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Symposium P: Materials and Strategies for Lab-on-a-Chip--Biological Analysis, Microfactories, and Fluidic Assembly of Nanostructures

April 10 - 13, 2007

Chairs

Sonia Grego RTI International 3040 Cornwallis Rd. Research Triangle Park, NC 27709-2194 919-248-4181		Orlin Velev Dept. of Chemical and Biomolecular Engineering North Carolina State University COE 1 Raleigh, NC 27695-7905 919-513-4318
J. M. Ramsey Dept. of Chemistry University of North Carolina-Chapel Hill Kenan Labs B030 Chapel Hill, NC 27599-3290 919-962-7492		Sabeth Verpoorte Institute for Drug Exploration University of Groningen P.O. Box 196 Groningen, 9700 AD The Netherlands 31-50-363-3337

Symposium Support
American Institute of Physics
Interdisciplinary Network of Emerging Science & Technologies
NIH, National Institute of Biomedical Imaging & Bioengineering
Procter & Gamble


Symposium P presentations will appear in the "Proceedings Library"
of the MRS Website
(www.mrs.org/publications_library)

---

* Invited paper

SESSION P1: Device Surface Modification
Chair: Carlos Martinez
Tuesday Morning, April 10, 2007
Room 2005 (Moscone West)
8:30 AM *P1.1
Preventing Nonspecific Adsorption in Plastic Microfluidic Devices Using Photoinitiated Grafting Frantisek Svec¹ and Timothy B. Stachowiak²; ¹The Molecular Foundry, LBNL, Berkeley, California; ²Chemical Engineering, University of California, Berkeley, California.

Microanalytical systems require that chemical functionality be accurately patterned and positioned within a microfluidic device. Numerous techniques exist for introducing or attaching chemical functionality into such devices, but preventing unwanted nonspecific adsorption of chemical or biological species often poses a difficult challenge. We fabricate microfluidic chips from cyclic olefin copolymer. Since this plastic is highly hydrophobic, it tends to adsorb a variety of compounds including proteins and peptides. Therefore, we first modify all surfaces that can come in contact with these compounds using UV initiated photografting of neutral, hydrophilic poly(ethylene glycol) methacrylate (PEGMA). This modification significantly decreased contact angle for water and minimizes the adsorption of proteins within the chip. We also prepared porous polymer monoliths inside the channel. These materials are useful for creating a variety of analytical components needed in an integrated system such as preconcentrators, chromatographic stationary phases, and immobilized enzymatic microreactors. Since the monoliths can also exhibit nonspecific adsorption in regions where surface interactions should be avoided, such as valves, filters, or supports for immobilized enzymatic reactors, we control the surface polarity of the monoliths via a patternable photografting process in order to prepare again hydrophilic surfaces that prevent protein adsorption. Photografting is a powerful technique for in situ surface modification of polymers and allows multiple different chemistries to be introduced into a single device with the use of appropriate photomasks. Here we utilize both single-step and sequential photografting techniques to graft patterns of neutral, hydrophilic monomers such as PEGMA, acrylamide, 2-hydroxyethyl methacrylate, and vinyl pyrrolidinone. Using these photografting processes, adsorption of model proteins, such as bovine serum albumin, can be reduced by two orders of magnitude compared with ungrafted regions. The ability to prepare patterned hydrophilic regions on polymer monoliths is useful for creating multifunctional devices for many chemical and biological applications.


9:00 AM *P1.2
Tailoring Topography and Chemistry of Surfaces for Detection and Separation. Jan Genzer, Chemical & Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina.

We will discuss several strategies aiming at physical and chemical modification of surfaces. Specifically, we will introduce a simple method leading to the formation of corrugated silicone elastomer surfaces made of hierarchically-buckled topographical features comprising self-similar wrinkles of multiple wavelengths and amplitudes; buckles with smaller wavelengths (and amplitudes) rest parallel to and within larger buckles, forming a nested structure. We will demonstrate how these hierarchically-buckled surfaces can be utilized for separation of objects based on their size. In addition, we will discuss several recent developments in the area of functionalized surface-anchored surfaces. We will show how one prepare functional surface comprising thermoresponsible polymers and used them for detection of small particles and proteins. Furthermore, we will discuss methodologies leading to the formation of surface-anchored random copolymers with adjustable co-monomer sequences (so-called, random-blocky copolymers, RBCs). For the latter systems, we will present recent results from forming RBCs from poly(styrene-co-4-bromostyrene) and functionalized poly(dimethylaminoethyl methacrylate) with anchored functional moieties.


