Matching Items (48)
Description
Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated membrane filter that will sample, lyse, and extract the DNA from microorganisms in aqueous environments. An off-the-shelf membrane filter disc was embedded in a PDMS layer and sequentially bound with other PDMS channel layers. No leakage was observed during filtration. This device was validated by concentrating a large amount of cyanobacterium Synechocystis in simulated sample water with consistent performance across devices. After accumulating sufficient biomass on the filter, a sequential electrochemical lysing process was performed by applying 5VDC across the filter. This device was further evaluated by delivering several samples of differing concentrations of cyanobacterium Synechocystis then quantifying the DNA using real-time PCR. Lastly, an environmental sample was run through the device and the amount of photosynthetic microorganisms present in the water was determined. The major breakthroughs in this design are low energy demand, cheap materials, simple design, straightforward fabrication, and robust performance, together enabling wide-utility of similar chip-based devices for field-deployable operations in environmental micro-biotechnology.
ContributorsLecluse, Aurelie (Author) / Meldrum, Deirdre (Thesis advisor) / Chao, Joseph (Thesis advisor) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2011
Description
Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study of cellular morphology, a single cell three-dimensional (3D) imaging system can be used to diagnose fatal diseases, such as cancer, at an early stage. One proven method, CellCT, accomplishes 3D imaging by rotating a single cell around a fixed axis. However, some existing cell rotating mechanisms require either intricate microfabrication, and some fail to provide a suitable environment for living cells. This thesis develops a microvorterx chamber that allows living cells to be rotated by hydrodynamic alone while facilitating imaging access. In this thesis work, 1) the new chamber design was developed through numerical simulation. Simulations revealed that in order to form a microvortex in the side chamber, the ratio of the chamber opening to the channel width must be smaller than one. After comparing different chamber designs, the trapezoidal side chamber was selected because it demonstrated controllable circulation and met the imaging requirements. Microvortex properties were not sensitive to the chambers with interface angles ranging from 0.32 to 0.64. A similar trend was observed when chamber heights were larger than chamber opening. 2) Micro-particle image velocimetry was used to characterize microvortices and validate simulation results. Agreement between experimentation and simulation confirmed that numerical simulation was an effective method for chamber design. 3) Finally, cell rotation experiments were performed in the trapezoidal side chamber. The experimental results demonstrated cell rotational rates ranging from 12 to 29 rpm for regular cells. With a volumetric flow rate of 0.5 µL/s, an irregular cell rotated at a mean rate of 97 ± 3 rpm. Rotational rates can be changed by altering inlet flow rates.
ContributorsZhang, Wenjie (Author) / Frakes, David (Thesis advisor) / Meldrum, Deirdre (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2011
Description
Laboratory automation systems have seen a lot of technological advances in recent times. As a result, the software that is written for them are becoming increasingly sophisticated. Existing software architectures and standards are targeted to a wider domain of software development and need to be customized in order to use them for developing software for laboratory automation systems. This thesis proposes an architecture that is based on existing software architectural paradigms and is specifically tailored to developing software for a laboratory automation system. The architecture is based on fairly autonomous software components that can be distributed across multiple computers. The components in the architecture make use of asynchronous communication methodologies that are facilitated by passing messages between one another. The architecture can be used to develop software that is distributed, responsive and thread-safe. The thesis also proposes a framework that has been developed to implement the ideas proposed by the architecture. The framework is used to develop software that is scalable, distributed, responsive and thread-safe. The framework currently has components to control very commonly used laboratory automation devices such as mechanical stages, cameras, and also to do common laboratory automation functionalities such as imaging.
