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Following its first observation 50 years ago Raman optical activity (ROA), which refers to a circular polarization dependence of Raman scattering from chiral molecules, has evolved into a powerful chiroptical spectroscopy for studying a large range of biomolecules in aqueous solution. Among other things ROA provides information about motif and fold as well as secondary structure of proteins; structure of carbohydrates and nucleic acids; polypeptide and carbohydrate structure of intact glycoproteins; and protein and nucleic acid structure of intact viruses. Quantum chemical simulations of observed Raman optical activity spectra can provide complete three-dimensional structures of biomolecules, together with information about conformational dynamics. This article reviews how ROA has provided new insight into the structure of unfolded/disordered states and sequences, ranging from the complete disorder of the random coil to the more controlled type of disorder exemplified by poly L-proline II helix in proteins, high mannose glycan chains in glycoproteins and constrained dynamic states of nucleic acids. Possible roles for this 'careful disorderliness' in biomolecular function, misfunction and disease are discussed, especially amyloid fibril formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Optical spectroscopy can be used to quickly characterise the structural properties of individual molecules. However, it cannot be applied to biological assemblies because light is generally blind to the spatial distribution of the component molecules. This insensitivity arises from the mismatch in length scales between the assemblies (a few tens of nm) and the wavelength of light required to excite chromophores (≥150 nm). Consequently, with conventional spectroscopy, ordered assemblies, such as the icosahedral capsids of viruses, appear to be indistinguishable isotropic spherical objects. This limits potential routes to rapid high-throughput portable detection appropriate for point-of-care diagnostics. Here, we demonstrate that chiral electromagnetic (EM) near fields, which have both enhanced chiral asymmetry (referred to as superchirality) and subwavelength spatial localisation (∼10 nm), can detect the icosahedral structure of virus capsids. Thus, they can detect both the presence and relative orientation of a bound virus capsid. To illustrate the potential uses of the exquisite structural sensitivity of subwavelength superchiral fields, we have used them to successfully detect virus particles in the complex milieu of blood serum.

Manipulating symmetry environments of metal ions to control functional properties is a fundamental concept of chemistry. For example, lattice strain enables control of symmetry in solids through a change in the nuclear positions surrounding a metal centre. Light-matter interactions can also induce strain but providing dynamic symmetry control is restricted to specific materials under intense laser illumination. Here, we show how effective chemical symmetry can be tuned by creating a symmetry-breaking rotational bulk polarisation in the electronic charge distribution surrounding a metal centre, which we term a meta-crystal field. The effect arises from an interface-mediated transfer of optical spin from a chiral light beam to produce an electronic torque that replicates the effect of strain created by high pressures. Since the phenomenon does not rely on a physical rearrangement of nuclear positions, material constraints are lifted, thus providing a generic and fully reversible method of manipulating effective symmetry in solids.

Protein-protein interactions (PPIs) play a pivotal role in many biological processes. Discriminating functionally important well-defined protein-protein complexes formed by specific interactions from random aggregates produced by nonspecific interactions is therefore a critical capability. While there are many techniques which enable rapid screening of binding affinities in PPIs, there is no generic spectroscopic phenomenon which provides rapid characterization of the structure of protein-protein complexes. In this study we show that chiral plasmonic fields probe the structural order and hence the level of PPI specificity in a model antibody-antigen system. Using surface-immobilized Fab' fragments of polyclonal rabbit IgG antibodies with high specificity for bovine serum albumin (BSA), we show that chiral plasmonic fields can discriminate between a structurally anisotropic ensemble of BSA-Fab' complexes and random ovalbumin (OVA)-Fab' aggregates, demonstrating their potential as the basis of a useful proteomic technology for the initial rapid high-throughput screening of PPIs.

Structural analysis of carbohydrates is a complicated endeavour, due to the complexity and diversity of the samples at hand. Herein, we apply a combined computational and experimental approach, employing molecular dynamics (MD) and density functional theory (DFT) calculations together with NMR and Raman optical activity (ROA) measurements, in the structural study of three mannobiose disaccharides, consisting of two mannoses with varying glycosidic linkages. The disaccharide structures make up the scaffold of high mannose glycans and are therefore important targets for structural analysis. Based on the MD population analysis and NMR, the major conformers of each mannobiose were identified and used as input for DFT analysis. By systematically varying the solvent models used to describe water interacting with the molecules and applying overlap integral analysis to the resulting calculational ROA spectra, we found that a full quantum mechanical/molecular mechanical approach is required for an optimal calculation of the ROA parameters. Subsequent normal mode analysis of the predicted vibrational modes was attempted in order to identify possible marker bands for glycosidic linkages. However, the normal mode vibrations of the mannobioses are completely delocalised, presumably due to conformational flexibility in these compounds, rendering the identification of isolated marker bands unfeasible., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The structural order of biopolymers, such as proteins, at interfaces defines the physical and chemical interactions of biological systems with their surroundings and is hence a critical parameter in a range of biological problems. Known spectroscopic methods for routine rapid monitoring of structural order in biolayers are generally only applied to model single-component systems that possess a spectral fingerprint which is highly sensitive to orientation. This spectroscopic behavior is not a generic property and may require the addition of a label. Importantly, such techniques cannot readily be applied to real multicomponent biolayers, have ill-defined or unknown compositions, and have complex spectroscopic signatures with many overlapping bands. Here, we demonstrate the sensitivity of plasmonic fields with enhanced chirality, a property referred to as superchirality, to global orientational order within both simple model and "real" complex protein layers. The sensitivity to structural order is derived from the capability of superchiral fields to detect the anisotropic nature of electric dipole-magnetic dipole response of the layer; this is validated by numerical simulations. As a model study, the evolution of orientational order with increasing surface density in layers of the antibody immunoglobulin G was monitored. As an exemplar of greater complexity, superchiral fields are demonstrated, without knowledge of exact composition, to be able to monitor how qualitative changes in composition alter the structural order of protein layers formed from blood serum, thereby establishing the efficacy of the phenomenon as a tool for studying complex biological interfaces.

The structure adopted by biomaterials, such as proteins, at interfaces is a crucial parameter in a range of important biological problems. It is a critical property in defining the functionality of cell/bacterial membranes and biofilms (i.e., in antibiotic-resistant infections) and the exploitation of immobilized enzymes in biocatalysis. The intrinsically small quantities of materials at interfaces precludes the application of conventional spectroscopic phenomena routinely used for (bio)structural analysis due to a lack of sensitivity. We show that the interaction of proteins with superchiral fields induces asymmetric changes in retardation phase effects of excited bright and dark modes of a chiral plasmonic nanostructure. Phase retardations are obtained by a simple procedure, which involves fitting the line shape of resonances in the reflectance spectra. These interference effects provide fingerprints that are an incisive probe of the structure of interfacial biomolecules. Using these fingerprints, layers composed of structurally related proteins with differing geometries can be discriminated. Thus, we demonstrate a powerful tool for the bioanalytical toolbox.

In the past few decades, Raman optical activity (ROA) spectroscopy has been shown to be very sensitive to the solution structure of peptides and proteins. A major and urgent challenge remains the need to make detailed assignments of experimental ROA patterns and relate those to the solution structure adopted by the protein. In the past few years, theoretical developments and implementations of ROA theory have made it possible to use quantum chemical methods to compute the ROA spectra of peptides. In this work, a large database of ROA spectra of peptide model structures describing the allowed backbone conformations of proteins was systematically calculated and used to make unprecedented detailed assignments of experimental ROA patterns to the conformational elements of the peptide in solution. By using a similarity index to compare an experimental spectrum to the database spectra (2902 theoretical spectra), the conformational preference of the peptide in solution can be assigned to a very specific region in the Ramachandran space. For six (poly)peptides this approach was validated and gives excellent agreement between experiment and theory. Additionally, hydrogen/deuterium exchanged structures and the conformational dependence of the amide modes in Raman spectra can be analysed using the new database. The excellent agreement between experiment and theory demonstrates the power of the newly developed database as a tool to study Raman and ROA patterns of peptides and proteins. The interpretation of experimental ROA patterns of different proteins published in the scientific literature is discussed based on the spectral trends observed in the database.

The refractive index sensitivity of plasmonic fields has been exploited for over 20 years in analytical technologies. While this sensitivity can be used to achieve attomole detection levels, they are in essence binary measurements that sense the presence/absence of a predetermined analyte. Using plasmonic fields, not to sense effective refractive indices but to provide more "granular" information about the structural characteristics of a medium, provides a more information rich output, which affords opportunities to create new powerful and flexible sensing technologies not limited by the need to synthesize chemical recognition elements. Here we report a new plasmonic phenomenon that is sensitive to the biomacromolecular structure without relying on measuring effective refractive indices. Chiral biomaterials mediate the hybridization of electric and magnetic modes of a chiral solid-inverse plasmonic structure, resulting in a measurable change in both reflectivity and chiroptical properties. The phenomenon originates from the electric-dipole-magnetic-dipole response of the biomaterial and is hence sensitive to biomacromolecular secondary structure providing unique fingerprints of α-helical, β-sheet, and disordered motifs. The phenomenon can be observed for subchiral plasmonic fields (i.e., fields with a lower chiral asymmetry than circularly polarized light) hence lifting constraints to engineer structures that produce fields with enhanced chirality, thus providing greater flexibility in nanostructure design. To demonstrate the efficacy of the phenomenon, we have detected and characterized picogram quantities of simple model helical biopolymers and more complex real proteins.

Optimal performance of nanophotonic devices, including sensors and solar cells, requires maximizing the interaction between light and matter. This efficiency is optimized when active moieties are localized in areas where electromagnetic (EM) fields are confined. Confinement of matter in these 'hotspots' has previously been accomplished through inefficient 'top-down' methods. Here we report a rapid 'bottom-up' approach to functionalize selective regions of plasmonic nanostructures that uses nano-localized heating of the surrounding water induced by pulsed laser irradiation. This localized heating is exploited in a chemical protection/deprotection strategy to allow selective regions of a nanostructure to be chemically modified. As an exemplar, we use the strategy to enhance the biosensing capabilities of a chiral plasmonic substrate. This novel spatially selective functionalization strategy provides new opportunities for efficient high-throughput control of chemistry on the nanoscale over macroscopic areas for device fabrication.

Development of low-cost disposable plasmonic substrates is vital for the applicability of plasmonic sensing. Such devices can be made using injection-molded templates to create plasmonic films. The elements of these plasmonic films are hybrid nanostructures composed of inverse and solid structures. Tuning the modal coupling between the two allows optimization of the optical properties for nanophotonic applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Optical spectroscopic methods do not routinely provide information on higher order hierarchical structure (tertiary/quaternary) of biological macromolecules and assemblies. This necessitates the use of time-consuming and material intensive techniques, such as protein crystallography, NMR, and electron microscopy. Here we demonstrate a spectroscopic phenomenon, superchiral polarimetry, which can rapidly characterize ligand-induced changes in protein higher order (tertiary/quaternary) structure at the picogram level, which is undetectable using conventional CD spectroscopy. This is achieved by utilizing the enhanced sensitivity of superchiral evanescent fields to mesoscale chiral structure.