Rapid Detection of Mycotoxin Contamination

A compilation of 12 original research articles and a review on the development of instrumental and immunoanalytical methods for mycotoxins; on the enhancement of sample preparation and selection to improve method applicability; and on practical applications of analytical methods in laboratory fungal cultures, cereal and feed samples, surface water (as a novel matrix of mycotoxins as emerging surface water contaminants), and during mycotoxin decontamination by bacteria. Target analyte mycotoxins include aflatoxins, deoxynivalenol, diacetoxyscirpenol, fumonisins, fusarenone-X, HT-2 toxins, nivalenol, ochratoxins, sterigmatocystin, T-2 toxin, and zearalenone

Development of a series of modified acridinium esters for use in clinical diagnostics

Acridinium esters (AE) have been traditionally used as chemiluminescent reporter molecules in clinical diagnostic assays due to their high quantum yields and detection limits in the attomole range. The aim was to produce a family of novel acridinium esters with modified chemiluminescent properties for use in clinical diagnostics. The hypothesis was that, by changing the substituents on the acridinium ring and the phenoxy ring of the acridinium ester molecule, a series of compounds with distinct chemiluminescent characteristics could be generated which would support dual analyte measurement. Two series of compounds were synthesized based on the addition of two methoxy (electron donating) or two bromo (electron withdrawing) groups to the 2 and 7 positions of the acridinium ring. Additionally, the effect of adding methyl or methoxy groups to the 2- and 6-positions of the phenoxy ring of the acridinium ester molecule was compared to the unsubstituted state. Five distinct acridinium esters were synthesized and their excitation and emission spectra determined. Two of the compounds were linked to a DNA oligonucleotide and incorporated into a Hybridisation Protection Assay (HPA), a direct and highly specific nucleic acid assay. The performance of these chemiluminescent labelled probes within the HPA (kinetics of light output and the rate of hydrolysis) was characterised. Major findings included a shift in the fluorometric spectra resulting from the substitution on the acridinium ring, resulting in the peak emission wavelengths of Nmethylacridone, 2,7-dibromo-10-methyl-9-acridone and 2,7-dimethoxy-10-methyl-9- acridone being observed at 430, 440 and 480 nm, respectively. Furthermore, the optimal pH for discrimination between hybridised and unhybridised probes for the dimethoxysubstituted acridinium ester 34cx was between 9.2 and 9.6 whilst the pH optimum of the routinely used unsubstituted compound 15 ranged from 7.5 to 8.5. Theoretically, the differential properties of dimethoxy-substituted AE DNA probes, the shift in emission wavelength and hydrolysis pH, may form the basis for a novel dual analyte HPA format to be developed

A microarray format for multi-parameter blood group serology

In this thesis multiple factors affecting probe-ligand interactions in a microarray format were investigated using blood group antigen and antibody sets for the development of a microarray based serology platform. Initial experiments focused on multiple parameters affecting immobilisation and functionality of monoclonal and polyclonal antibodies on various microarray surfaces. A range of critical parameters affecting protein-ligand interactions, such as efficient blocking of non-specific binding, detergent type, RBC concentration, reaction volume and mixing, were optimised in a series of experiments with labelled anti-species antibodies. Selected microarray surface and optimised spotting and reaction conditions were then used for studies of antibody-RBC antigen interactions in situ. A 'dual' solid-phase approach was investigated for blood group serology reactions where probe antibodies were immobilised on the microarray and target antigens were carried on the RBC surface, which can be considered as the second solid-phase. In summary this investigation has 1) established a microarray format and the reaction conditions for antibody-antigen interaction studies in blood group serology; 2) for the first time successfully exploited microarray format for comprehensive blood typing; 3) examined the novel technique of membrane fragment microarray immobilisation for a reverse, antibody screen reaction; 4) verified the findings and quantitatively characterised the interactions on Biacore, a surface plasmon resonance real-time detection system. This provides a strong basis for the development of microarray based multi-parameter blood group serology diagnostic platforms.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Integrated modular microfluidic system for forensic Alu DNA typing

Driven by the numerous applications of genome-related research, fully integrated microfluidic systems have been developed that have advanced the capabilities of molecular and, in particular, genetic analyses. A brief overview on integrated microfluidic systems for DNA analysis is given in Chapter 1 followed by a report on micro-capillary electrophoresis (µCE) of Alu elements with laser-induced fluorescence (LIF) detection, in which the monomorphic Alu insertions on the X and Y chromosomes were utilized to detect male DNA in large female DNA background (Y: X = 1:19) without cell sorting prior to the determination. The polymorphic Alu loci with known restricted geographical distribution were used for ethnicity determination. A valveless integrated microsystem that consists of three modules is discussed as well: (1) A solid-phase extraction (SPE) module microfabricated on polycarbonate, for DNA extraction from whole cell lysates (extraction bed capacity ~209 ±35.6 ng/cm² of total DNA). (2) A continuous-flow polymerase chain reaction (CFPCR) module fabricated in polycarbonate (Tg ~150 ºC) in which selected gene fragments were amplified using biotin and fluorescently-labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones. (3) µCE module fabricated in poly(methylmethacrylate), which utilized a bioaffinity selection and purification bed (2.9-µL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to µCE. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed where they were extracted onto the surface of the poly(methylmethacrylate) micropillars (50-µm width; 100-µm height; total surface area of ~117 mm²). This SPE process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (Kd =10-15M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single stranded DNA released for size-based separations and LIF detection. The multiplexed SPE-CFPCR-µCE yielded detectable fluorescence signal (S/N≥3; LOD ~75 cells) for Alu DNA amplicons for gender and ethnicity determinations with a separation efficiency of ~1.5 x105 plates/m. Compared to traditional cross-T injection procedures typically used for µCE, the affinity preconcentration and injection procedure generated signal enhancements of 17-40 fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation