Conical Intersection Accessibility Dictates Brightness in Red Fluorescent Proteins.

Red fluorescent protein (RFP) variants are highly sought after for in vivo imaging since longer wavelengths improve depth and contrast in fluorescence imaging. However, the lower energy emission wavelength usually correlates with a lower fluorescent quantum yield compared to their green emitting counterparts. To guide the rational design of bright variants, we have theoretically assessed two variants (mScarlet and mRouge) which are reported to have very different brightness. Using an α-CASSCF QM/MM framework (chromophore and all protein residues within 6 Å of it in the QM region, for a total of more than 450 QM atoms), we identify key points on the ground and first excited state potential energy surfaces. The brighter variant mScarlet has a rigid scaffold, and the chromophore stays largely planar on the ground state. The dimmer variant mRouge shows more flexibility and can accommodate a pretwisted chromophore conformation which provides easier access to conical intersections. The main difference between the variants lies in the intersection seam regions, which appear largely inaccessible in mScarlet but partially accessible in mRouge. This observation is mainly related with changes in the cavity charge distribution, the hydrogen-bonding network involving the chromophore and a key ARG/THR mutation (which changes both charge and steric hindrance).

Do the properties of gels constructed by interlinking triply-responsive microgels follow from those of the building blocks?

Microgels (MGs) are swellable crosslinked polymer colloids. They can also be used as the only building block to construct nanostructured hydrogels which are denoted as doubly crosslinked microgels (DX MGs). Here, new triply responsive DX MGs comprised of interlinked MGs of oligo(ethylene glycol)methacrylate (OEGMA), 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA), methacrylic acid (MAA) and a o-nitrobenzyl-based UV photocleavable crosslinker are investigated. The MGs swelled or collapsed in response to temperature and pH changes. These behaviours were rationalised with a generic model using Monte Carlo simulations. The MGs also degraded when UV irradiated due to photocleavage of nPh. DX MGs were assembled from the MGs to give injectable gels that were not cytotoxic to nucleus pulposus cells. Comparison of the responsive properties of the DX MGs and MGs showed that the temperature and pH responses of the former were mostly governed by the latter. However, two key differences were found. Firstly, whilst increasing the crosslinker mol% in the MG building blocks (x) did not change MG particle swelling, the compression modulus (E) and swelling of the DX MG gels were strongly affected by x. The E value for the gels was tuneable using x which is a potentially useful new observation for DX MGs. Secondly, UV irradiation of the DX MGs enhanced gel mechanical photostability in contrast to the behaviour of the MGs. We find that the properties of the DX MGs do not simply follow those of the parent MGs and propose mechanisms to account for the differences. The new family of multi-responsive DX MGs presented in this study have potential application for soft tissue repair as injectable gels or as gel implants which report sterilisation.

Highly deformable hydrogels constructed by pH-triggered polyacid nanoparticle disassembly in aqueous dispersions.

Most hydrogels are prepared using small-molecule monomers but unfortunately this approach may not be feasible for certain biomaterial applications. Consequently, alternative gel construction strategies have been established, which include using covalent inter-linking of preformed gel particles, or microgels (MGs). For example, covalently interlinking pH-responsive MGs can produce hydrogels comprising doubly crosslinked microgels (DX MGs). We hypothesised that the deformability of such DX MGs was limited by the presence of intra-MG crosslinking. Thus, in this study we designed new nanoparticle (NP)-based gels based on pH-swellable NPs that are not internally crosslinked. Two polyacid NPs were synthesised containing methacrylic acid (MAA) and either ethyl acrylate (EA) or methyl methacrylate (MMA). The PMAA-EA and PMAA-MMA NPs were subsequently vinyl-functionalised using glycidyl methacrylate (GMA) prior to gel formation via free-radical crosslinking. The NPs mostly disassembled on raising the solution pH but some self-crosslinking was nevertheless evident. The gels constructed from the EA- and MMA-based NPs had greater breaking strains than a control DX MG. The effect of varying the solution pH during curing on the morphology and mechanical properties of gels prepared using PMAA-MMA-GMA NPs was studied and both remarkable deformability and excellent recovery were observed. The gels were strongly pH-responsive and had tensile breaking strains of up to 420% with a compressive strain-at-break of more than 93%. An optimised formulation produced the most deformable and stretchable gel yet constructed using NPs or MGs as the only building block.

