Two-Dimensional and High-Throughput Electrophoretic Separation of Proteins Using Polymeric Microchips

A major task in proteomics is to identify proteins from a biological sample using two-dimensional (2-D) separation prior to mass spectrometry of peptides generated via proteolytic digestion of the proteins. For 2-D separations, microfluidic devices are superior to bench top and capillary-based systems since they potentially provide higher separation efficiencies due to the minimal dead volumes produced during peak transfer between the two separation dimensions. In addition, fast separations can be envisioned because the column lengths are typically shorter in microfluidic platforms without scarifying peak capacity. High-throughput capabilities are extremely desirable for many types of bio-analytical analyses, such as understanding molecular interactions and the role they play in cellular functioning and drug discovery. Polymeric microchips possess a variety of physiochemical properties to match the intended application and their ease of fabrication increases the accessibility of technology to a large research base. In this dissertation, a comprehensive 2-D separation platform for proteins using a polymeric microchip with the ability to perform high performance separations within a few minutes was established. The system combined sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS µ-CGE) with micellar electrokinetic chromatography (MEKC) in a poly(methyl methacrylate), PMMA, microchip and was reported with a programmed pulse injection/separation protocol with laser-induced fluorescence for detection. A novel sixteen-channel polycarbonate (PC) microfluidic device for high-throughput separations of proteins was also presented using a process to pattern gold features as microelectrode array for sixteen parallel channels on microchips. The system was able to simultaneously analyze sixteen different samples in parallel consisting of native proteins, amino acids, peptides, and oligonucleotides with conductivity. Finally, due to the diverse nature of polymer properties and the large number of potential applications for microfluidic chips, the physiochemical properties of various polymers were investigated to guide researchers in selecting the best material for a given application including protein analysis

In situ Roughening of Polymeric Microstructures

A method to perform in-situ roughening of arrays of microstructures weakly adherent to an underlying substrate was presented. SU8, 1002F, and polydimethylsiloxane (PDMS) microstructures were roughened by polishing with a particle slurry. The roughness and the percentage of dislodged or damaged microstructures was evaluated as a function of the roughening time for both SU8 and 1002F structures. A maximal RMS roughness of 7-18 nm for the surfaces was obtained within 15 to 30 s of polishing with the slurry. This represented a 4-9 fold increase in surface roughness relative to that of the native surface. Less than 0.8% of the microstructures on the array were removed or damage after 5 min of polishing. Native and roughened arrays were assessed for their ability to support fibronectin adhesion and cell attachment and growth. The quantity of adherent fibronectin was increased on roughened arrays by two-fold over that on native arrays. Cell adhesion to the roughened surfaces was also increased compared to native surfaces. Surface roughening with the particle slurry also improved the ability to stamp molecules onto the substrate during microcontact printing. Roughening both the PDMS stamp and substrate resulted in up to a 20-fold improvement in the transfer of BSA-Alexa Fluor 647 from the stamp to the substrate. Thus roughening of micron-scale surfaces with a particle slurry increased the adhesion of biomolecules as well as cells to microstructures with little to no damage to large scale arrays of the structures

Enrichment and expansion of cells using antibody-coated micropallet arrays

Positive selection, sorting, and collection of single cells from within a heterogeneous population are required for many biological studies. We recently demonstrated a miniaturized cell array for this purpose; however, on-chip pre-enrichment and isolation of specific target cells would provide significant value for cell isolation

Sorting and expansion of murine embryonic stem cell colonies using micropallet arrays

Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronection coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4 fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties

Micropallet arrays with poly(ethylene glycol) walls

Arrays of releasable micropallets with surrounding walls of poly(ethylene glycol) (PEG) were fabricated for the patterning and sorting of adherent cells. PEG walls were fabricated between the SU-8 pallets using a simple, mask-free strategy. By utilizing the difference in UV-transmittance of glass and SU-8, PEG monomer was selectively photopolymerized in the space surrounding the pallets. Since the PEG walls are composed of a cross-linked structure, the stability of the walls is independent of the pallet array geometry and the properties of the overlying solution. Even though surrounded with PEG walls, the individual pallets were detached from the array by the mechanical force generated by a focused laser pulse, with a release threshold of 6 μJ. Since the PEG hydrogels are repellent to protein adsorption and cell attachment, the walls localized cell growth to the pallet top surface. Cells grown in the microwells formed by the PEG walls were released by detaching the underlying pallet. The released cells/pallets were collected, cultured and clonally expanded. The micropallet arrays with PEG walls provide a platform for performing single cell analysis and sorting on chip

Reaction of aromatic carboxylic acids with isocyanates using ionic liquids as novel and efficient media

Ionic liquids (ILs) based on 1,3-dialkylimidazolium and ammonium have been used as an efficient reaction media in the amidation of several carboxylic acids with isocyanates. The method has wide applicability, and the protocol is mild and efficient compared to the existing methods based on conventional solvents. Proper 'design' of the ILs allows us to obtain amides in good to excellent yields. In this reaction, both aromatic and aliphatic isocyanates could be used

Coupled Fluid-Wall Modelling Of Steady Flow In Stenotic Carotid Arteries

Arterial stenoses may cause critical blood flow and wall conditions leading to clinical complications. In this paper computational models of stenotic carotid arteries are proposed and the vessel wall collapse phenomenon is studied. The models are based on fluid-structure interactions (FSI) between blood and the arterial walls. Coupled finite element and computational fluid dynamics methods are used to simultaneously solve for stress and displacement in the solid, and for pressure, velocity and shear stress in the fluid domain. Results show high wall shear stress at the stenosis throat and low (negative) values accompanied by disturbed flow patterns downstream of the stenosis. The wall circumferential stress varies abruptly from tensile to compressive along the stenosis with high stress concentration on the plaque shoulders showing regions of possible plaque rupture. Wall compression and collapse are observed for severe cases. Post-stenotic collapse of the arterial wall occurs for stenotic severity as low as 50%, with the assumption that a given amount of blood flow needs to pass the stenotic artery; whereas if constant pressure drop should be maintained across a constriction, then collapse happens at severity of 75% and above. The former assumption is based on the requirement of adequate blood supply to the downstream organs/tissue, while the latter stems from the fact that the pumping mechanism of the body has a limited capacity in regulating blood pressure, in case a stenosis appears in the vasculature. © 2009 Informa UK Ltd All rights reserved