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- Kim, Dokyoon.
- Aug. 2013.
- Description
- Book — online resource (xiii, 89 pages) : illustrations (some color)
- Summary
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This dissertation presents the basic principles of the magnetic protein chip immunoassay and applications of the magnetic protein chip for the detection of protein biomarkers. The magnetic protein chip is based on the giant magnetoresistive (GMR) spin-valve sensors, and electrical resistance changes of the sensors are measured as signals. Immunoassays were developed using the magnetic protein chips to quantitate protein biomarkers with high sensitivity. The magnetic protein chip immunoassay has several advantages over conventional non-magnetic techniques, such as multiplex capability, smaller sample volume requirement, enhanced signal-to-noise ratio, and facile integration with electronics. Ionizing radiation is high energy radiation that can remove electrons from atoms, and it causes various cellular damages some of which are lethal. Using an in vivo mouse radiation model, we developed protocols for measuring fms-related tyrosine kinase 3 ligand (Flt3lg) and serum amyloid A1 (Saa1) in small amounts of blood collected during the first week after X-ray exposures of sham, 0.1, 1, 2, 3, or 6 Gy. Flt3lg concentrations showed excellent dose discrimination at >= 1 Gy in the time window of 1 to 7 days after exposure except 1 Gy at day 7. Saa1 dose response was limited to the first two days after exposure. A discriminant analysis using both proteins could show improved dose classification accuracy. Our magnetic protein chip immunoassay demonstrated the dose and time responses and low-dose sensitivity that have important advantages in radiation triage biodosimetry. Necrotizing enterocolitis (NEC) is an inflammatory bowel disease often observed in pre-term babies and has high mortality rate. However, timely diagnosis of NEC has been hampered due to its unspecific symptoms and ineffective clinical tests currently available. We developed a magnetic protein chip immunoassay for the validation of NEC biomarkers. Three biomarkers, C-reactive protein (CRP), matrix metalloproteinase-7 (MMP7), and epithelial cell adhesion molecule (EpCAM) were quantitated using a small amount of blood samples. Receiver operating characteristic (ROC) curve analysis combined with bootstrapping technique showed excellent discrimination of NEC from healthy control and NEC from sepsis. Given the generality of the detection scheme used in the magnetic protein chip immunoassay, the magnetic protein chips are expected to hold great potential for medical diagnosis and clinical research.
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- Hager-Barnard, Elizabeth Ann.
- Dec. 2011.
- Description
- Book — online resource (xviii, 172 pages) : illustrations (some color)
- Summary
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Recent successes in the pharmaceutical industry have yielded a variety of new drugs based on proteins and nucleic acids. While these drugs are very promising, they are only effective once they are inside cells. Unfortunately, transporting drugs into cells tends to be challenging because cell membranes provide a protective barrier. However, there is hope for these drugs since researchers have identified many drug delivery agents that can shuttle drugs past this barrier. Cell penetrating peptides (CPPs) are particularly promising delivery agents due to their low toxicity and ability to deliver a wide number of therapeutic agents. However there are challenges to using CPPs, because their delivery mechanisms cannot yet be controlled. Engineering new CPPs that use specific, known translocation mechanisms would be a key achievement that could increase delivery efficiency and prevent unwanted side effects. Accomplishing this goal requires new experimental methods for determining the factors that control a CPP's translocation mechanisms. In this thesis I present new atomic force microscopy (AFM) methods for studying CPPs and other cell membrane active species. I first present a theoretical model that shows how results from AFM can indicate whether CPPs change the energy barrier to bilayer penetration. I then describe new experimental AFM methods we developed for examining CPP transduction mechanisms.
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Online 3. Interrogating, manipulating, and controlling nano-bio interfaces [2011]
- VanDersarl, Jules John.
- Aug. 2011.
- Description
- Book — online resource (xix, 122 pages) : illustrations (some color)
- Summary
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Cells communicate through direct contact and soluble chemical signals. Mimicking an extracellular environment requires controlling these signals at micron length scales. Integrated circuits make electronic control at these scales trivial, but fluidic control at these length scales requires very different principles. Standard microfluidic devices can finely control flowing fluids, but fluid flow affects cells in a myriad of ways. Alternatively, diffusion based chemical delivery methods tend to be crude, ill defined systems that offer very limited control. This thesis describes three distinctive platforms that combine the active spatial and temporal control of microfluidic systems with a delivery system that relies purely on diffusion. First, we detail a silicon based array of nanoreservoirs underneath the cell culture surface which are used to store and release bioactive molecules. These reservoirs are opened and closed with electrochemical dissolution and deposition at a narrow reservoir opening. Next, we describe an adaptation of traditional, elastomer based microfluidics. In these devices the cell culture area is separated from a microfluidic channel located directly underneath the chamber by a nanoporous membrane. The desirable microfluidic properties, including temporal and spatial control, are preserved, while fluidic flow over the cells is eliminated. Finally, we demonstrate a novel "nanostraw" culture surface, which is combined with the previous device to offer fluidic access directly to the cell cytosol, creating a powerful tool with implications for cell delivery and sampling. Additional work on probing the assembly of protein structures is also detailed. Clathrin 2-dimensional lattice assembly on lipid monolayers, serving as cell membrane mimics, was monitored and studied through surface rheological techniques. Rheological measurements elucidated important network properties, and the formation process was compared to various models for clathrin network assembly.
