Nucleic-based acid technology (NAT) is a reliable and well-established method in molecular diagnosis for the detection of bacterial infection. Specifically, PCR (polymerase chain reaction) is the most popular technique to amplify the number of DNA or RNA copies in the sample. However, due to the thermal cycles in the PCR method, advanced equipment and technologies are required to precisely control the temperature during the cycles. To overcome this limitation, isothermal amplification methods have been developed which function at constant temperatures and help reduce the need for state-of-the-art machines to perform the amplification. Among isothermal amplification methods, LAMP (loop mediated isothermal amplification) has demonstrated robustness and sensitivity compared to PCR. Additionally, microfluidic lab-on-a-chip (LOC) technology can facilitate the intensive processes which have been used traditionally in laboratories by automating the required procedures, reducing the volume of the reagents and minimizing the cost and the time of experiments. Although many microfluidic LOC devices have been developed in order to be used in resource poor settings, there is still a need for a simple setup which is inexpensive, accurate and can be performed without the need for a trained technician. In this thesis, a disposable microfluidic device was developed which is capable of performing high-throughput DNA amplification by using a simple segmentation method in order to digitize the sample into multiple micro-wells. Moreover, design and fabrication of a disposable, inexpensive flexible heater which is an inevitable part of the setup using a direct write process was introduced in order to provide the required energy for the LAMP reaction. Parallel real-time DNA amplification with limit of detection down to few copies per micro-well in less than an hour was illustrated. Using E. coli 0157, it was demonstrated that the detection time of E.coli can be as quick as 11 to 55 minutes with sample concentrations varying from 700,000 copies/micro-well (11 minutes), 70,000 copies/micro-well (18 minutes), 700 copies/micro-well (31 minutes), 7 copies/micro-well (40 minutes) and 0.07 copies/micro-well (55 minutes). Finally, the capability of the device for on chip reagent storage up to 3 days without using any coating methods was illustrated. / Thesis / Master of Applied Science (MASc)

Rapid and sensitive detection of biomolecules is an important issue in nanomedicine. Many disorders are manifested by changes in protein levels of serum and other biofluids. Rapid and effective differentiation between normal and cancerous cells is an important challenge for the diagnosis and treatment of tumor. Likewise, rapid and effective identification of pathogens is a key target in both biomedical and environmental monitoring. Most biological recognition processes occur via specific interactions. Gold nanoparticles (AuNPs) feature sizes commensurate with biomacromolecules, coupled with useful physical and optical properties. A key issue in the use of nanomaterials is controlling the interfacial interactions of these complex systems. Modulation of these physicochemical properties can be readily achieved by engineering nanoparticles surface. Inspired by the idea of mimicking nature, a convenient, precise and rapid method for sensing proteins, cancerous cells and bacteria has been developed by overtaking the superb performance of biological olfactory systems in odor detection, identification, tracking, and location. On the fundamental side, an array-based/`chemical nose' sensor composed of cationic functionalized AuNPs as receptors and anionic fluorescent conjugated polymers or green fluorescent proteins or enzyme/substrates as transducers that can properly detect and identify proteins, bacteria, and cancerous cells has been successfully fabricated.

In chronic lymphocytic leukemia (CLL), the presence of multiple subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has implicated antigen(s) in leukemogenesis. These stereotyped subsets display similar immunoglobulin (IG) gene usage, almost identical complementarity determining region 3’s and may share clinical features. For instance, subsets #1 (IGHV1/5/7/IGKV1-39) and #2 (IGHV3-21/IGLV3-21) have inferior outcome compared to non-subset patients, whereas subset #4 (IGHV4-34/IGKV2-30) display a favourable prognosis. The aim of this thesis was to investigate genomic aberrations, gene expression patterns and methylation profiles in stereotyped subsets and compare epigenetic profiles in CLL and mantle cell lymphoma (MCL). In paper I, we investigated genomic aberrations in subsets #2, #4 and #16 and in non-stereotyped samples (n=101) using high-density 250K SNP arrays. Subset #2 and non-subset #2 IGHV3-21 cases displayed a higher frequency of aberrations than subset #4 cases. The high incidence of del(11q) in both subset #2/non-subset #2 may reflect the adverse survival reported for IGHV3-21 patients. In contrast, the lower frequency of genetic events and lack of poor-prognostic aberrations in subset #4 may partially explain their indolent disease. In paper II, we analysed the global RNA expression in subset #4, #16 and non-subset IGHV4-34 CLL patients (n=25). Subsets #4 and 16 showed distinct gene expression profiles, where genes involved in cell regulatory pathways were significantly lower expressed in subset #4, in line with their low-proliferative disease. In paper III, a genome-wide methylation array was applied to investigate methylation profiles in subsets #1, #2 and #4 (n=39). We identified differential methylation patterns for all subsets and found affected genes to be involved in e.g. apoptosis and therapy resistance. When performing functional annotation, a clear enrichment of genes involved in adaptive immunity was observed. These genes were preferentially methylated in subset #1 when compared to either subset #2 or #4, possibly due to different antigen responses. In paper IV, the genome-wide methylation profiles for 30 CLL and 20 MCL patients were investigated. Distinct methylation profiles were observed, where MCL displayed a more homogeneous profile. Homeobox transcription factor genes showed a higher degree of methylation in MCL, while apoptosis-related genes and proliferation-associated genes were methylated in CLL. In summary, this thesis demonstrates that stereotyped CLL subsets display differences in gene expression profiles, genetic aberrations and methylation patterns, underscoring the functional relevance of subgrouping according to BCR stereotypy. The distinct methylation profiles of CLL and MCL suggests that different epigenetic mechanisms are involved in the pathogenesis of these B-cell malignancies.