High-throughput biosensing using chiral plasmonic nanostructures

The object of this thesis, is to demonstrate the potential capabilities of injection moulded chiral plasmonic nanostructures for enhanced sensing in biological systems. The key phenomenon employed throughout this thesis is the generation of electromagnetic fields, that produce a greater chiral asymmetry than that of circularly polarised light, termed ‘superchiral’ fields. These superchiral fields will be demonstrated as being an incisive probe into the structure, conformation, and orientation of proteins immobilised on the nanostructure surface of these injection moulded substrates. Initially, it will be shown how this phenomenon is sensitive to higher order changes in protein structure induced upon ligand binding, using an asymmetry parameter extracted from the optical rotatory dispersion (ORD) spectra. Where these changes would not be routinely detected by conventional chiroptical spectroscopy techniques, such as circular dichroism (CD). Further to this, as these nanostructures display the plasmonic analogue of the interference effect, electromagnetically induced transparency (EIT), a narrow transparency window is created within a broad reflectance spectrum. Where the spectra can be modelled using a simple coupled oscillator model, and the retardation phase effects extracted. This allows two new asymmetry parameters to be introduced for characterising any changes induced by the biological samples, the experimental separation parameter ∆∆S, and the modelled retardation phase asymmetries. These will be used to characterise the orientation of three structurally similar protein fragments, called Affimers, with the modelled phase asymmetries being shown as a particularly incisive probe into the surface immobilised orientation. Furthermore, conformational changes in the cancer relevant protein, HSP90, will be characterised upon the addition of increasing concentrations of the inhibitor molecule 17-AAG. With the orientation of the immobilised HSP90 protein being shown to influence the sensitivity observed for any protein-ligand interactions that occur. Finally, this phenomenon will be used to quantitatively detect elevated protein levels in a complex solution. Elevated levels of IgG will be measured in human blood serum solutions, utilising the isoelectric point of the proteins in solution to enhance the level of IgG adsorbed in the protein corona. This will demonstrate for the first time, the use of superchiral fields generated around injection moulded chiral nanostructures, to detect protein changes in complex real life solutions, such as human blood serum. Representing the first step in creating a high-throughput ultrasensitive system for a range of diagnostic applications

Biomacromolecular stereostructure mediates mode hybridization in chiral plasmonic nanostructures

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

Chiral plasmonic fields probe structural order of biointerfaces

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

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Available from TIB Hannover: FR 4753 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Chiral Plasmonic Fields Probe Structural Order of Biointerfaces

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

Superchiral Plasmonic Phase Sensitivity for Fingerprinting of Protein Interface Structure

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>i.e.</i>, 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

Lymph node targeting of BCG vaccines amplifies CD4 and CD8 T-cell responses and protection against Mycobacterium tuberculosis

Vaccination with Mycobacterium bovis BCG provides limited protection against pulmonary tuberculosis and a risk of dissemination in immune-compromised vaccinees. For the development of new TB vaccines that stimulate strong T-cell responses a variety of strategies is being followed, especially recombinant BCG and attenuated M. tuberculosis. The objective of the current study was to test potential benefits of vaccination through direct lymph-node targeting of wildtype BCG; the recommended route of vaccination with BCG is intradermal. C57BL/6 mice were immunised with BCG by intradermal, subcutaneous or intralymphatic injections. Cellular immune responses and protection against M. tuberculosis were determined. Intralymphatic vaccination was 100-1000 times more effective in stimulating BCG-specific immune responses than intradermal or subcutaneous immunisation. Intralymphatic administration stimulated high frequencies of mycobacterium-specific lymphocytes with strong proliferating capacity and production of TNF-α, IL-2, IL-17 and, especially, IFN-γ secretion by. CD4 and CD8 T cells. Most importantly, intralymphatic vaccination with 2×10(3)CFU BCG induced sustained protection against M. tuberculosis in intratracheally challenged C57BL/6 mice, whereas subcutaneous vaccination with 2×10(5)CFU BCG conferred only a transient protection. Hence, direct administration of M. bovis BCG to lymph nodes demonstrates that efficient targeting to lymph nodes may help to overcome the efficacy problems of vaccination with BCG