Surface structure, optoelectronic properties and charge transport in ZnO nanocrystal/MDMO-PPV multilayer films.

Blends of semiconducting nanocrystals and conjugated polymers continue to attract major research interest because of their potential applications in optoelectronic devices, such as solar cells, photodetectors and light-emitting diodes. In this study we investigate the surface structure, morphological and optoelectronic properties of multilayer films constructed from ZnO nanocrystals (NCs) and poly[2-methoxy-5-(3',7'-dimethyloctyloxy)-1,4-phenylenevinylene] (MDMO-PPV). The effects of layer number and ZnO concentration (CZnO) used on the multilayer film properties are investigated. An optimised solvent blend enabled well-controlled layers to be sequentially spin coated and the construction of multilayer films containing six ZnO NC (Z) and MDMO-PPV (M) layers (denoted as (ZM)6). Contact angle data showed a strong dependence on CZnO and indicated distinct differences in the coverage of MDMO-PPV by the ZnO NCs. UV-visible spectroscopy showed that the MDMO-PPV absorption increased linearly with the number of layers in the films and demonstrates highly tuneable light absorption. Photoluminescence spectra showed reversible quenching as well as a surprising red-shift of the MDMO-PPV emission peak. Solar cells were constructed to probe vertical photo-generated charge transport. The measurements showed that (ZM)6 devices prepared using CZnO = 14.0 mg mL-1 had a remarkably high open circuit voltage of ∼800 mV. The device power conversion efficiency was similar to that of a control bilayer device prepared using a much thicker MDMO-PPV layer. The results of this study provide insight into the structure-optoelectronic property relationships of new semiconducting multilayer films which should also apply to other semiconducting NC/polymer combinations.

Pickering Emulsions Stabilized by pH-Responsive Microgels and Their Scalable Transformation to Robust Submicrometer Colloidoisomes with Selective Permeability.

Colloidosomes are micrometer-sized hollow particles that have shells consisting of coagulated or fused colloid particles. While many large colloidosomes with sizes well above 1.0 µm have been prepared, there are fewer examples of submicrometer colloidosomes. Here, we establish a simple emulsion templating-based method for the preparation of robust submicrometer pH-responsive microgel colloidosomes. The colloidosomes are constructed from microgel particles based on ethyl acrylate and methacrylic acid with peripheral vinyl groups. The pH-responsive microgels acted as both a Pickering emulsion stabilizer and macro-cross-linker. The emulsion formation studies showed that the minimum droplet diameter was reached when the microgel particles were partially swollen. Microgel colloidosomes were prepared by covalently interlinking the microgels adsorbed at the oil-water interface using thermal free-radical coupling. The colloidosomes were prepared using a standard high-shear mixer with two different rotor sizes that corresponded to high shear (HS) and very high shear (VHS) mixing conditions. The latter enabled the construction of submicrometer pH-responsive microgel-colloidosomes on the gram scale. The colloidosomes swelled strongly when the pH increased to above 6.0. The colloidosomes were robust and showed no evidence of colloidosome breakup at high pH. The effect of solute size on shell permeation was studied using a range of FITC-dextran polymers, and size-selective permeation occurred. The average pore size of the VHS microgel-colloidosomes was estimated to be between 6.6 and 9.0 nm at pH 6.2. The microgel-colloidosome properties suggest that they have the potential for future applications in cosmetics, photonics, and delivery.

Bioinspired Multistimuli-Induced Synergistic Changes in Color and Shape of Hydrogel and Actuator Based on Fluorescent Microgels.

