensing, biomaterials, biomedicine, energy conversion, catalysis, etc. Nonetheless, the rational design of the current state-of-the-art of supramolecular hydrogels is limited due to a lack of quantitatively systematic studies of structure-property relationships between microscopic supramolecular motifs and the macroscopic mechanical behaviour. In order to overcome this limitation, the direct visualization and resolution of single molecules by super-resolution microscopy (SRM) can enable in-depth investigations into the structure-property relationships of supramolecular hydrogels with unprecedented temporal and spatial resolution. Through the deep understanding of structure-property relationships, the rational design of supramolecular hydrogels with controlled architecture and function will become possible, bringing new potential for the development of supramolecular materials with desirable applications in many different fields.
This project represents a new research strategy at the frontier of supramolecular chemistry, polymer chemistry and material science, aiming to develop an innovative methodology to probe dynamic host-guest complexation as individual entities and within hydrogels at the molecular level with temporal and spatial resolution; design of supramolecular hydrogels with tuneable dynamics, morphologies and optical properties; gain fundamental knowledge that will serve to the development of technologies in sensing, biomedical and pharmaceutical sector. In this way, the ability to create desirable supramolecular hydrogels with tuneable properties and functions will draw enormous attention from scientists in different fields of chemistry, materials science, engineering and biomedicine, and will also inspire new collaboration within our scientific community. It is also noteworthy that the profound knowledge that we will acquire by studying the rheological behaviour of a range of supramolecular hydrogels will contribute fundamental knowledge to the field of polymer rheology, especially for the class of supramolecular polymers formed from noncovalent interactions.
Up to now, fluorescent aryl viologen (ArV) and extended viologen (EV) derivatives have been developed with different molecular geometries and with photoluminescence (PL) emission over a broad range of the visible electromagnetic spectrum. Upon complexation within cucurbituril (CB[n]) cavities, the local environment of these viologens was affected by changes of the charge transfer (CT) interactions and possible excimer formation, altering their optical properties, such as an increase in the fluorescence quantum yield and fluorescence lifetime. Differences in the PL intensity of EVs in the presence of CB[8] and CB[7] were identified at very low concentrations (nM) by implementing single molecule TIRF (total internal reflection fluorescence) microscopy to reveal distinct guest-host motifs. Additionally, monomers bearing fluorescent viologen derivatives have been synthesized and incorporated in hydrogels in order to investigate their effect in the dynamics of supramolecular hydrogels and viscoelastic properties by the piezo axial vibrator and rotational rheometer. The next step of this project will be to simultaneously carry out super-resolution microscopy with rheological analysis in order to probe the physical crosslinking dynamics of CB[8] host–guest complexation in hydrogels developed with different polymer backbones and fluorescent guests, and correlate with their viscoelastic properties.
Key outcomes of CAM-RIG:
• Successful sourcing and set up of the bespoke CAM-RIG set up coupling super resolution microscopy with rheology.
• Testing and validation of the CAM-RIG set up
• Synthesis and characterisation of novel fluorophore guests as well as studying their binding to CB[n].
• Characterisation of the fluorophore guests on CAM-RIG set up.
• Initial synthesis of hydrogels containing the viologen fluorophore guest molecules as well as characterisation of the resultant fluorescent properties.
• Investigation of binding of small molecule fluorophores with CB[n] on a single molecule level
Several novel methodologies have been developed to date in an effort to image hydrogels by superresolution microscopy (SRM) in which fluorophores should be present at nM concentrations. Single molecule TIRF microscopy was set up and used to interrogate 2:2 host-guest binding at nM concentrations by PDRA 2 (3 March 2019 – 31 October 2019). This was the first time, to the best of our knowledge, that CB[n] host-guest complexes would be stable at such levels of dilution. A manuscript will soon be prepared to disseminate these findings.

This work was supported by PDRA 3 (5 June 2019 – 31 October 2019). The guest fluorophores can also be incorporated into the dynamic crosslinked hydrogels through 2:2 binding with CB[8]. PDRA 3 worked on the synthesis of fluorescently labelled CB[8]-mediated hydrogels. Guests binding to CB[8] through heteroternary host-guest interactions give rise to hydrogels which exhibit different viscoelastic properties. The synthesis of such hydrogels which also incorporate a small quanitity of the fluorescent 2:2 complexes is still being optimized and we hope that we will soon be able to resolve how the supramolecular interactions which drive the viscoelastic properties of hydrogels can be imaged by SRM whilst simultaneously measuring their rheological properties using the PAV.

RA 1 was responsible for the installation of new equipment which is able to pull fibers from hydrogels helping with reproducible and controlled fiber formation. The equipment was designed, developed and built in the lab.
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