ContributorsKuppuswamy, Venkataramanan (Author) / Meldrum, Deirdre (Thesis advisor) / Collofello, James (Thesis advisor) / Sarjoughian, Hessam S. (Committee member) / Johnson, Roger (Committee member) / Arizona State University (Publisher)
Created2012
Description
Salmonella enterica is a gastrointestinal (GI) pathogen that can cause systemic diseases. It invades the host through the GI tract and can induce powerful immune responses in addition to disease. Thus, it is considered as a promising candidate to use as oral live vaccine vectors. Scientists have been making great efforts to get a properly attenuated Salmonella vaccine strain for a long time, but could not achieve a balance between attenuation and immunogenicity. So the regulated delayed attenuation/lysis Salmonella vaccine vectors were proposed as a design to seek this balance. The research work is progressing steadily, but more improvements need to be made. As one of the possible improvements, the cyclic adenosine monophosphate (cAMP) -independent cAMP receptor protein (Crp*) is expected to protect the Crp-dependent crucial regulator, araC PBAD, in these vaccine designs from interference by glucose, which decreases synthesis of cAMP, and enhance the colonizing ability by and immunogenicity of the vaccine strains. In this study, the cAMP-independent crp gene mutation, crp-70, with or without araC PBAD promoter cassette, was introduced into existing Salmonella vaccine strains. Then the plasmid stability, growth rate, resistance to catabolite repression, colonizing ability, immunogenicity and protection to challenge of these new strains were compared with wild-type crp or araC PBAD crp strains using western blots, enzyme-linked immunosorbent assays (ELISA) and animal studies, so as to evaluate the effects of the crp-70 mutation on the vaccine strains. The performances of the crp-70 strains in some aspects were closed to or even exceeded the crp+ strains, but generally they did not exhibit the expected advantages compared to their wild-type parents. Crp-70 rescued the expression of araC PBAD fur from catabolite repression. The strain harboring araC PBAD crp-70 was severely affected by its slow growth, and its colonizing ability and immunogenicity was much weaker than the other strains. The Pcrp crp-70 strain showed relatively good ability in colonization and immune stimulation. Both the araC PBAD crp-70 and the Pcrp crp-70 strains could provide certain levels of protection against the challenge with virulent pneumococci, which were a little lower than for the crp+ strains.
ContributorsShao, Shihuan (Author) / Curtiss, Roy (Thesis advisor) / Arizona State University (Publisher)
Created2012
Description
Single cell analysis has become increasingly important in understanding disease onset, progression, treatment and prognosis, especially when applied to cancer where cellular responses are highly heterogeneous. Through the advent of single cell computerized tomography (Cell-CT), researchers and clinicians now have the ability to obtain high resolution three-dimensional (3D) reconstructions of single cells. Yet to date, no live-cell compatible version of the technology exists. In this thesis, a microfluidic chip with the ability to rotate live single cells in hydrodynamic microvortices about an axis parallel to the optical focal plane has been demonstrated. The chip utilizes a novel 3D microchamber design arranged beneath a main channel creating flow detachment into the chamber, producing recirculating flow conditions. Single cells are flowed through the main channel, held in the center of the microvortex by an optical trap, and rotated by the forces induced by the recirculating fluid flow. Computational fluid dynamics (CFD) was employed to optimize the geometry of the microchamber. Two methods for the fabrication of the 3D microchamber were devised: anisotropic etching of silicon and backside diffuser photolithography (BDPL). First, the optimization of the silicon etching conditions was demonstrated through design of experiment (DOE). In addition, a non-conventional method of soft-lithography was demonstrated which incorporates the use of two positive molds, one of the main channel and the other of the microchambers, compressed together during replication to produce a single ultra-thin (<200 µm) negative used for device assembly. Second, methods for using thick negative photoresists such as SU-8 with BDPL have been developed which include a new simple and effective method for promoting the adhesion of SU-8 to glass. An assembly method that bonds two individual ultra-thin (<100 µm) replications of the channel and the microfeatures has also been demonstrated. Finally, a pressure driven pumping system with nanoliter per minute flow rate regulation, sub-second response times, and < 3% flow variability has been designed and characterized. The fabrication and assembly of this device is inexpensive and utilizes simple variants of conventional microfluidic fabrication techniques, making it easily accessible to the single cell analysis community.
ContributorsMyers, Jakrey R (Author) / Meldrum, Deirdre (Thesis advisor) / Johnson, Roger (Committee member) / Frakes, David (Committee member) / Arizona State University (Publisher)
Created2012
DescriptionA novel and unconventional approach for delivering a eukaryotic apoptosis factor, TNF-related apoptosis-inducing ligand (TRAIL), to cancer cells within and around necrotizing tumors by utilizing a S. Typhimurium purine requiring auxotroph as a biological vector to develop two anticancer therapies with multiple modality and broad economic feasibility.
