No description available.

No description available.

No description available.

Inductively coupled plasma mass spectrometry (ICP-MS) is a well established and versatile technique for the elemental analysis of a wide spectrum of samples. For a majority of the elements that can be analysed by ICP-MS limits of detection in the order of sub ng 1 1 levels can be attained. However, a number of these elements have problems associated with them that lead to a restriction of the limits of detection that can be achieved. Phosphorus and Sulphur are two such elements that exhibit poorer limits of detection, the improvement of which would be highly desirable to the ICPMS analyst. Six methods for the measurement of 31p and 32S isotopes have been developed with the aim of avoiding the spectroscopic interferences at the native m/z ratios of 31 and 32 respectively. These approaches have utilised a hexapole collision cell, a 'cold/cool plasma' and an experimental ICP torch bonnet. Via the collision cell and 'cold/cool plasma' approaches the isotopes of interest were either converted to different species for successful measurement at an alternative m/z ratio or interfering species were removed allowing measurement at the native m/z. Limits of detection achieved by these approaches were comparable with those quoted in the literature and by ICP-MS instrument manufacturers. The approach using the torch bonnet was not successful in attenuating spectroscopic interferences; however, it did show potential for continuing as an area of research. The development of these six methods is discussed in Chapter 2. As an application of the successful methods developed for the measurement Of 31p and 328 isotopes, DNA (and its associated components) was selected for study as this biomolecule is comprised of approximately IO % phosphorus. DNA in solution was successfully quantified by these methods and DNA components, studied during polymerase chain reaction processes and in single nucleotide polymorphisms, were qualified. The application of these methods to the study of DNA and its components is discussed in Chapter 3. As part of a collaboration between the Loughborough University Atomic Spectroscopy Research Group and the Cancer Biomarkers and Prevention Group at the University of Leicester, ICP-MS was employed in the investigation of the interactions between two Pt based anti-cancer drugs (cisplatin and oxaliplatin) and their biological target DNA. For this collaboration, DNA was the interest common to both groups. The interaction of each of these drugs with known quantities of DNA was measured by ICP-MS and binding constant data was calculated for use as the basis of a clinical test for drug efficacy in cancer patients. The binding constant data showed that the interaction between drug and target is particularly inefficient. This area of research is discussed in Chapter 4. The potential for ICP-MS interface modification was also explored. Two modified designs are discussed that may prove to be advantageous for the transport of ions between the atmospheric pressure conditions of the ICP ion source and the vacuum conditions of the mass spectrometer. One of these designs was successfully manufactured and produced positive data. Research into this design is being furthered by the Thermo Electron Corporation, the discussion of which is in Chapter 5. A further collaboration was established with both the Biomaterials-related Infection Group of the School of Medical and Surgical Sciences and the Polymer Group of the School of Mechanical, Materials and Manufacturing Engineering at the University of Nottingham. Here ICP-MS was employed in the measurement of silver leaching from a silver nano-particle impregnated polymer material that could be used in the production of catheters. Silver leaching from a catheter is potentially desirable due to its anti-microbial properties. The study of this leaching revealed that significant quantities of silver were being transferred from the polymer into surrounding human serum media over the period of seven days and beyond. For the most part this work was routine ICP-MS measurement, and did not involve research or development, so does not take part in the main body of this thesis. This work is discussed in Appendix 5.