Fluorescent hydrogels have emerged as one of the most promising candidates for developing biomimetic materials and artificial intelligence owing to their unique fluorescence and responsive properties. However, it is still challenging to fabricate hydrogel that exhibits synergistic changes in fluorescence color and shape in response to multistimulus via a simple method. Herein, blue- and orange-emitting fluorescent microgels (MGs) both are designed and synthesized with pH-, thermal-, and cationic-sensitivity via one-step polymerization, respectively. The two fluorescent MGs are incorporated into transparent doubly crosslinked microgel (DX MG) hydrogels with a preset ratio. The DX MG hydrogels can tune the fluorescent color accompanied by size variation via subjecting to external multistimulus. Thus, DX MG hydrogels can be exploited for multiresponsive fluorescent bilayer actuators. The actuators can undergo complex shape deformation and color changes. Inspired by natural organisms, an artificial morning glory with color and size changes are showcased in response to buffer solutions of different pH values. Besides, an intelligent skin hydrogel, imitating natural calotes versicolor, by assembling four layers of DX MG with different ratios of MGs, is tailored. This work serves as an inspiration for the design and fabrication of novel biomimetic smart materials with synergistic functions.

Comparing pH-responsive nanogel swelling in dispersion and inside a polyacrylamide gel using photoluminescence spectroscopy and small-angle neutron scattering.

Nanosized probes that report their changes in dimensions within networks in response to environmental stimuli are potentially important for applications such as drug delivery, load-supporting hydrogels and soft robotics. Recently, we developed a fluorescent pH-responsive nanogel (NG) that used Förster-resonance energy transfer (FRET) to report changes in the probe separation and NG swelling within hydrogels using photoluminescence (PL) spectroscopy. However, FRET cannot measure nanoparticle dimensions and is subject to artefacts. Here, we report the use of small-angle neutron scattering (SANS) to study both the NGs in dispersion and in polyacrylamide (PAAm) gels as a function of pH. We compare the PL and SANS data for both systems and as a function of pH. The SANS data for the dispersed NGs indicate that they have a core-shell structure with a swollen mesh size of ∼1.0 nm. We hypothesized that the NGs inside the PAAm gel would show the same general changes in scattering as the pH is increased, as observed for the dispersed NGs, and this is confirmed by the data. In summary, the data confirm that PL is a suitable (accessible) method for reporting internal environmental changes within gels using NG probes.

Highly swelling pH-responsive microgels for dual mode near infra-red fluorescence reporting and imaging.

Near infra-red (NIR) fluorescence is a desirable property for probe particles because such deeply penetrating light enables remote reporting of the local environment in complex surroundings and imaging. Here, two NIR non-radiative energy transfer (NRET) fluorophores (Cy5 and Cy5.5) are coupled to preformed pH-responsive poly(ethylacrylate-methacrylic acid-divinylbenzene) microgel particles (PEA-MAA-5/5.5 MGs) to obtain new NIR fluorescent probes that are cytocompatible and swell strongly. NIR ratiometric photoluminescence (PL) intensity analysis enables reporting of pH-triggered PEA-MAA-5/5.5 MG particle swelling ratios over a very wide range (from 1-90). The dispersions have greatly improved colloidal stability compared to a reference temperature-responsive NIR MG based on poly(N-isopropylacrylamide) (PNP-5/5.5). We also show that the wavelength of maximum PL intensity (λ max) is a second PL parameter that enables remote reporting of swelling for both PEA-MAA-5/5.5 and PNP-5/5.5 MGs. After internalization the PEA-MAA-5/5.5 MGs are successfully imaged in stem cells using NIR light. They are also imaged after subcutaneous injection into model tissue using NIR light. The new NIR PEA-MAA-5/5.5 MGs have excellent potential for reporting their swelling states (and any changes) within physiological settings as well as very high ionic strength environments (e.g., waste water).

Using Soft Polymer Template Engineering of Mesoporous TiO2 Scaffolds to Increase Perovskite Grain Size and Solar Cell Efficiency.

