Waterborne or foodborne diseases are caused by consuming contaminated fluids or foods. The presence of pathogenic microorganisms can contaminate food or drinking water. These microorganisms can cause sickness even if they are present in minimal concentrations. The World Health Organization (WHO) has defined the standards for clean drinking water as the absence of E. coli in a 100 mL collected volume. Contaminated water or food can cause many diseases, and diarrhea is one of a prominent disease. Early detection of contamination in food or drinking water is critical. Conventional culture-based methods are time-consuming, labour intensive, and not suitable for on-site testing. Nucleic acid-based tests are sensitive and can rapidly detect pathogens. Microfluidic technology can play a significant role to develop low-cost, rapid, integrated, and portable nucleic acid-based detection devices. Microfluidic systems for isothermal amplification assays can be classified into two groups such as droplet-based and chamber-based systems. In this thesis, both droplet-based and chamber-based approaches were used to build the microfluidic methods for isothermal amplification assays.
First, a simple electromechanical probe (tweezers) was developed that can manipulate a small aqueous droplet in a bi-layer oil phase. The tweezer consisted of two needles positioned close to each other and used polarization of the aqueous droplet in an applied electrical field to confine the droplet between the needles with minimal solid contact. AC electric potential was applied to the two metal electrodes. Droplet acquired a charge from the high voltage electrode and consequently performed an oscillatory motion with the same electrode. This droplet motion was controlled using two parameters of electric potential and frequency of the applied signal. Initially, electrically actuated droplet (0.3 µL) motion was investigated for a range of applied potential (400-960 Volts) and frequencies (0.1-1000 Hz). The droplet motion with high voltage electrode was characterized into three modes such as detachment, oscillation, and attachment.
Mechanical motion of tweezer was used to transport droplet to various positions. Consequently, operations such as transportation, extraction, and merging were demonstrated. First, droplet (5 µL) transportation was characterized under the applied potential of 2000 Volts at various frequencies (5 to 1000 Hz). The droplet was successfully transported to the speed of 15 mm/s at higher frequencies (100 or 1000 Hz). Droplets of various volumes (12-80 µL) were extracted by increasing applied electric potential, from 0 to 6000 Volts at 5 Hz. Then, the operation of droplets merging was demonstrated using operational conditions for electrical tweezer.
Finally, electrical tweezer was used to prepare samples for isothermal amplification assays. Two droplets consisted of various reagents of isothermal amplification assays, were transported and merged using the electrical tweezer. Then, a merged droplet (25 µL) was transported and immobilized in the amplification zone. The temperature of the amplification zone (~65°C) was maintained using an in-situ heater. DNA amplification was verified by measuring the off-chip end-point fluorescence intensity of isothermal assays.
Second, an integrated microfluidic device has been developed to prepare a sample for isothermal amplification assays. And in-situ real-time amplification assays were performed to detect bacteria. The device consisted of two chambers (lysis and amplification) connected through a microchannel. A low-cost fabrication method was introduced to embed two resistive wire heaters around both chambers. Initially, bacteria cells were thermally lysed in the lysis chamber at 92°C for 5 min. Then, DNA was electrophoretically transported from lysis to the amplification chamber. The electric potential of 10 Volts was applied for 10 min for DNA transportation. Next, transported DNA was amplified at 65°C and DNA amplification was detected by measuring in-situ fluorescence intensity in the real-time format. The operation of the integrated microfluidic device was demonstrated in three steps. 1) Operation of individual components. 2) Operation of two components in a coupled format. 3) Integrated operation of three components with measurement of fluorescent intensity in a real-time format. The bacteria samples with the concentration of 100 CFU/mL were detected in less than one hour. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24017 |
| Date | January 2018 |
| Creators | Shahid, Ali |
| Contributors | Selvaganapathy, Ponnambalam (Ravi), Mechanical Engineering |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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