Matching Items (3)
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- All Subjects: Microfluidics
- Creators: Herckes, Pierre
Description
Dielectrophoresis (DEP) is a technique that influences the motion of polarizable particles in an electric field gradient. DEP can be combined with other effects that influence the motion of a particle in a microchannel, such as electrophoresis and electroosmosis. Together, these three can be used to probe properties of an analyte, including charge, conductivity, and zeta potential. DEP shows promise as a high-resolution differentiation and separation method, with the ability to distinguish between subtly-different populations. This, combined with the fast (on the order of minutes) analysis times offered by the technique, lend it many of the features necessary to be used in rapid diagnostics and point-of-care devices.
Here, a mathematical model of dielectrophoretic data is presented to connect analyte properties with data features, including the intercept and slope, enabling DEP to be used in applications which require this information. The promise of DEP to distinguish between analytes with small differences is illustrated with antibiotic resistant bacteria. The DEP system is shown to differentiate between methicillin-resistant and susceptible Staphylococcus aureus. This differentiation was achieved both label free and with bacteria that had been fluorescently-labeled. Klebsiella pneumoniae carbapenemase-positive and negative Klebsiella pneumoniae were also distinguished, demonstrating the differentiation for a different mechanism of antibiotic resistance. Differences in dielectrophoretic behavior as displayed by S. aureus and K. pneumoniae were also shown by Staphylococcus epidermidis. These differences were exploited for a separation in space of gentamicin-resistant and -susceptible S. epidermidis. Besides establishing the ability of DEP to distinguish between populations with small biophysical differences, these studies illustrate the possibility for the use of DEP in applications such as rapid diagnostics.
Here, a mathematical model of dielectrophoretic data is presented to connect analyte properties with data features, including the intercept and slope, enabling DEP to be used in applications which require this information. The promise of DEP to distinguish between analytes with small differences is illustrated with antibiotic resistant bacteria. The DEP system is shown to differentiate between methicillin-resistant and susceptible Staphylococcus aureus. This differentiation was achieved both label free and with bacteria that had been fluorescently-labeled. Klebsiella pneumoniae carbapenemase-positive and negative Klebsiella pneumoniae were also distinguished, demonstrating the differentiation for a different mechanism of antibiotic resistance. Differences in dielectrophoretic behavior as displayed by S. aureus and K. pneumoniae were also shown by Staphylococcus epidermidis. These differences were exploited for a separation in space of gentamicin-resistant and -susceptible S. epidermidis. Besides establishing the ability of DEP to distinguish between populations with small biophysical differences, these studies illustrate the possibility for the use of DEP in applications such as rapid diagnostics.
ContributorsHilton, Shannon (Author) / Hayes, Mark A. (Thesis advisor) / Borges, Chad (Committee member) / Herckes, Pierre (Committee member) / Arizona State University (Publisher)
Created2019
Description
Microfluidic systems have gained popularity in the last two decades for their potential applications in manipulating micro- and nano- particulates of interest. Several different microfluidics devices have been built capable of rapidly probing, sorting, and trapping analytes of interest. Microfluidics can be combined with separation science to address challenges of obtaining a concentrated and pure distinct analyte from mixtures of increasingly similar entities. Many of these techniques have been developed to assess biological analytes of interest; one of which is dielectrophoresis (DEP), a force which acts on polarizable analytes in the presence of a non-uniform electric fields. This method can achieve high resolution separations with the unique attribute of concentrating, rather than diluting, analytes upon separation. Studies utilizing DEP have manipulated a wide range of analytes including various cell types, proteins, DNA, and viruses. These analytes range from approximately 50 nm to 1 µm in size. Many of the currently-utilized techniques for assessing these analytes are time intensive, cost prohibitive, and require specialized equipment and technical skills.
The work presented in this dissertation focuses on developing and utilizing insulator-based dielectrophoresis (iDEP) to probe a wide range of analytes; where the intrinsic properties of an analyte will determine its behavior in a microchannel. This is based on the analyte’s interactions with the electrokinetic and dielectrophoretic forces present. Novel applications of this technique to probe the biophysical difference(s) between serovars of the foodborne pathogen, Listeria monocytogenes, and surface modified Escherichia coli, are investigated. Both of these applications demonstrate the capabilities of iDEP to achieve high resolution separations and probe slight changes in the biophysical properties of an analyte of interest. To improve upon existing iDEP strategies a novel insulator design which streamlines analytes in an iDEP device while still achieving the desirable forces for separation is developed, fabricated, and tested. Finally, pioneering work to develop an iDEP device capable of manipulating larger analytes, which range in size 10-250 µm, is presented.
The work presented in this dissertation focuses on developing and utilizing insulator-based dielectrophoresis (iDEP) to probe a wide range of analytes; where the intrinsic properties of an analyte will determine its behavior in a microchannel. This is based on the analyte’s interactions with the electrokinetic and dielectrophoretic forces present. Novel applications of this technique to probe the biophysical difference(s) between serovars of the foodborne pathogen, Listeria monocytogenes, and surface modified Escherichia coli, are investigated. Both of these applications demonstrate the capabilities of iDEP to achieve high resolution separations and probe slight changes in the biophysical properties of an analyte of interest. To improve upon existing iDEP strategies a novel insulator design which streamlines analytes in an iDEP device while still achieving the desirable forces for separation is developed, fabricated, and tested. Finally, pioneering work to develop an iDEP device capable of manipulating larger analytes, which range in size 10-250 µm, is presented.
ContributorsCrowther, Claire Victoria (Author) / Hayes, Mark A. (Thesis advisor) / Gile, Gillian H (Committee member) / Ros, Alexandra (Committee member) / Herckes, Pierre (Committee member) / Arizona State University (Publisher)
Created2018
Description
A major goal of the Center for Biosignatures Discovery Automation (CBDA) is to design a diagnostic tool that detects novel cancer biosignatures at the single-cell level. We designed the Single-cell QUantitative In situ Reverse Transcription-Polymerase Chain Reaction (SQUIRT-PCR) system by combining a two-photon laser lysis (2PLL) system with a microfluidic reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) platform. It is important to identify early molecular changes from intact tissues as prognosis for premalignant conditions and develop new molecular targets for prevention of cancer progression and improved therapies. This project analyzes RNA expression at the single-cell level and presents itself with two major challenges: (1) detecting low levels of RNA and (2) minimizing RNA absorption in the polydimethylsiloxane (PDMS) microfluidic channel. The first challenge was overcome by successfully detecting picogram (pg) levels of RNA using the Fluidigm (FD) BioMark™ HD System (Fluidigm Corporation, South San Francisco, CA) for RT-qPCR analysis. This technology incorporates a highly precise integrated fluidic circuit (IFC) that allows for high-throughput genetic screening using microarrays. The second challenge entailed collecting data from RNA flow-through samples that were passed through microfluidic channels. One channel was treated with a coating of polyethylene glycol (PEG) and the other remained untreated. Various flow-through samples were subjected to RT-qPCR and analyzed using the FD FLEXsix™ Gene Expression IFC. As predicted, the results showed that the treated PDMS channel absorbed less RNA than the untreated PDMS channel. Once the optimization of the PDMS microfluidic platform is complete, it will be implemented into the 2PLL system. This novel technology will be able to identify cell populations in situ and could have a large impact on cancer diagnostics.
ContributorsBlatt, Amy Elissa (Author) / Meldrum, Deirdre R. (Thesis director) / Tran, Thai (Committee member) / Chao, Joseph (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05