Matching Items (11)
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- All Subjects: Microfluidics
- Creators: Buttry, Daniel
- Creators: Hayes, Mark A.
Description
This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS.
ContributorsBhattacharya, Sanchari (Author) / Ros, Alexandra (Committee member) / Ros, Robert (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2013
Description
DNA and DNA nanoassemblies such as DNA origamis have large potential in biosensing, drug delivery, nanoelectronic circuits, and biological computing requiring suitable methods for migration and precise positioning. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro-and nanometer-sized objects. In order to exploit iDEP for naturally formed DNA and DNA nanoassemblies, a detailed understanding of the underlying polarization and dielectrophoretic migration is essential. The shape and the counterion distribution are considered two essential factors in the polarization mechanism. Here, the dielectrophoretic behavior of 6-helix bundle (6HxB) and triangle DNA origamis with identical sequences but substantial topological differences was explored. The polarizability models were discussed for the two species according to their structural difference. The experimental observations reveal distinct iDEP trapping behavior in low frequency AC electric fields in addition to numerical simulations showing a considerable contribution of the electrophoretic transport of the DNA origami species in the DEP trapping regions. Furthermore, the polarizabilities of the two species were determined by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the DNA origamis according to an adapted Kramer’s rate model. The orientations of both species in the escape process were studied. Finally, to study the counterion distribution around the DNA molecules, both λ-DNA and 6HxB DNA were used in a phosphate buffer containing magnesium, revealing distinctive negative dielectrophoretic trapping behavior as opposed to positive trapping in a sodium/potassium phosphate buffer system.
ContributorsGan, Lin (Author) / Ros, Alexandra (Thesis advisor) / Buttry, Daniel (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2015
Description
Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among all microfluidic techniques, dielectrophoresis (DEP) is one of the most effective and efficient techniques to quickly isolate and separate polarizable particles under inhomogeneous electric field. To date, extensive studies have demonstrated that DEP devices are able to precisely manipulate cells ranging from over 10 μm (mammalian cells) down to about 1 μm (small bacteria). However, very limited DEP studies on manipulating submicron bioparticles, such as viruses, have been reported.
In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition.
DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step.
In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition.
DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step.
ContributorsDing, Jie (Author) / Hayes, Mark A. (Thesis advisor) / Ros, Alexandra (Committee member) / Buttry, Daniel A (Committee member) / Arizona State University (Publisher)
Created2017
Description
Disease prevention and personalized treatment will be impacted by the continued integration of protein biomarkers into medical practice. While there are already numerous biomarkers used clinically, the detection of protein biomarkers among complex matrices remains a challenging problem. One very important strategy for improvements in clinical application of biomarkers is separation/preconcentration, impacting the reliability, efficiency and early detection. Electrophoretic exclusion can be used to separate, purify, and concentrate biomarkers. This counterflow gradient technique exploits hydrodynamic flow and electrophoretic forces to exclude, enrich, and separate analytes. The development of this technique has evolved onto an array-based microfluidic platform which offers a greater range of geometries/configurations for optimization and expanded capabilities and applications. Toward this end of expanded capabilities, fundamental studies of subtle changes to the entrance flow and electric field configurations are investigated. Three closely related microfluidic interfaces are modeled, fabricated and tested. A charged fluorescent dye is used as a sensitive and accurate probe to test the concentration variation at these interfaces. Models and experiments focus on visualizing the concentration profile in areas of high temporal dynamics, and show strong qualitative agreement, which suggests the theoretical assessment capabilities can be used to faithfully design novel and more efficient interfaces. Microfluidic electrophoretic separation technique can be combined with electron microscopy as a protein concentration/purification step aiding in sample preparation. The integrated system with grids embedded into the microdevice reduces the amount of time required for sample preparation to less than five minutes. Spatially separated and preconcentrated proteins are transferred directly from an upstream reservoir onto grids. Dilute concentration as low as 0.005 mg/mL can be manipulated to achieve meaningful results. Selective concentration of one protein from a mixture of two proteins is also demonstrated. Electrophoretic exclusion is also used for biomarker applications. Experiments using a single biomarker are conducted to assess the ability of the microdevice for enrichment in central reservoirs. A mixture of two protein biomarkers are performed to evaluate the proficiency of the device for separations capability. Moreover, a battery is able to power the microdevice, which facilitates the future application as a portable device.
ContributorsZhu, Fanyi (Author) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2019
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
X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm – ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm – ~20 μm crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion. Additionally, a passive mixer was created to generate unique solution concentrations within isolated nanowells to crystallize phycocyanin and lysozyme. Crystal imaging with brightfield microscopy, UV fluorescence, and SONICC coupled with numerical modeling allowed quantification of crystal growth conditions for efficient phase diagram development. The developed microfluidic tools demonstrated the capability of improving samples for protein crystallography, offering a foundation for continued development of platforms to aid protein structure determination.
ContributorsAbdallah, Bahige G (Author) / Ros, Alexandra (Thesis advisor) / Buttry, Daniel (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2016
Description
Locomotion of microorganisms is commonly observed in nature. Although microorganism locomotion is commonly attributed to mechanical deformation of solid appendages, in 1956 Nobel Laureate Peter Mitchell proposed that an asymmetric ion flux on a bacterium's surface could generate electric fields that drive locomotion via self-electrophoresis. Recent advances in nanofabrication have enabled the engineering of synthetic analogues, bimetallic colloidal particles, that swim due to asymmetric ion flux originally proposed by Mitchell. Bimetallic colloidal particles swim through aqueous solutions by converting chemical fuel to fluid motion through asymmetric electrochemical reactions. This dissertation presents novel bimetallic motor fabrication strategies, motor functionality, and a study of the motor collective behavior in chemical concentration gradients. Brownian dynamics simulations and experiments show that the motors exhibit chemokinesis, a motile response to chemical gradients that results in net migration and concentration of particles. Chemokinesis is typically observed in living organisms and distinct from chemotaxis in that there is no particle directional sensing. The synthetic motor chemokinesis observed in this work is due to variation in the motor's velocity and effective diffusivity as a function of the fuel and salt concentration. Static concentration fields are generated in microfluidic devices fabricated with porous walls. The development of nanoscale particles that swim autonomously and collectively in chemical concentration gradients can be leveraged for a wide range of applications such as directed drug delivery, self-healing materials, and environmental remediation.