9:30 AM P1.3
Surface Engineering in Microfluidic Devices for the Isolation of Smooth Muscle Cells and Endothelial Cells. Shashi Murthy¹, Brian Plouffe¹ and Milica Radisic^2,3; ¹Dept of Chemical Engineering, Northeastern University, Boston, Massachusetts; ²Institute of Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; ³Dept of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.

Microfluidic cell separation systems have emerged as attractive alternatives to traditional techniques in recent years. These systems offer the advantages of being able to handle small sample volumes and at the same time achieve highly selective separation. Conventional separation techniques, including both fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), typically require a pre-processing incubation step to attach ligated tags (such as fluorescent dyes or magnetic beads) to cell surfaces prior to separation. These techniques are also constrained by infrastructure and high cost. Microfluidic devices with surface-immobilized adhesion molecules eliminate the need for pre-processing incubation and are a low cost alternative. We describe the selective adhesion of smooth muscle cells and endothelial cells in microfluidic devices coated with adhesion peptides. The device geometry is such that the shear stress varies linearly as a function of flow channel length, allowing simultaneous evaluation of the effects of surface chemistry and fluid shear on cell adhesion. The adhesion peptides, val-ala-pro-gly (VAPG) and arg-glu-asp-val (REDV), are known to bind selectively to smooth muscle cells and endothelial cells, respectively. These peptides were tethered to the device surface using silane chemistry and NHS-ester coupling. Cell adhesion was examined in a shear stress range of 1.3-4.0 dyn/cm². Under these conditions, endothelial cells show significantly higher adhesion to REDV-coated devices compared to smooth muscle cells and fibroblasts. Correspondingly, smooth muscle cell adhesion in VAPG-coated devices is much greater than that of endothelial cells and fibroblasts. This selective binding behavior is also observed when mixed suspensions of the three cell types are flowed into both types of peptide-coated microfluidic devices. These results suggest that microfluidic devices coated with REDV and VAPG can be used as effective separation tools in various applications, such as tissue engineering. Specific examples of applications in cardiac and skin tissue engineering will be discussed.


9:45 AM P1.4
Electrochemical Biolithography for Micropatterning Proteins and Cells within Three-Dimensional Microstructures. Matsuhiko Nishizawa, Hirokazu Kaji, Masahiko Hashimoto, Takeaki Kawashima, Soichiro Sekine and Takashi Abe; Tohoku Univ., Sendai, Japan.

Microfluidic systems have been widely investigated for biological applications and hold great promise in the development of diagnostic assays and bioreactors. The combination of the surface patterning techniques with the microfluidic systems paves the way to multi-functional, high-throughput, and cost-effective analysis, in which the “real-time” and “on-demand” micropatterning of delicate bioelements is strongly required. However, most of the photolithography-based techniques are unable to be applied within the sealed microchannels. We would like to report a novel technique “electrochemical bio-lithography”, which enables the localized immobilization of proteins and cells within 3D microstructures such as microfluidic channels. The principle of the technique is based on our finding that the albumin- or heparin-coated surfaces, initially anti-biofouling, rapidly becomes protein- and cell-adhesive upon exposure to the reactive oxidizing agent such as hypobromous acid, which can be produced by the electrochemical oxidation of bromide ion in a biological buffer solution. Since this lithography can be conducted under typical physiological conditions, it enables the spatiotemporal control of cell adhesion and growth on substrates; it facilities the stepwise immobilization of multitype protein arrays and multiphenotype cell arrays. And importantly, this technique is simple enough to be integrated into the miniaturized and semi-closed systems such as microfluidic devices, indicating the possible “on-demand” immobilization of proteins and living cells just prior to use of the microfluidic biodevices.


