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Abstract: Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180–250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption.

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Abstract: Circular Dichroism imaging in the 190–650 nm spectral region pionered at Diamond Light Source B23 beamline, has been made possible by the highly collimated microbeam generated at the beamline and has been used to study the homogeneity of the supramolecular structures of thin films of chiral materials deposited on fused quartz substrates. This facility has been expanded with the installation of a Mueller Matrix Polarimeter, MMP, coupled to the beamlight, of which a preliminary data will be discussed. In the solid state, the measurement of CD related to the supramolecular structure is hampered by the presence of circular birefringence, linear dichroism, and linear birefringence that can only be evaluated using the MMP technique. The ability to characterize the chiroptical property of thin chiral films prepared under a variety of conditions and protocols such as drop cast, spin coating, spray at different temperatures and concentrations will enable the determination of the critical parameters for reproducible, uniform and homogeneous specimen preparation, which is the sine qua non for any commercial application. This is of particular importance for optoelectronic materials, but it can also be extended to a broad variety of materials with applications from biosensors to biological tissues.

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Abstract: We report on the fabrication and characterisation of graphene field-effect transistor (GFET) biosensors for the detection of Clusterin, a prominent protein biomarker of Alzheimer’s disease (AD). The GFET sensors were fabricated on Si/SiO2 substrate using photolithographic patterning and metal lift-off techniques with evaporated chromium and sputtered gold contacts. Raman Spectroscopy was performed on the devices to determine the quality of the graphene. The GFETs were annealed to improve their performance before the channels were functionalized by immobilising the graphene surface with linker molecules and anti-Clusterin antibodies. Concentration of linker molecules was also independently verified by absorption spectroscopy using the highly collimated micro-beam light of Diamond B23 beamline. The detection was achieved through the binding reaction between the antibody and varying concentrations of Clusterin antigen from 1 to 100 pg/mL, as well as specificity tests using human chorionic gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFETs were characterized using direct current (DC) 4-probe electrical resistance (4-PER) measurements, which demonstrated a limit of detection of the biosensors to be ∼ 300 fg/mL (4 fM). Comparison with back-gated Dirac voltage shifts with varying concentration of Clusterin show 4-PER measurements to be more accurate, at present, and point to a requirement for further optimisation of the fabrication processes for our next generation of GFET sensors. Thus, we have successfully fabricated a promising set of GFET biosensors for the detection of Clusterin protein biomarker. The developed GFET biosensors are entirely generic and also have the potential to be applied to a variety of other disease detection applications such as Parkinson’s, cancer, and cardiovascular.

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Abstract: Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.

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Abstract: Polymer thin films that emit and absorb circularly polarised light have been demonstrated with the promise of achieving important technological advances; from efficient, high-performance displays, to 3D imaging and all-organic spintronic devices. However, the origin of the large chiroptical effects in such films has, until now, remained elusive. We investigate the emergence of such phenomena in achiral polymers blended with a chiral small-molecule additive (1-aza[6]helicene) and intrinsically chiral-sidechain polymers using a combination of spectroscopic methods and structural probes. We show that – under conditions relevant for device fabrication – the large chiroptical effects are caused by magneto-electric coupling (natural optical activity), not structural chirality as previously assumed, and may occur because of local order in a cylinder blue phase-type organisation. This disruptive mechanistic insight into chiral polymer thin films will offer new approaches towards chiroptical materials development after almost three decades of research in this area.