Application of dielectrophoretic field-flow fractionation in environmental studies | |
| Author | Sittisak Pui-ock |
| Call Number | AIT Diss. no.EV-08-14 |
| Subject(s) | Dielectrophoresis Human ecology--Study and teaching |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Technical Science in Environmental Engineering and Management Inter-University Program on Environmental Toxicology, Technology and Management |
| Publisher | Asian Institute of Technology |
| Series Statement | Dissertation ; no. EV-08-14 |
| Abstract | Earlier studies suggest dielectrophoretic techniques may be applicable for rapid detection of toxicity by exploiting changes induced in the dielectric properties of cells as a result of exposure to toxic agents. However, existing dielectric methods, including electrorotation and dielectrophoretic crossover frequency measurements, are inconvenient, tedious and lengthy. The aim of this study was to develop a practical and speedy dielectrophoretic approach to in vitro toxicity testing. To achieve this, the current study developed several innovations in dielectrophoresis combined with field-flow fractionation (dFFF) using flow cytometry for cell detection. In dFFF, a micro-patterned, nano-film electrode imposes electric forces on cells suspended in a parabolic flow profile as they pass through a thin chamber. Relative changes in the dFFF peak elution times, expressed as a dielectric response index D;, reveal the responses that occur in cell membrane area (capacitance) and ion permeability (conductivity) caused by toxicant exposure. For our purposes, cells were exposed to eight toxicants, each of different single or mixed modes of action. Cell responses were studied over the first 150 minutes of exposure. The selected toxicants include acrylonitrile, actinomycin D, carbon tetrachloride, endosulfan, N-nitroso-N-methylurea (NMU), paraquat dichloride, puromycin, and styrene oxide. The human-derived leukemia cell line HL-60 was used as the in vitro mammalian cell toxicity detection model. For all chemical treatments except actinomycin D, the Di of the HL-60 cells increased in a dose- and time- dependent manner. The sensitivity of the dFFF method was compared with both the cell viability assay for all toxicants studied and either mitochondrial potentiometric dye assay or DNA alkaline comet assay, depending on each toxicant's specific mode of action. Except for low doses of nucleic acid-targeting agents (actinomycin D and NMU), the dFFF method detected all toxicants with more sensitivity than the other assays, often up to 10⁵ times more sensitive than the viability approach. Based on the octanollwater partition (Kow) characteristics of the chemicals tested, the lipophilic chemicals (those with high log Kow) proved to be more easily detected in terms of the dFFF response index Di; than the hydrophilic chemicals (those with low log Kow). The dFFF method appeared to be especially sensitive to agents that directly damaged the plasma membrane or induced apoptosis in the cells. Notably, dFFF detected significant changes in the cell plasma membrane even at the earliest stage of apoptosis induction. In addition, dFFF more acutely detected changes in the plasma membrane of cells exposed to toxicants at doses too low to induce apoptosis or loss of cell viability. Intriguingly, the cells were still stimulated, leading to an increased cell membrane area, perhaps in the form of additional microvilli. A dFFF sample preparation protocol was also developed for testing the toxicity of environmental water samples. Finally, the flow cytometer was replaced by a laser light scatter detector. This device allows the dFFF method to be realized in a more portable, accessible, and cost-effective configuration. Based upon all the findings of this study, it is concluded that the dFFF technique merits further study as a potential rapid in vitro toxicant screening tool |
| Year | 2008 |
| Corresponding Series Added Entry | Asian Institute of Technology. Dissertation ; no. EV-08-14 |
| Type | Dissertation |
| 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 (EV) |
| Chairperson(s) | Mathuros Ruchirawat; |
| Examination Committee(s) | Gascoyne, Peter R.C. ;Preeda Parkpian |
| Scholarship Donor(s) | Chulabhorn Research Institute ;Mahidol University ;Asian Institute of Technology Fellowship |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology - Chulabhorn Research Institute - Mahidol University, 2008 |