10:30 AM *P1.5
Curable Perfluoropolyethers for Microfluidic Devices and as Enabling Materials for Molding and Harvesting Complex Nano-objects Joseph DeSimone, Junhoe Cha, Jason Rolland, Zhaokang Hu and Michael Ramsey; University of North Carolina, Chapel Hill, Chapel Hill, North Carolina.

Photocurable perfluoropolyethers (PFPEs) have been developed for use as novel elastomeric, chemically resistant microfluidic devices and as high performance molding materials for us in imprint lithographic applications. The PFPE-based materials employed herein have Young's modulus values that range from 1.5 to 90 MPa and are tunable throughout that range based on changes in the molecular weight between crosslinks. With photochemically and thermally curable PFPE materials in hand, PFPE-based microfluidic devices could easily be fabricated by soft lithography using an SU-8 master. Since the absorption or partitioning of analytes into bulk PDMS have been reported, small molecule partitioning behavior for PFPE- and PDMS-based devices was investigated. PDMS chips showed a strong fluorescence signal due to Nile Red and Rhodamine B base partitioning into it, while no partitioning was observed in PFPE-based devices as indicated by the lack of fluorescence. An electrokinetic property of PFPE was investigated by measuring electroosmotic mobility (EOM). To demonstrate electrokinetic control of solutions in these microchips, pinch flow and injection of Lissamine Rhodamine B sulfonyl chloride and Oregon green 488 carboxylic acid was successfully performed in PFPE chips. Due to the excellent resistance of small molecule partition and chemical and electrokinetic properties, the PFPE material has the potential to expand the field of microfluidics to many novel applications such as chemically-intensive lab-on-a-chip devices. The PFPE-based materials make for excellent imprint lithography molding materials. We are adapting and merging the precision, uniformity and mass production concepts associated with imprint lithography to generate and solution harvest extremely versatile organic carriers having specific chemical functionality and tailored mechanical properties for application in nano-medicine. We have been able to fabricate and harvest using PFPE-based molding materials monodisperse, shape-specific nano-particles that can be made from any organic matrix material (e.g. PEG, polylactide, cationic hydrogels, degradable di-sulfide linked materials), containing any cargo (e.g. therapeutics, contrast agents, linker groups), and conjugated with any ligand (e.g. integrin receptor peptide, melanocyte stimulating hormone, vasoactive intestinal peptide, anti-Her2 mouse antibodies, cell-penetrating peptides, and a variety of vitamins). This paper will also discuss our recently initiated in vitro and in vivo studies with these harvested particles for the detection, imaging and treatment of various diseases.


11:00 AM P1.6
Biofunctionalizing Nitride Surfaces without Silanes. Rory Stine, Kendra M. McCoy, Shawn P. Mulvaney and Lloyd J. Whitman; U. S. Naval Research Lab, Washington, District of Columbia.

Silicon nitride is widely used as a coating in the microelectronics industry because of its ability to resist penetration by contaminants such as water, oxygen, and ionic species. This property also makes silicon nitride a common terminal passivation layer for chip-based biosensors and bioMEMS devices, all of which come into contact with aqueous saline solutions. Current methods for biofunctionalizing silicon nitride rely almost exclusively on silane-based films, both for direct functionalization and as bifunctional linkers. However, even under stringent controls, the chemistry of silane films on silicon nitride surfaces is notoriously inconsistent and suffers from degradation over time when used in aqueous environments. We have developed an alternate, silane-free, method for functionalizing silicon nitride surfaces. The native oxide is first stripped via HF solution, and then treated with a plasma that makes the surface reactive to aldehydes. Using a bifunctional aldehyde coupler, we then adsorb a robust NeutrAvidin layer that can be used to immobilize any biotinylated biomolecule and has excellent nonfouling properties. We will describe the surface chemistry and compare our approach with silane-based methods as analyzed by XPS and radiolabeling experiments. We will also show that this chemistry can be successfully applied to GaN surfaces, and used for immunoassays and DNA hybridization assays in a range of sample matrices on both surfaces.


11:15 AM P1.7
Micro-channel Patterning for Preparing a Self-referencing Surface for the Detection of Cancer Antigens using Surface Plasmon Resonance Biosensor. Fengyu Su, Chunye Xu and Minoru Taya; Mechanical Engineering, University of Washington, Seattle, Washington.