A series of bipyridine and pyridine ligands have been prepared and fully characterised. These are 5- bromobutyr-2,2'-bipyridine (L 1), (2,2'-Bipyridin-5-ylmethyl)-5-(1 ,2-dithiolan-3-yl)pentanoate (L2) N-(2,2'- bipyridin-5-ylmethyl)-5-(1 ,2-dithiolan-3-yl)pentanamide (L3), pyridin-3-ylmethyl-5-(1,2-dithiolan-3- yl)pentanoate (L4) pyridin-4-ylmethyl-5-(1 ,2-dithiolan-3-yl)pentanamide (LS) and ethyl 5-(1,2-dithiolan-3- yl)pentanoate (L6). Their Re(l) and RU(lI) complexes have also been prepared and characterised in most cases. The photophysical properties of these complexes have been investigated showing expected intense . emission. The behaviour of a variety of these ligands and complexes has been studied on a colloidal silver surface, analysed by Surface Enhanced (Resonance) Raman Spectroscopy (SE(R)RS). Concentration dependent behaviour was observed for L2 and L3. A relatively low intensity of the SERS signal at the highest concentrations was attributed to a combination of interference of fluorescence from species in solution and high surface coverage of the nanoparticles preventing the formation of silver-bipyridine complexes. At the optimum concentration we propose the formation of silver-bipyridine complexes which display a resonance enhanced component of the SERS signal. At lower concentrations a simple lack of substrate capable of giving a signal results in a reduction in the observed peak intensity again. Similar behaviour was also observed for the respective rhenium(l) complexes, [Re(COh(L2)Br] and [Re(COh(l3) Br] while preliminary work also indicated the same behaviour in the charged rhenium(l) complexes [Re(COh(bpy)(py)f and [Re(COh(bpy)(LS)r. These insights may enable the development of the system for future sensing applications. Complex [Re(COh(L2)Br] and [Re(COh(bpy)Br] showed selective changes in its photophysical properties in the presence of Hg2+ ions when experiments were carried out in acetonitrile solution. Detailed studies of the system suggest that an exchange of the Br ligand fora CH3CN ligand takes place through an intermediate species, with formation of the charged complex [Re(COh(L2)(CH3CN)f and [Re(COh(bpy) (CH3CN)1 irrespective of the counter-ion (either CI04- or N03-). Similar behaviour was not observed with a range of other metal cations.

The work described in this thesis is to develop a microscope into a high resolution bio-molecular interaction sensor. A "prism less" widefield surface plasmon microscope has been constructed and applied to imaging of interactions of protein and its antibody in aqueous media through a high NA objective. There are two main parts in this thesis: (1) design and layout of a high resolution angle scanning widefield surface plasmon resonance microscope; and (2) the application to bio-molecular interactions. In the first part, an angle-scanning widefield surface plasmon imaging (AW-SPRI) system consisting of an optical system, a liquid handling system and a data processing system is described. In the optical system, surface plasmons are excited by objective coupling. A spatial light modulator in a conjugate back focal plane of the objective lens allows dynamic control of illumination angle. The reflected bright-field widefield images, encoded with SPR signals, are detected by a CCD. The SPR signals in the images are decoded by a signal processing algorithm. AW-SPRI also combines well-controlled liquid handling units in order to monitor bio-molecular interactions or detect analytes in water-based solvent. The system shows high sensor resolution ( 5 x 10-5 RIU) as a biosensor. In addition, the edge response of A W -SPRI images with the BSA grating vector parallel to the incident polarization direction is 6.5 flm in air and 7.6 urn in water and the edge response with the BSA grating vector perpendicular to the incident polarization direction is 4.3 urn in air and 4.8 urn in water. The system presents high spatial resolution, too. The second part introduces sensor chip preparation and bio-molecular interactions. The sensor chip is where the bio-molecular interaction takes place and where the biochemical binding event is transduced into SPR signals. There are three types of sensor chips mentioned in this thesis which are bare gold-coated coverslips, protein grating patterns on a gold surface created by micro-contact printing, and protein grating patterns covalently immobilised on a gold surface created by photolithography. The patterned proteins on gold surfaces as our sensor chips are used to perform bio-molecular interactions. The results show our system can be used for comparison or determination of the concentration of ligands on the sensor chip or the affinities of the analyte with different samples on sensor chips. In addition, the measurement of affinity and rate constants show that the AW-SPRI can be used to measuring binding processes and carry out kinetic analysis of macromolecular interactions with standard interaction cycle method. Although the errors are larger than with the commercial SPR machines (SR 7000DC), they are nevertheless of similar order of magnitudes. To the authors' knowledge, this is the first demonstration of such high spatial resolution for quantitative, label-free, real-time detection of bio-molecule interaction cycles.

No description available.

No description available.