ContributorsKoons, Andrew (Author) / Curtiss, Roy (Thesis director) / Lake, Douglas (Committee member) / Janthakahalli, Nagaraj Vinay (Committee member) / Barrett, The Honors College (Contributor)
Created2013-12
Description
Background
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.
Methodology
We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.
Principal Findings
We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.
Conclusions
Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.
Methodology
We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.
Principal Findings
We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.
Conclusions
Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.
ContributorsNandakumar, Vivek (Author) / Kelbauskas, Laimonas (Author) / Hernandez, Kathryn (Author) / Lintecum, Kelly (Author) / Senechal, Patti (Author) / Bussey, Kimberly (Author) / Davies, Paul (Author) / Johnson, Roger (Author) / Meldrum, Deirdre (Author) / Ira A. Fulton Schools of Engineering (Contributor) / School of Electrical, Computer and Energy Engineering (Contributor) / Biodesign Institute (Contributor) / Center for Biosignatures Discovery Automation (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Department of Physics (Contributor)
Created2012-01-05
Description
A single cell is the very fundamental element in an organism; however, it contains the most complicated and stochastic information, such as DNA, RNA, and protein expression. Thus, it is a necessity to study stochastic gene expression in order to discover the biosignatures at the single-cell level. The heterogeneous gene expression of single cells from an isogenic cell population has already been studied for years. Yet to date, single-cell studies have been confined in a fashion of analyzing isolated single cells or a dilution of cells from the bulk-cell populations. These techniques or devices are limited by either the mechanism of cell lysis or the difficulties to target specific cells without harming neighboring cells.
This dissertation presents the development of a laser lysis chip combined with a two-photon laser system to perform single-cell lysis of single cells in situ from three-dimensional (3D) cell spheroids followed by analysis of the cell lysate with two-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The 3D spheroids were trapped in a well in the custom-designed laser lysis chip. Next, each single cell of interest in the 3D spheroid was identified and lysed one at a time utilizing a two-photon excited laser. After each cell lysis, the contents inside the target cell were released to the surrounding media and carried out to the lysate collector. Finally, the gene expression of each individual cell was measured by two-step RT-qPCR then spatially mapped back to its original location in the spheroids to construct a 3D gene expression map.
This novel technology and approach enables multiple gene expression measurements in single cells of multicellular organisms as well as cell-to-cell heterogeneous responses to the environment with spatial recognition. Furthermore, this method can be applied to study precancerous tissues for a better understanding of cancer progression and for identifying early tumor development.
This dissertation presents the development of a laser lysis chip combined with a two-photon laser system to perform single-cell lysis of single cells in situ from three-dimensional (3D) cell spheroids followed by analysis of the cell lysate with two-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The 3D spheroids were trapped in a well in the custom-designed laser lysis chip. Next, each single cell of interest in the 3D spheroid was identified and lysed one at a time utilizing a two-photon excited laser. After each cell lysis, the contents inside the target cell were released to the surrounding media and carried out to the lysate collector. Finally, the gene expression of each individual cell was measured by two-step RT-qPCR then spatially mapped back to its original location in the spheroids to construct a 3D gene expression map.
This novel technology and approach enables multiple gene expression measurements in single cells of multicellular organisms as well as cell-to-cell heterogeneous responses to the environment with spatial recognition. Furthermore, this method can be applied to study precancerous tissues for a better understanding of cancer progression and for identifying early tumor development.