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Online 4. Nanopillar structures for cellular interface [2011]
- Xie, Chong.
- Sept. 2011.
- Description
- Book — online resource (xv, 73 pages) : illustrations (some color)
- Summary
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Abstract: The small scale of nano-materials makes them one of the best man-made candidates to interact with biological systems at subcellular or even molecular level. It has been the focal point of the research interests to interfacing live cells with one dimensional nanostructures, such as nanowires and nanopillars. In my Phd research, I have utilized nanopillar based structures and devices to interface biological cells electrically, optically and mechanically. 1. We achieve improved electric interface between biological cells and solid state device by using arrays of vertically aligned nanopillar electrodes. Their tight attachment to the cell membrane allows us to acquire intracellular-like action potential signals non-destructively from cultured cardiomyocytes, which is responsible for various important cellular functions. 2. We demonstrate below-the-diffraction-limit observation volume in vitro and inside live cells by using vertically aligned silicon dioxide nanopillars. With a diameter much smaller than the wavelength of visible light, a transparent silicon dioxide nanopillar embedded in a nontransparent substrate restricts the propagation of light and affords evanescence wave excitation along its vertical surface. This effect creates highly-confined illumination volume that selectively excites fluorescence molecules in the vicinity of the nanopillar. We show that this nanopillar illumination can be used for in vitro single molecule detection with high fluorescence background. In addition, we demonstrate that vertical nanopillars interface tightly with live cells and function as highly localized light sources inside the cell. Furthermore, chemical modification of the nanopillar surface provides a unique way to locally recruit proteins of interest and simultaneously observe their behavior within the complex, crowded environment of the cell. 3. We engineer and fabricate vertically nanopillar arrays, and culture various types of cells atop. We study the cell growth pattern in presence of nanopillar arrays, including attachment, migration, etc. We also design patterned nanopillar arrays and utilized them to guide and control cell growth via cell-nanopillar interaction.
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Online 5. Tunable bio-inorganic interfaces for intracellular access [2011]
- Almquist, Benjamin David.
- July 2011.
- Description
- Book — online resource (xviii, 83 pages) : illustrations (some color)
- Summary
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Electrophysiological tools and biologic delivery systems generally rely on non-optimal methods for gaining access through cellular membranes. Electrophysiological techniques that provide intracellular access, such as patch clamping, result in membrane holes and cell death in a matter of hours, while the delivery of bioactive materials are hampered by low bioavailability following passage through the endosomal pathways. In each case, the lipid bilayer backbone of the cellular membrane presents a formidable barrier to intracellular access. As biological gatekeepers, cell membranes not only physically define everything from whole organisms to individual organelles, they also prevent unobstructed flow of molecules between the inner and outer regions of the membrane. This occurs since the hydrophobic lipid acyl tails form a narrow hydrophobic layer a few nanometers thick, which is highly unfavorable for the passage of most hydrophilic molecules. It is this region that is one of the greatest obstacles to the dream of biotechnology seamlessly and non-destructively integrating synthetic components with biological systems. This thesis contributes to the understanding of how to rationally design devices that interact specifically with this hydrophobic region. In turn, this work begins to establish design guidelines for creating non-destructive, membrane-penetrating bio-inorganic interfaces. The beginning chapters focus on the development of the "stealth" probe platform. In nature, there exist specialized transmembrane proteins capable of incorporating into lipid bilayers by replicating the lipid hydrophilic-hydrophobic-hydrophilic structure. The stealth probe design mimics this structure by creating 2-10nm hydrophobic bands on otherwise hydrophilic structures. However, since current lithographic methods do not possess the necessary resolution, a new fabrication technique using a combination of top-down fabrication with bottom-up self-assembly methods was developed. This approach uses an evaporated chrome-gold-chrome stack and focused ion beam (FIB) milling, where the exposed edge of the embedded gold layer can be specifically functionalized with a hydrophobic thiol-mediated self-assembled monolayer. Chapter 3 explores the propensity for insertion and specific interaction of the stealth probe hydrophobic band with the hydrophobic lipid bilayer core. In order to gain quantitative insight into the interaction behavior, atomic force microscopy was used in conjunction with a new, stacked lipid bilayer testing platform. By using stacks of 100's to 1000's of lipid bilayers, substrate-probe interaction artifacts can be removed while simultaneously allowing precise determination of probe location within a lipid bilayer. It was found that completely hydrophilic probes reside