Despite the abundance of the templates, both human and rodent SINEs are normally expressed at a very low level. DNA methylation-mediated silencing has been proposed as a possible cause of their transcriptional repression. The effect of DNA methylation and the effect of DNA methylation-dependent methyl-CpG-binding domain proteins (MBD proteins) on SINE transcription were studied here. It was shown that both human and rodent SINEs are bound by MeCP2, MBD1 and MBD2. Both human and rodent SINEs were also shown to be occupied by HDAC1, HDAC2 and a component of SWI/SNF complex, Brahma. Human Alus were also found to be occupied by components of two corepressor complexes, SIN3 and NuRD. Whether MBD proteins repress SINE transcription in a DNA methylation-dependent manner was further investigated using systems with low or near absent DNA methylation and, in the case of MeCP2 protein, by its direct removal. MeCP2 was found to have no repressive effect on B1 and B2 expression. RT-PCR analysis showed no increase in B1 and B2 RNA levels in MeCP2 null mice kidneys. ChIP analysis of Dnmt1n/n p53-/- embryonic fibroblasts, which have less than 5% of the normal DNA methylation level, showed significant reduction in MeCP2 and MBD2 binding, confirming that their presence is DNA methylation-dependant. RT-PCR comparison of Dnmt1+/+ p53-/- and Dnmt1n/n p53-/- cells, however, detected no increase in B1 or B2 RNA levels. This was consistent with results obtained from MeCP2 null mice, where lack of MeCP2 did not result in increased B1 and B2 expression and with a previous study involving human Alus (Yu et al., 2001). MBD2 also does not seem to repress SINE activity, as its release following loss of DNA methylation did not result in increased SINE RNA levels. Strikingly, all human and rodent SINEs studied here were found to be bound by transcription factors TFIIIB and TFIIIC at comparable levels with actively transcribed genes. Some RNA polymerase III was also detected, but at levels significantly lower than on active genes, suggesting a defect in RNA polymerase III loading onto SINEs. This occupancy of the transcriptional complex was comparable in cells with normal levels of DNA methylation and in cells with significantly reduced levels of DNA methylation, suggesting that the occupancy is not affected by methylated DNA or DNA methylation-dependent components of chromatin. Indeed, removal of 50% of histone H1 did not result in increased B1 or B2 expression in this study. The fact that all tested SINEs are occupied by TFIIIB and TFIIIC also brings an unprecedented insight into the number of these transcription factors present in the cell.

Starting with a comprehensive review of both surface plasmon resonance (SPR) based and localized surface plasmon resonance (LSPR) based sensors, this thesis reports the studies on the development of a novel sensitive gold nanorod (GNR) based label-free LSPR optical fibre biosensor, and the development of a novel robust method for effectively modifying the surface of cetyl-trimethyl ammonium bromide (CTAB) capped GNRs and their LSPR biosensing applications. A novel GNR-based LSPR optical fibre sensor was fabricated and evaluated in this work. The sensor probe was prepared by covalently immobilizing GNRs, synthesized using a seed-mediated growth method, on the decladed surface of a piece of multimode optical fibre. In order to operate the LSPR sensor as a reflective sensor, a silver mirror was also coated at one distal end of the sensor probe by a dip coating method. In the refractive index sensitivity test, it was found that the longitudinal plasmon band (LPB) of GNRs is highly sensitive to the refractive index change close to the GNRs surface, and the sensitivity of the LSPR optical fibre sensor increases with the increase of the aspect ratio of GNRs. The results showed that the GNR-based LSPR optical fibre sensors prepared in this work have linear and high refract index sensitivities. For sensors based on GNRs with aspect ratios of 2.6, 3.1, 3.7 and 4.3, their refractive index sensitivities were found to be 269, 401, 506 and 766 nm/RIU (RIU = refractive index unit), respectively, in the refractive index range from 1.34 to 1.41. In order to evaluate the biosensing performance, the GNR-based LSPR optical fibre sensor with aspect ratio of 4.1 and a 2 cm sensing length was further functionalized with human IgG to detect the specific target — anti-human IgG, and a detection limit of 1.6 nM was observed using a wavelength-based interrogation approach. In another study, in order to overcome the drawbacks of the CTAB-capped GNRs found in biosensing and biomedical applications, a simple yet robust pH-mediated method for effectively modifying the surface of CTAB-capped GNRs synthesized by the seed-mediated growth method was developed. This method allows the complete replacement of the CTAB molecules attached on the GNRs surface with the 11-mercaptoundecaonic acid (MUA) molecules to take place in a total aqueous environment by controlling the pH of the MUA aqueous solution, thus avoiding the irreversible aggregation of GNRs during the complex surface modification process observed in the previous reported methods. The success of the complete replacement of CTAB with MUA was confirmed by the surface elemental analysis using an X-ray photoelectron spectroscopy (XPS), and the MUA-modified GNRs created in this work demonstrated a high stability up to 4 months at least when stored in a buffer solution at pH 9 at 4°C. The MUA-modified GNRs with an aspect ratio of 3.9 were furthered developed as a solution-phase-based label-free LSPR biosensor by functionalizing the GNRs with human IgG. A detection limit as low as 0.4 nM for detecting anti-human IgG was achieved by this sensor. The achievements of this work are concluded and the directions of future work are also pointed out.