Papers

AVS2004 Session BI+MN-FrM: Bio-MEMS and Microfluidics

Friday, November 19, 2004 10:00 AM in Room 210D

Friday Morning

Start	Invited?	Item
10:00 AM		BI+MN-FrM-6 Creating Protein and Vesicle Arrays Using Designated Surface Chemistry in Combination with a Novel Microfluidic Pattering Device B. Niederberger, M. Dusseiller, D. Falconnet, B. Städler, G.L. Zhen, F. Rossetti, J. Vörös (ETH Zurich, Switzerland) Protein microarrays play a key-role in drug discovery, drug development and diagnostics by providing a highly sensitive, parallel analysis of the proteome of complex samples. The methods (e.g. spotting) that are currently available for the creation of DNA microarrays can not be directly used for proteins because they are subject to the loss of function upon contact with an ambient environment. In this work, we present a novel way to create arrays of different proteins or vesicles using a microfluidic device. The concept relies on a designated surface chemistry, which allows activation for subsequent binding events, in combination with crossing microfluidic channels for the local functionalization by separated laminar streams. Besides its simplicity and cost efficiency, this concept has the major advantage that it keeps the proteins in a hydrated environment throughout the experiment. The working principle of this arrayer is to activate spots by an activation stream and to do subsequent functionalization by reagent streams flowing perpendicular to the first stream. The surface pattern was provided by a MAPL-chip (Molecular Assembly Patterning by Lift-off) which consists of well defined areas (i.e. spots) of biotin or NTA functionalized PLL-g-PEG surrounded by a resistant surface of unfunctionalized PLL-g-PEG. The PDMS flow cell was fabricated by soft lithography and sealed to the sample surface by pressure. The position and the width of the streams containing the analytes could be adjusted using different flow rates in the microchannels. Fluorescent microscopy was used to monitor in situ the creation of a microarray consisting of alternating spots of streptavidin labeled with two different fluorophores. The concept was further extended to create heterogeneous arrays of his-tagged proteins and vesicles. This novel technique enables the creation of protein (including membrane-protein) microarrays in normal research labs in a simple and cost efficient way.
10:20 AM		BI+MN-FrM-7 Polymeric Materials for DNA Sensing and Integration into Microfluidic Channels R.A. Zangmeister, M.J. Tarlov (National Institute of Standards and Technology) Advances in microchip technology coupled with innovative bioassays are advancing the field of biosensing in microfluidics. We have previously reported a method for immobilizing single-stranded DNA (ss-DNA) probe molecules in polyacrylamide hydrogels within plastic microfluidic channels, creating a sensing matrix for target oligos. Spatially defined plugs are formed by photopolymerization of a solution containing 19:1 polyacrylamide/bisacrylamide and ss-DNA modified at the 5' end with an acrylic acid group. Low concentrations of ss-DNA targets can be electrophoresed into the hydrogels where complementary strands are captured by hybridization and are detected. We are interested in identifying and characterizing other polymeric materials that can be used as DNA sensing matrices for use in microchannel devices. Our goal is to identify polymeric materials that can be patterned within a microchannel, either by photochemical or electrochemical means, and that possess surface chemical groups that can be used to chemically graft probe oligos, or potentially other biological probe molecules, onto the surface. One such candidate that we are currently investigating is poly(3-aminophenol). Our strategy is to pattern a poly(3-aminophenol) thin film, modify it with probe oligos, and demonstrate a hybridization based DNA assay on that surface for use in a microfluidic format. We are able to selectively deposit poly(3-aminophenol) thin films onto gold electrodes under potential control. Surface pendant amine groups, as evidenced in infrared studies, allow for linkage of probe oligos to the polymer surface. Polymer deposition conditions, characterization, modification with probe oligos, and success of target hybridization detection will be discussed.
10:40 AM		BI+MN-FrM-8 Cell Biology On a Chip: Elastomeric Microfluidic Platforms for Cell Culture Applications A. Folch (University of Washington) The ability to culture cells in vitro has revolutionized hypothesis testing in basic cell and molecular biology research and has become a standard methodology in drug screening and toxicology assays. However, the traditional cell culture methodology - consisting essentially of the immersion of a large population of cells in a homogeneous fluid medium - has become increasingly limiting, both from a fundamental point of view (cells in vivo are surrounded by complex spatiotemporal microenvironments) and from a practical perspective (scaling up the number of fluid handling steps and cell manipulations for high-throughput studies in vitro is prohibitively expensive). The recent advances by our laboratory to address both limitations will be presented, including a microfluidic long-term cell culture platform that features cellular micropatterns and focal delivery of soluble factors to single cells. We are also developing elastomeric sensors and actuators for single-cell probing and manipulation by inexperienced users. These inexpensive technologies allow us to test novel hypotheses concerning neuromuscular development, chemotaxis, and neuronal axon guidance.
11:20 AM		BI+MN-FrM-10 Ultrasensitive MALDI MS Analysis of Peptides Separated in an RF Plasma Polymer Modified Microfluidic Device G.R. Kinsel, X. Li (University of Texas at Arlington) Rapid, information rich analysis of complex biological samples, such as the proteome of a given cellular system, represents a significant challenge for modern bioanalytical devices. A prototype open-channel microfluidic device under development in laboratory integrates an array of technologies available and/or developed in our laboratory to achieve efficient separation and ultrasensitive detection the components of peptide/protein mixtures. Specifically, separation of peptide mixtures is achieved through electroosmotic flow of the sample through 100 micron open-channels imprinted into a PMMA wafer. Modulation of the separation characteristics is achieved by either using the channels as formed or following coating of the channels by pulsed RF plasma polymerization of thin films having various chemical properties. Changes in peptide retention characteristics have been observed to correlate with changes in the column coating chemistry. Separation of simple mixtures can be achieved in minutes using this device. Following separation of the peptides, MALDI mass spectra of the isolated compounds is achieved by rastering the desorption / ionization laser down the open channel. This approach clearly allows the unambiguous assignment of the peptide molecular weight. In addition, because of the confinement of the sample to extremely small volumes, and the consequent high surface concentrations, extremely low limits of detection have been obtained for the separated peptides e.g. an LOD of 1.6 attomole of the peptide casomorphin has been observed. The coupling of this microfluidic device with MALDI mass spectrometry clearly holds enormous promise for substantially lowering the limits of detection and the requisite analysis time, while providing maximum information content for components in complex peptide / protein mixtures.
11:40 AM		BI+MN-FrM-11 Measurement and Analysis of Changes in EOF with Protein Adsorption using the Dynamic Current Monitoring Method. K. Lenghaus (Clemson University); M.J. Tarlov, L. Locascio (NIST); J. Jenkins, S. Sundaram, S. Krishnamoorthy (CFD Research Corporation); J.J. Hickman (Clemson University) The high surface to area ratio of MEMS devices places certain constraints upon their operation. One of these is that conventional, pressure driven flow is a relatively inefficient means of moving liquids through microfluidic channels, owing to the large backpressure encountered. The parabolic flow profile of pressure driven flow can also be undesirable in certain applications, especially in regards to sample separation and delivering analytes to detectors. Electro-osmotic flow (EOF), providing that conditions are conducive to its operation, can thus be a preferable option, since it doesnâ?Tt have the same problems with high backpressures, and its top hat flow profile, as shown by capillary electrophoresis, is well suited to separations and analysis. However EOF is sensitive to the type and density of electrical charges at the wall, and the adsorption of molecules or biomolecular species can substantially alter the EOF characteristics of the system. Using the dynamic current monitoring method, the change in EOF with protein exposure was tracked on the timescale of minutes, and the effect of changing the driving voltage, buffer composition, capillary surface and other parameters was obtained. Building on our previous protein adsorption work, we show that under some circumstances changes in EOF with exposure to different proteins can be extremely rapid. Whether or not desorption and recovery of the original EOF characteristics occurs depends on the specific protein/surface combination, as does the final EOF reached. The rates of adsorption and desorption were also determined using finite element analysis methods, compared with those obtained under pressure driven flow conditions, and a hypothesis of the method of interaction has been postulated.