in the hydrophilic hydration region between bilayers, whereas hydrophobically functionalized stealth probes preferred to reside in the bilayer core. This behavior was found to be independent of hydrophobic functionalization, with butanethiol and dodecanethiol both displaying preferential localization. The subsequent chapters explore how the molecular structure of the hydrophobic band and the band thickness affect membrane-probe interface stability. The lipid stack platform provides an easy method of force-clamp testing, which enabled quantitative extrapolation of the unstressed interface strength. A series of tests with various length alkanethiols found that the crystallinity of the molecules in the hydrophobic band is the dominant factor influencing interfacial stability. Surprisingly, hydrophobicity was found to be a secondary factor, although necessary to drive spontaneous membrane integration. Molecular length was also found to play a role in determining the ultimate interfacial strength, with short chain molecules similar in length to amino acid side chains promoting the most stable interfaces. The thickness of the hydrophobic band was found to regulate the interface structure. Bands with thicknesses comparable to that of the host lipid bilayer core likely promote a fused interface geometry, similar in structure to that of transmembrane protein-lipid bilayer interfaces. Thicker bands began to transition to a 'T-junction' interface that is characterized by a lower interface stability. Interestingly, the behavior of 10nm bands were indistinguishable from completely hydrophobic probes, reinforcing the importance of nanoscale patterning for stable membrane integration. Chapter 6 builds on the results of the previous chapters by exploring how various stealth probe geometries influence adhesion behavior. In agreement with force clamp testing, short disordered monolayers displayed strong integration into the bilayer core, while crystalline monolayers displayed extremely weak integration. Preliminary adhesion testing results with human red blood cells demonstrate that the stealth probe geometry holds promise for in vitro and in vivo platforms, expanding the results of this work from simply a biophysical test system to a real world example. Finally, the behavior of two hydrophobic bands either commensurately spaced with the hydrophobic core spacing in the bilayer stack, or incommensurately spaced in order to force one band to reside in the hydrophilic hydration layer, is explored. It was found that the commensurately spaced bands display superior strength to single band tips, which is attributed to the necessity to simultaneously rupture both membrane-hydrophobic band interfaces. Conversely, the incommensurately spaced probes display a significant destabilization of the interface. This is thought to be due to the forced residence of one hydrophobic band in a hydrophilic hydration layer. This result is intriguing for biologic delivery systems, as the nuclear double membrane presents a unique barrier geometry, and a double band system may provide a facile means for penetration.
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Online 6. A Microfabricated magnetic sifter and high throughput physical fabrication of magnetic nanoparticles for applications in protein and cell separation [2010]
- Earhart, Christopher Michael.
- Aug. 2010.
- Description
- Book — online resource (xxiv, 170 pages) : illustrations (some color)
- Summary
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Nanoscience and nanotechnology have been applied in recent years to cancer research, with the goal of bringing about a revolutionary change in the ways in which cancer is diagnosed and treated. Magnetic nanotechnologies, in particular, have shown significant potential in several areas, such as imaging, therapeutics, early detection, and point-of-care therapy monitoring. Most applications of magnetic nanotechnology in cancer research involve the use of nanoscale magnetic carriers. Depending on the properties of the particular carrier and the manner in which they are employed, magnetic carriers can act as MRI contrast enhancing agents, drug delivery vehicles, bio-labels for detection with magnetic biosensors, or selection agents in immunomagnetic separation platforms. The small sizes (10-100 nm), functional surface chemistries, and controllable magnetic responses of nanoscale magnetic carriers allows for interaction with and control of biological entities in both novel and powerful ways. The focus of this thesis is on a specific application of magnetic carriers -- magnetic separation of biomolecules and rare cells. After an introduction and an overview of magnetic separation principles are given in Chapters 1 and 2, Chapters 3-6 discuss the design, fabrication, and use of a novel magnetic separation device, the magnetic sifter. The magnetic sifter is a microfabricated, 7 x 7 mm planar die containing a dense array of pores (~200-5000/mm^2) in a magnetically soft membrane. When magnetized by an external field, the sifter pores generate large magnetic field gradients (~10^6 T/m) near the pore edges, which can capture nanoscale magnetic carriers with high efficiency and throughput. The gradients can be turned off by removing the external field, and the magnetic carriers can be released. The magnetic sifter is a microfluidic device, in the sense that it contains microfabricated, micron-scale pores which generate large field gradients. It is also a macrofluidic device, in that high volume throughput is achieved by parallel flow through the dense array