The mesoporous (meso)-TiO2 layer is a key component of high-efficiency perovskite solar cells (PSCs). Herein, pore size controllable meso-TiO2 layers are prepared using spin coating of commercial TiO2 nanoparticle (NP) paste with added soft polymer templates (SPT) followed by removal of the SPT at 500 °C. The SPTs consist of swollen crosslinked polymer colloids (microgels, MGs) or a commercial linear polymer (denoted as LIN). The MGs and LIN were comprised of the same polymer, which was poly(N-isopropylacrylamide) (PNIPAm). Large (L-MG) and small (S-MG) MG SPTs were employed to study the effect of the template size. The SPT approach enabled pore size engineering in one deposition step. The SPT/TiO2 nanoparticle films had pore sizes > 100 nm, whereas the average pore size was 37 nm for the control meso-TiO2 scaffold. The largest pore sizes were obtained using L-MG. SPT engineering increased the perovskite grain size in the same order as the SPT sizes: LIN < S-MG < L-MG and these grain sizes were larger than those obtained using the control. The power conversion efficiencies (PCEs) of the SPT/TiO2 devices were ∼20% higher than that for the control meso-TiO2 device and the PCE of the champion S-MG device was 18.8%. The PCE improvement is due to the increased grain size and more effective light harvesting of the SPT devices. The increased grain size was also responsible for the improved stability of the SPT/TiO2 devices. The SPT method used here is simple, scalable, and versatile and should also apply to other PSCs.

Using green emitting pH-responsive nanogels to report environmental changes within hydrogels: a nanoprobe for versatile sensing.

Remotely reporting the local environment within hydrogels using inexpensive laboratory techniques has excellent potential to improve our understanding of the nanometer-scale changes that cause macroscopic swelling or deswelling. Whilst photoluminescence (PL) spectroscopy is a popular method for such studies this approach commonly requires bespoke and time-consuming synthesis to attach fluorophores which may leave toxic residues. A promising and more versatile alternative is to use a pre-formed nanogel probe that contains a donor/acceptor pair and then "dope" that into the gel during gel assembly. Here, we introduce green-emitting methacrylic acid-based nanogel probe particles and use them to report the local environment within four different gels as well as stem cells. As the swelling of the nanogel probe changes within the gels the non-radiative energy transfer efficiency is strongly altered. This efficiency change is sensitively reported using the PL ratiometric intensity from the donor and acceptor. We demonstrate that our new nanoprobes can reversibly report gel swelling changes due to five different environmental stimuli. The latter are divalent cations, gel degradation, pH changes, temperature changes and tensile strain. In the latter case, the nanoprobe rendered a nanocomposite gel mechanochromic. The results not only provide new structural insights for hierarchical natural and synthetic gels, but also demonstrate that our new green-fluorescing nanoprobes provide a viable alternative to custom fluorophore labelling for reporting the internal gel environment and its changes.

Self-curing super-stretchable polymer/microgel complex coacervate gels without covalent bond formation.

Elastic physical gels are highly desirable because they can be conveniently prepared and readily shaped. Unfortunately, many elastic physical gels prepared in water require in situ free-radical polymerization during the gel formation stage. In contrast, complex coacervate gels are physical gels that can be prepared by simply mixing two pre-formed oppositely-charged polyelectrolytes. However, as far as we are aware, highly elastic complex coacervate gels have not yet been reported. Herein, we combine polyanionic microgel particles with a well-known commercially-available cationic polyelectrolyte to prepare polymer/microgel complex coacervate (PMCC) physical gels. This new family of gels requires annealing at only 37 °C and behaves like a covalent gel but does not form covalent bonds. Thermal reconfiguration of the dynamic ionic bonds transforms the shapeable pre-gel into a highly elastic gel that is super-stretchable, adhesive, self-healing, highly swellable and can be further toughened using Ca2+ as an ionic crosslinker. Our PMCC gels have excellent potential for applications as engineering gels and structural biomaterials, as well as for wound healing and water purification.

Plasmonic and colloidal stability behaviours of Au-acrylic core-shell nanoparticles with thin pH-responsive shells.

The localised surface plasmon resonance (LSPR) of Au nanoparticles (NPs) as well as its interaction with nearby entities provides a wealth of fundamental and practical information at the nanometre scale. A number of studies have investigated core-shell NPs with Au cores and polymer shells that are temperature-responsive. However, there are very few studies of pH-responsive Au-polymer NP shells. Precipitation polymerisation is a scalable method and here we establish such a method to synthesise pH-responsive Au-poly(methyl methacrylate) copolymer core-shell NPs without the need for pre-functionalisation. The comonomers used were methacrylic acid (MAA) or 2-carboxyethyl acrylate (CEA) and the shells were crosslinked with ethylene glycol dimethacrylate. A series of five core-shell systems with collapsed shell thicknesses less than 30 nm are studied. The shell-thicknesses for the CEA-based core-shell NPs are relatively thin (≤5 nm) compared to related Au-polymer core-shell NPs prepared using precipitation polymerisation. The LSPR properties of the core-shell NPs were dependent on the shell thickness and were successfully simulated using finite difference time domain (FDTD) calculations. Two systems are considered further as exemplars. The MAA-based core-shell system with the thickest shell exhibited enhanced colloidal stability to added electrolyte. The CEA-based core-shell dispersion with the thinnest shells displayed reversible pH-triggered aggregation and was cytocompatible for HeLa cells. Proof-of-concept data are presented that demonstrate intracellular pH reporting.

Core-shell-shell cytocompatible polymer dot-based particles with near-infrared emission and enhanced dispersion stability.

Polymer dots (PDs) are promising fluorescent probes for biomaterials applications. Here, novel cytocompatible composite PD particles have been synthesised with a core-shell-shell morphology. The particles show near-infrared emission, improved fluorescent brightness and enhanced colloidal stability compared to pure PDs. The particles also show non-radiative resonance energy transfer (NRET) with a model dye.

Reducing hole transporter use and increasing perovskite solar cell stability with dual-role polystyrene microgel particles.

Perovskite solar cells (PSCs) are a disruptive technology that continues to attract considerable attention due to their remarkable and sustained power conversion efficiency increase. Improving PSC stability and reducing expensive hole transport material (HTM) usage are two aspects that are gaining increased attention. In a new approach, we investigate the ability of insulating polystyrene microgel particles (MGs) to increase PSC stability and replace the majority of the HTM phase. MGs are sub-micrometre crosslinked polymer particles that swell in a good solvent. The MGs were prepared using a scalable emulsion polymerisation method. Mixed HTM/MG dispersions were subsequently spin-coated onto PSCs and formed composite HTM-MG layers. The HTMs employed were poly(triaryl amine) (PTAA), poly(3-hexylthiophene) (P3HT) and Spiro-MeOTAD (Spiro). The MGs formed mechanically robust composite HTMs with PTAA and P3HT. In contrast, Spiro-MG composites contained micro-cracks due the inability of the relatively small Spiro molecules to interdigitate. The efficiencies for the PSCs containing PTAA-MG and P3HT-MG decreased by only ∼20% compared to control PSCs despite PTAA and P3HT being the minority phases. They occupied only ∼35 vol% of the composite HTMs. An unexpected finding from the study was that the MGs dispersed well within the PTAA matrix. This morphology aided strong quenching of the CH3NH3PbI3-xClx fluorescence. In addition, the open circuit voltages for the PSCs prepared using P3HT-MG increased by ∼170 mV compared to control PSCs. To demonstrate their versatility the MGs were also used to encapsulate P3HT-based PSCs. Solar cell stability data for the latter as well as those for PSCs containing composite HTM-MG were both far superior compared to data measured for a control PSC. Since MGs can reduce conjugated polymer use and increase stability they have good potential as dual-role PSC additives.

Responsive Nanogel Probe for Ratiometric Fluorescent Sensing of pH and Strain in Hydrogels.

In this study a new pH-responsive nanogel probe containing a complementary nonradiative resonance energy transfer (NRET) fluorophore pair is investigated and its ability to act as a versatile probe of network-related changes in three hydrogels demonstrated. Fluorescent sensing using NRET is a powerful method for studying relationships between Angstrom length-scale structure and macroscopic properties of soft matter. Unfortunately, inclusion of NRET fluorophores into such materials requires material-specific chemistry. Here, low concentrations of preformed nanogel probes were included into hydrogel hosts. Ratiometric photoluminescence (PL) data for the gels labeled with the nanogel probes enabled pH-triggered swelling and deswelling to be studied as well as Ca2+-triggered collapse and solute release. PL measurements during compression of a nanogel probe-labeled nanocomposite gel demonstrated mechanochromic behavior and strain sensing. The new nanogel probes have excellent potential for investigating the internal structures of gels and provide a versatile ratiometric fluorescent platform for studying pH and strain.