A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Environmental Engineering and Management Inter-University Program on Environmental Toxicology, Technology and Management

Thesis (M.Sc.) - Asian Institute of Technology - Chulabhorn Research Institute - Mahidol University, 2008

Fluorescence in situ hybridization (FISH) and amplification by polymerase chain reaction (PCR) are two of the most commonly applied molecular microbiological methods. A major limitation of FISH is its dependence on organic fluorophores whose susceptibility to light-induced degradation forces researchers to work in the dark. Frequent inefficiency and non-specificity are the major drawbacks of PCR. The main goal of this study was to develop alternative methodologies overcoming shortcomings of these two methods through an application of nanoparticles. An innovative FISH methodology with superior photostability and biological compatibility was developed through the use of the chitosan-capped Mn-doped ZnS quantum dots (QDs) as fluorescence label for the phylogenetic oligonucleotide probes. Stringent hybridization between the QD-labeled oligonucleotide probes and target rRNA site in E. coli was carried out in a formamide-free buffer under high temperature (55-65°C) incubation. Highly luminescent, the QDs enabled the efficient detection by epifluorescence microscopy of E. coli cells which were 75% brighter than the background fluorescence. Extremely photostable, the QDs eliminated the need to work in the dark and the use of antifade reagents. In its current form, the QD-FISH methodology is somewhat limited by the interference from autofluorescence, which requires additional research. Meanwhile, image processing and analysis can help overcome this limitation and demonstrate the effectiveness of the QD-FISH technique in detecting E. coli from a mixture of bacteria. Case-specific improvement of the efficiency and specificity of amplification of nucleic acids was attained through the addition of either 20-nm silica (SiO₂) or 15-nm gold (Au) nanoparticles in PCR systems (--10¹² particles in 25-uL PCR mixture). One of the proposed mechanisms for the enhancement of nucleic acid amplification by Au nanoparticles revolves around the excellent heat conductivity of Au nanoparticles which leads to efficient thermal cycling in PCR. In this study, similar patterns of inhibition and enhancement of nucleic acid amplification were observed in PCR systems supplemented with either Au or SiO₂ nanoparticles, despite the difference in ability of these nanoparticles to conduct heat. Thus, the role of thermal conductivity on nanoparticle-PCR, though important, only provides a partial explanation and the interaction between the nanoparticles and the components of the PCR system appears to play a more significant role in the nanoparticle-based enhancement of nucleic acid amplification