Date of Award
Master of Applied Science (MASc)
P. Ravi Selvaganapathy
Microbiological contamination from bacteria such as Escherichia coli and Salmonella is one of the main reasons for waterborne illness. Real time and accurate monitoring of water is needed in order to alleviate this human health concern. Performing multiple and parallel analysis of biomarkers such as DNA and mRNA that targets different regions of pathogen functionality provides a complete picture of its presence and viability in the shortest possible time. These biomarkers are present inside the cell and need to be extracted for analysis and detection. Hence, lysis of these pathogenic bacteria is an important part in the sample preparation for rapid detection. In addition, collecting a small amount of bacteria present in a large volume of sample and concentrating them before lysing is important as it facilitates the downstream assay. Various techniques, categorized as mechanical, chemical, thermal and electrical, have been used for lysing cells. In the electrical method, cells are lysed by exposure to an external electric field. The advantage of this method, in contrast to other methods, is that it allows lysis without the introduction of any chemical and biological reagents and permits rapid recovery of intercellular organelles. Despite the advantages, issues such as high voltage requirement, bubble generation and Joule heating are associated with the electrical method.
To alleviate the issues associated with electrical lysis, a new design and associated fabrication process for a microfluidic cell lysis device is described in this thesis. The device consists of a nanoporous polycarbonate (PCTE) membrane sandwiched between two PDMS microchannels with electrodes embedded at the reservoirs of the microchannels. Microcontact printing is used to attach this PCTE membrane with PDMS.
By using this PCTE membrane, it was possible to intensify the electric field at the interface of two channels while maintaining it low in the other sections of the device. Furthermore, the device also allowed electrophoretic trapping of cells before lysis at a lower applied potential. For instance, it could trap bacteria such as E. coli from a continuous flow into the intersection between two channels for lower electric field (308 V/cm) and lyse the cell when electric field was increased more than 1000 V/cm into that section.
Application of lower DC voltage with pressure driven flow alleviated adverse effect from Joule heating. Moreover, gas evolution and bubble generation was not observed during the operation of this device.
Accumulation and lysis of bacteria were studied under a fluorescence microscope and quantified by using intensity measurement. To observe the accumulation and lysis, LIVE/DEAD BacLight Bacterial Viability Kit consisting of two separate components of SYTO 9 and propidium iodide (PI) into the cell suspension in addition to GFP expressed E. coli were used. Finally, plate counting was done to determine the efficiency of the device and it was observed that the device could lyse 90% of bacteria for an operation voltage of 300V within 3 min.
In conclusion, a robust, reliable and flexible microfluidic cell lysis device was proposed and analyzed which is useful for sample pretreatment in a Micro Total Analysis System.
Islam, Md. Shehadul, "A Low Power Electrical Method for Cell Accumulation and Lysis Using Microfluidics" (2012). Open Access Dissertations and Theses. Paper 7405.
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