of pores. Magnetic modeling of the magnetic sifter and a method for its fabrication are presented in Chapters 3 and 4. When paired with magnetic carriers functionalized with recognition moieties, the magnetic sifter can be used in both protein and cell enrichment schemes. The focus of Chapter 5 is on using the magnetic sifter to capture individual magnetic nanoparticles. The intended application is for enrichment of cancer protein markers prior to detection with a magnetic spin-valve biosensor. High capture efficiencies (80-100%) of magnetic nanoparticles have been achieved for a single pass through the magnetic sifter. Magnetic nanoparticles can also be released from the magnetic sifter with nearly 100% efficiency. The focus of Chapter 6 is the use of the magnetic sifter for rare cell enrichment, for applications in enumeration and enrichment of circulating tumor cells. It is shown that the magnetic sifter can capture tumor cells from whole blood with high efficiency and throughput (~60% at 5 ml/hr). Furthermore, with its planar structure and presentation of captured cells, the magnetic sifter doubles as a cell imaging platform, allowing for identification and quantification with optical microscopy. In addition to its high capture efficiency and gentle release of captured cells, the small size and scalable fabrication of the magnetic sifter are attractive for applications in point-of-care cancer diagnostics for early detection and monitoring of metastatic disease. In Chapter 7, a method for high-throughput fabrication of novel magnetic carriers, synthetic antiferromagnetic (SAF) nanoparticles is presented. The method has enabled production of large quantities of SAF nanoparticles, which have desirable properties for applications in magnetic separation. The capture and release behavior of SAF nanoparticles with the magnetic sifter has been demonstrated. High capture efficiencies are achieved at flow rates 10-20x higher than what was previously possible with commercially available magnetic carriers. Both the magnetic sifter and physically fabricated SAF nanoparticles offer unique advantages over traditional technologies. They are each, in their own right, promising new technologies for applications in cancer detection and therapy monitoring.
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Online 7. Biomechanics of human stratum corneum : dry skin conditions, tissue damage and alleviation [2010]
- Levi, Kemal.
- Nov. 2009, c2010.
- Description
- Book — online resource (xviii, 155 pages) : illustrations (some color)
- Summary
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The outermost layer of human skin, the stratum corneum (SC), is subject daily to variable ambient moisture and temperature conditions as well as application of potentially damaging cleansing agents. The inevitable results of these exposures are "tightness" of the skin which is directly related to the buildup of tensile residual drying stresses in the SC layer. In this work, we first describe the application of the substrate curvature technique to quantitatively measure the magnitude of these stresses and their relationship to selected drying environments and times. The SC drying stresses were observed to be very sensitive to the relative humidity and temperature of the drying environment as well as harshness of the chemical treatment. There was a strong correlation with the SC drying stresses and the chemical potential of water in the drying environment. The evolution of drying stresses in SC is discussed in relation to the effects of hydration and damage caused by chemical treatments on the underlying SC structure. We also describe the application of the substrate curvature technique to characterize stresses in occlusive topical coatings. We then extend the substrate curvature technique to measure the combined effects of the coating applied to human stratum corneum (SC) where the overall drying stresses may have contributions from the coating, the SC and the interaction of the coating with the SC. We show how these separate contributions in the coating and SC layers can be differentiated. Using this methodology, we characterize the effect of a range of moisturizing treatments on the drying stresses in human stratum corneum. Following moisturizer treatment, the SC was observed to have distinctive stress profiles with drying time depending on the effectiveness of the treatment. The stress values of specimens treated with the humectant moisturizers were observed to increase and stabilize after a few hours in the drying environment where they remained relatively constant until the end of exposure to the drying environment whereas the stress values of specimens treated with the emollient treatments were observed to rise rapidly to a peak stress value and relax to a final stress value. The effect of moisturizing treatments on the SC drying stresses was rationalized in terms of SC water loss and the chemical state of the SC components. Finally, we employ a fracture mechanics approach to understand the implications of the drying stresses in SC as a mechanical driving force for damage propagation (e.g. cracking and chapping) in the tissue. The crack driving force G was found for several cracking configurations and compared with the intercellular delamination energy, Gc, which is a property of the tissue that provides a measure of the resistance to cracking. Using this approach, we demonstrate how damaging treatments enhance and moisturizing treatments alleviate the propensity for dry skin damage.
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