Improvement of the Quantum Dot-based FISH technique and its application for identification of microorganisms in water | |
| Author | Das, Quail |
| Call Number | AIT Thesis no.EV-08-30 |
| Subject(s) | Fluorescence in situ hybridization Microorganisms--Environmental aspects |
| Note | A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Environmental Engineering and Management |
| Publisher | Asian Institute of Technology |
| Abstract | After the discovery of DNA, microorganism identification technique is moved toward to nucleic acid -based molecular techniques. Over the years, various techniques have been developed where fluorescent in situ hybridization (FISH) is one of the most extensively used techniques. Despite its usefulness, conventional FISH technique also holds some disadvantages, for example, an organic dye which is used to provide fluorescence signal is susceptible to photobleaching. One of the solutions to address this problem is the use of Quantum Dot (QD) nanoparticles. QDs have the unparallel ability to produce size-tune fluorescence emission and the broad-excitation spectra. QDs are also stable in presence of light. In the present study, a QD-based FISH technique was optimized with the use of chitosan capped ZnS:Mn²+ QD synthesized by AIT's Center of Excellence in Nanotechnology. The QD-based FISH technique was used for the identification of three Proteobacteria from different groups. The specificity of this technique was validated by hybridizing bacteria with specific and non-specific QD-oligonucleotide probes. Finally, the applicability of the chitosan capped Mn doped ZnS QD-oligonucleotide probe was tested for detection of E. coli in the bacteria-spiked water samples. To date there is no information available in literature on the QD-based FISH technique for identification of whole cell of bacteria. Similar to conventional FISH, the QD-based FISH technique is also comprised with five steps: cell preparation, conjugation of QD with oligonucleotides, hybridization, washing and microscopic observation. Bioconjugation is occurred between positively charged QD and negatively charged oligonucleotides. The suitable pH condition to keep the conjugation stable throughout the hybridization was optimized. Permeabilization of QDs was also investigated with different surfactant treatment of cell wall. Hybridization was done with different temperature setting in presence of buffer with formamide. Wasing was also done to get rid of any unattached and non-specific QD-probes. Finally, cells were observed under fluorescence microscope without use of any staining dye or anti-fading agent. The protocol developed was optimized by treating the fixed bacterial cells with 1 % nonidet P40 substitute at room temperature and hybridization is occurred at 55°C for 3 hours in acidic conditions (pH 5). The specificity tests of the QD-FISH protocol were validated by hybridizing the bacterial cells with both specific and non-specific QD-conjugated probes; and the specificity were achieved with the use of 20% of formamide for ALFbI and 40% of formamide for BET42a and GAM42a probes at 55°C. With this formamide condition the targeted cells were brightly fluorescence than non-targeted cells but the non-targeted cells were still visible. The false positive detection of microorganisms is still the disadvantage of QD-FISH protocol. The optimized QD-FISH protocol is successfully applied to E, coli-spiked in milliQ water where the E. coli populations are well-separated. The developed QD-FISH protocol is efficient enough that bacterial cells were detected even in low concentrations (~7X10³/ml). It could be concluded that QD-based FISH technique appears to be promising in identification of microorganisms, however, more research works need to be done before applying the developed protocol in environmental sample. |
| Year | 2008 |
| Type | Thesis |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Energy and Climate Change (Former title: Department of Energy, Environment, and Climate Change (DEECC)) |
| Academic Program/FoS | Environmental Engineering and Management (EV) |
| Chairperson(s) | Shipin, Oleg. V. ;Dutta, Joydeep; |
| Examination Committee(s) | Annachhatre, Ajit P.; |
| Scholarship Donor(s) | ADB-Japan; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2008 |