The bacterial meningitis caused by Neisseria meningitidis is responsible for considerable morbidity and mortality throughout the world. Given the limitations of existing diagnostic tests and the severity of the illness associated with the disease, there is a clear requirement for a rapid and specific diagnostic assay. This thesis describes the development of nanoparticle based tests for the detection of Neisseria meningitidis specific cell surface markers. As an initial target antigen, a recombinant form of highly conserved outer membrane protein, OMP85 was used. Within the OMP85 protein sequence, a predicted antigenic sequence between residues 720 and 745 was identified and found to be unique to this organism. This amino acid sequence was synthesised as peptide (SR1) with a gly-gly-cysteine spacer sequence at the N-terminus using t-boc chemistry. Also, the major virulence factor, capsular polysaccharide of N. meningitidis serogroup B bacteria was purified. Polyclonal antibodies were raised against purified OMP85 antigen in rabbits and against SR1 peptide and also against formalin inactivated N. meningitidis serogroup B whole cell bacteria in sheep. This panel of different antibodies including the commercial anti-capsular monoclonal antibodies were examined for cross reactivity against a range of closely related Gram negative bacteria. Based on these cross-reactivity studies, a highly specific anti-NM antibody was developed following purification of the anti-SR1 antiserum by immuno-affinity chromatography. Purified OMP85 antigen and anti-OMP85 antibody were successfully conjugated on 13, 30, 40, 50 and 60 nm gold nanoparticles by an electrostatic adsorption method. Coupling of the gold nanoparticles results in a shift of the respective surface plasmon peak toward longer wavelengths (in the range of 600-800 nm) resulting in a change of the colour of the colloidal suspension from red to purple to blue. An attempt was made to develop a rapid diagnostic assay based on gold nanoparticle induced colour shift assay for N. meningitidis by utilising the specific interaction of OMP85 and anti-OMP85 antibody conjugated to gold nanoparticles as a model system. However, this system was not reproducible and is likely to be due to problems with stability of gold nanoparticles during the conjugation process. As an alternative approach, a highly selective quartz crystal microbalance (QCM)-based immunosensor was designed using the same OMP85/anti-OMP85 antibody system. A method was developed using polyvinylidene fluoride (PVDF) coated QCM crystals with protein A for the directional orientation of the antibodies. To further enhance the sensitivity of the test, OMP85-conjugated gold nanoparticles were used as signal amplification probes for the reproducible detection of the target down to 300 ng/mL, corresponding to a five fold increase in sensitivity compared to detection of OMP85 antigen alone. Also, this sensor has successfully been employed to detect whole cell bacteria at a concentration as low as 100 cfu/mL. Thus, in this study using the real-time QCM measurements, a novel strategy has been developed for the sensitive detection of both N. meningitidis bacteria and the protein antigen at very low concentrations, using gold nanoparticles as signal amplification probes.

The majority of chloroplast proteins are nuclear-encoded and post-translationally imported into the chloroplast. These newly imported proteins are translocated from the cytosolic compartment to the stroma by the Translocons of the Outer/Inner membranes of Chloroplast (TOC/TIC). In order to understand protein transport across the chloroplast outer membrane, it is crucial to investigate the structure and function of these complexes. The TOC complex is composed of the beta-barrel channel protein Toc75 and the GTPase receptors Toc34 and Toc159. Toc75 is a member of the OMP85 (Outer Member Protein, 85 kDa) superfamily. Other proteins of the OMP85 superfamily also exist in Gram-negative bacteria and mitochondria. The members of this family contain a C-terminal transmembrane beta-barrel and a soluble N-terminus with a varying numbers of POTRA (POlypeptide TRansport Associated) domains. The recent crystal structures of the POTRA domains of Gram-negative bacteria reveal that these domains are localized in the periplasmic side. This thesis identifies the orientation of the POTRA domains as being localized in the cytosol and provides initial evidence for their involvement in the protein import. Three POTRA domains of psToc75 were identified, purified in E. coli and characterized by MALDI-TOF mass spectrometry and circular dichroism. Using variety of immunofluorescence methods, such as flow cytometry and LSCM, the topology of the POTRA domains was investigated. Chloroplast agglutination assays were used to assess the location of immuno-reactive fragments of the POTRA domains, which supported the results from the flow cytometry and LSCM. Finally, thermolysin was used to probe the surface of the isolated intact chloroplasts. Proteolytic digestion along with the data obtained from flow cytometry, LSCM and agglutination assays suggested the orientation of the N-terminal POTRA domains facing the cytosol, followed by a C-terminal beta-barrel domain. The import competence of individual POTRA domains was determined by in vitro chloroplast import and binding competition assays. POTRA1 inhibited the binding of the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts, while POTRA3 inhibited the import of radiolabeled precursors into isolated chloroplasts; however, in both assays, the inhibition of precursor binding and import was to a lesser extent than non-labeled prSSU.