ContributorsWang, Guozhen (Author) / Meldrum, Deirdre R (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Hong (Committee member) / Goryll, Michael (Committee member) / Arizona State University (Publisher)
Created2016
Description
Within the last decade there has been remarkable interest in single-cell metabolic analysis as a key technology for understanding cellular heterogeneity, disease initiation, progression, and drug resistance. Technologies have been developed for oxygen consumption rate (OCR) measurements using various configurations of microfluidic devices. The technical challenges of current approaches include: (1) deposition of multiple sensors for multi-parameter metabolic measurements, e.g. oxygen, pH, etc.; (2) tedious and labor-intensive microwell array fabrication processes; (3) low yield of hermetic sealing between two rigid fused silica parts, even with a compliance layer of PDMS or Parylene-C. In this thesis, several improved microfabrication technologies are developed and demonstrated for analyzing multiple metabolic parameters from single cells, including (1) a modified "lid-on-top" configuration with a multiple sensor trapping (MST) lid which spatially confines multiple sensors to micro-pockets enclosed by lips for hermetic sealing of wells; (2) a multiple step photo-polymerization method for patterning three optical sensors (oxygen, pH and reference) on fused silica and on a polyethylene terephthalate (PET) surface; (3) a photo-polymerization method for patterning tri-color (oxygen, pH and reference) optical sensors on both fused silica and on the PET surface; (4) improved KMPR/SU-8 microfabrication protocols for fabricating microwell arrays that can withstand cell culture conditions. Implementation of these improved microfabrication methods should address the aforementioned challenges and provide a high throughput and multi-parameter single cell metabolic analysis platform.
ContributorsSong, Ganquan (Author) / Meldrum, Deirdre R (Thesis advisor) / Goryll, Michael (Committee member) / Wang, Hong (Committee member) / Tian, Yanqing (Committee member) / Arizona State University (Publisher)
Created2014
Description
In the past decades, single-cell metabolic analysis has been playing a key role in understanding cellular heterogeneity, disease initiation, progression, and drug resistance. Therefore, it is critical to develop technologies for individual cellular metabolic analysis using various configurations of microfluidic devices. Compared to bulk-cell analysis which is widely used by reporting an averaged measurement, single-cell analysis is able to present the individual cellular responses to the external stimuli. Particularly, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are two key parameters to monitor heterogeneous metabolic profiles of cancer cells. To achieve multi-parameter metabolic measurements on single cells, several technical challenges need to be overcome: (1) low adhesion of soft materials micro-fabricated on glass surface for multiple-sensor deposition and single-cell immobilization, e.g. SU-8, KMPR, etc.; (2) high risk of using external mechanical forces to create hermetic seals between two rigid fused silica parts, even with compliance layers; (3) how to accomplish high-throughput for single-cell trapping, metabolic profiling and drug screening; (4) high process cost of micromachining on glass substrate and incapability of mass production.
In this dissertation, the development of microfabrication technologies is demonstrated to design reliable configurations for analyzing multiple metabolic parameters from single cells, including (1) improved KMPR/SU-8 microfabrication protocols for fabricating microwell arrays that can be integrated and sealed to 3 × 3 tri-color sensor arrays for OCR and ECAR measurements; (2) design and characterization of a microfluidic device enabling rapid single-cell trapping and hermetic sealing single cells and tri-color sensors within 10 × 10 hermetically sealed microchamber arrays; (3) exhibition of a low-cost microfluidic device based on plastics for single-cell metabolic multi-parameter profiling. Implementation of these improved microfabrication methods should address the aforementioned challenges and provide a high throughput and multi-parameter single cell metabolic analysis platform.
In this dissertation, the development of microfabrication technologies is demonstrated to design reliable configurations for analyzing multiple metabolic parameters from single cells, including (1) improved KMPR/SU-8 microfabrication protocols for fabricating microwell arrays that can be integrated and sealed to 3 × 3 tri-color sensor arrays for OCR and ECAR measurements; (2) design and characterization of a microfluidic device enabling rapid single-cell trapping and hermetic sealing single cells and tri-color sensors within 10 × 10 hermetically sealed microchamber arrays; (3) exhibition of a low-cost microfluidic device based on plastics for single-cell metabolic multi-parameter profiling. Implementation of these improved microfabrication methods should address the aforementioned challenges and provide a high throughput and multi-parameter single cell metabolic analysis platform.
ContributorsSong, Ganquan (Author) / Meldrum, Deirdre R. (Thesis advisor) / Goryll, Michael (Committee member) / Kelbauskas, Laimonas (Committee member) / Wang, Hong (Committee member) / Arizona State University (Publisher)
Created2017