ContributorsWheat, Philip Matthew (Author) / Posner, Jonathan D (Thesis advisor) / Phelan, Patrick (Committee member) / Chen, Kangping (Committee member) / Buttry, Daniel (Committee member) / Calhoun, Ronald (Committee member) / Arizona State University (Publisher)
Created2011
Description
Understanding cellular processes can provide insight into disease pathogenesis and reveal critical information for prevention, diagnosis, and treatment. As key executors and signaling regulators, proteins carry relevant information not available from genomics and transcriptomics. Cell-to-cell differences significantly affect disease incidence and drug responses, generating a need for protein analysis at the single-cell level. However, quantitative protein analysis at the single-cell level remains challenging due to the low protein amount in a single cell and the proteome complexity. It requires sensitive detection techniques and appropriate sample preparation and delivery to the detection area. Here, a microfluidic platform in tandem with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has been developed for targeted intracellular protein analysis. The elastomeric multi-layer microfluidic platform, termed MIMAS, was designed as a series of 8.75 nL wells separated by pneumatic valves. The MIMAS platform allows cell loading, sample processing on-chip, and further in situ mass spectrometry analysis. The sample processing includes cell lysis, immunocapture, tryptic digestion and MALDI matrix solution loading for co-crystallization. This work demonstrates that the MIMAS approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 from MCF-7 breast cancer cells using an isotope-labeled peptide. The limit of detection was determined as 11.22 nM, equivalent to 5.91 x 10^7 protein molecules per well. Moreover, the MIMAS platform design was improved, allowing the successful quantification of Bcl-2 protein in small cell ensembles down to ~10 cells in 4 nL wells. Furthermore, the MIMAS platform was integrated with laser capture microdissection (LCM) for protein analysis from post-mortem human tissues. Intracellular amyloid-β peptide (Aβ), a hallmark of Alzheimer’s Disease, was assessed from human brain tissue using the LCM-MIMAS. The successful detection of Aβ from small cell ensembles (20 sliced pyramidal cells) demonstrated the LCM-MIMAS capability of assessing intracellular proteins from specific tissue cell subpopulations. The MIMAS approach is a promising tool for intracellular protein analysis from small cell ensembles, with the potential for single-cell analysis. It allows for protein analysis towards the understanding of biological phenomena for clinical and biological research.
ContributorsCruz Villarreal, Jorvani (Author) / Ros, Alexandra (Thesis advisor) / Borges, Chad R (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2022
Description
Microfluidics has enabled many biological and biochemical applications such as high-throughput drug testing or point-of-care diagnostics. Dielectrophoresis (DEP) has recently achieved prominence as a powerful microfluidic technique for nanoparticle separation. Novel electric field-assisted insulator-based dielectrophoresis (iDEP) microfluidic devices have been employed to fractionate rod-shaped nanoparticles like Single-walled carbon nanotubes (SWNTs) and manipulate biomolecules like Deoxyribonucleic acid (DNA) and proteins. This dissertation involves the development of traditional as well as 3D-printed iDEP devices for the manipulation of nm-to-µm scale analytes. First, novel iDEP microfluidic constriction-based sorting devices were developed to introduce inhomogeneous electric field gradients to fractionate SWNTs by length. SWNTs possess length-specific optical and electrical properties, expanding their potential applications for future nanoscale devices. Standard synthesis procedures yield SWNTs in large-length polydispersity and chirality. Thus, an iDEP-based fractionation tool for desired lengths of SWNTs may be beneficial. This dissertation presents the first study of DEP characterization and fractionation of SWNTs using an iDEP microfluidic device. Using this iDEP constriction sorter device, two different length distributions of SWNTs were sorted with a sorting efficiency of >90%. This study provides the fundamentals of fractionating SWNTs by length, which can help separate and purify SWNTs for future nanoscale-based applications. Manipulation of nm-scale analytes requires achieving high electric field gradients in an iDEP microfluidic device, posing one of the significant challenges for DEP applications. Introducing nm-sized constrictions in an iDEP device can help generate a higher electric field gradient. However, this requires cumbersome and expensive fabrication methods. In recent years, 3D printing has drawn tremendous attention in microfluidics, alleviating complications associated with complex fabrication methods. A high-resolution 3D-printed iDEP device was developed and fabricated for iDEP-based manipulation of analytes. A completely 3D-printed device with 2 µm post-gaps was realized, and fluorescent polystyrene (PS) beads, λ-DNA, and phycocyanin protein trapping were demonstrated. Furthermore, a nm-resolution 3D-printed iDEP device was successfully printed. In the future, these high-resolution 3D-printed devices may lead to exploring DEP characteristics of nanoscale analytes like single protein molecules and viruses. The electric field-assisted unique fractionation phenomena in microfluidic platforms will become a critical solution for nanoparticle separation and manipulating biomolecules.
ContributorsRabbani, Mohammad Towshif (Author) / Ros, Alexandra (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2022