Accurate referencing is very important in surface plasmon resonance (SPR) biosensor, since the SPR signal shift could be induced not only by specific bindings but also non-specific bindings and environmental changes. In this work, a self-referencing method is used to detect cancer antigens, by which the bulk effect could be eliminated efficiently because the environmental changes are almost identical for the sensing and referencing areas in the same channel when the analyte solution pass them at the same time. Striped pattern of sensing and referencing surfaces are prepared by using micro-channel patterning method. A micro-flow cell of poly(dimethylsiloxane) (PDMS) containing a group of 200 micrometer channels is placed onto the Au sensor chip. The materials flowing through the microchannels are adsorbed onto the surface via physical adsorption or chemical reaction. Through multi-step functionalization, sensing materials of anti-CEA antibodies and referencing materials of anti-FITC antibodies are immobilized on gold surface in a striped pattern. For detecting test, a PDMS flow cell with channels of 1000 micrometer wide is placed onto the patterned sensor chip. When the analyte solution of CEA antigen passes through the patterned surface, the signal changes on both anti-CEA antibodies and referencing surfaces are recorded by SPR simultaneously. By eliminating the SPR angle shift on referencing surface from sensing surface, accurate binding events between antibodies and antigens are obtained. Keywords: micro-channel patterning, surface plasmon resonance, self-referencing, cancer detection, biosensor.


11:30 AM P1.8
Merging Photoresist Lithography and Protein Microarraying to Design Depatocellular Microenvironment. Ji Youn Lee¹, Sunny Shah¹, Gang-Yu Liu² and Alexander Revzin¹; ¹Department of Biomedical Engineering, University of California, Davis, California; ²Deparatment of Chemistry, University of California, Davis, California.

Development of hepatocyte transplantation strategies or the artificial liver-assist devices requires an unlimited supply of functional hepatocytes. Therefore, efforts are underway to derive differentiated adult hepatocytes from stem cells or fetal hepatocytes. However, the mechanisms to direct those cells along a hepatocyte lineage and the optimized culture conditions are still unknown. Our research focuses on developing controllable cellular micropatterns to orchestrate cellular interactions with the surrounding environment in a precise and combinatorial fashion in order to expedite discovery of inducers of liver-specific differentiation. This presentation will describe a new surface micropatterning approach which combines photoresist lithography and protein microarraying to create arrays of multiple ECM proteins with precisely defined, single cell-level control over cellular contacts. Hepatocytes cultured on these surfaces could be exposed to multiple scenarios of cell-cell and cell-surface interactions in parallel. Functional analysis of cells cultured on micropatterned surfaces was performed by traditional immunostaining, as well as, laser-mediated cell retrieval followed by real-time RT-PCR analysis of liver-specific gene expression. Collection of cells from specific locations on a microfabricated surface allowed to retain local microenvironment context during cell analysis. Methods for creating complex, combinatorial micropatterns and for analyzing local tissue-specific gene expression will be particularly useful for studies aimed at converging on a microenvironment niche necessary for driving progenitor cells or fetal hepatocytes toward mature liver phenotype.


11:45 AM P1.9
Chemical Modifications of Inert Self-Assembled Monolayers with Oxygen Plasma for Biosensor Applications Kun-Lin Yang and Changying Xue; Chemical and Biomolecular Engineering, National University of Singapore, Singapore, Singapore.

We report a new approach to chemically modify inert self-assembled monolayers (SAMs) with oxygen plasma and generated functional groups which permit the immobilization of protein human IgG on the surface. The covalent attachment of IgG to the surface is possibly through the formation of Schiff base between the aldehyde functional groups generated in situ from the oxygen plasma treatment and the amine functional groups of proteins. However, only 1 to 2 s of treatment time is required to modify the surface of the SAMs. Longer treatment time will result in the etching of the SAMs and no protein can be immobilized on the surface. This approach may provide a new means of introducing functional groups to the inert SAMs and avoid the use of bifunctional linkers for protein immobilization.


SESSION P2: Device Fabrication Strategies
Chair: Jan Genzer
Tuesday Afternoon, April 10, 2007
Room 2005 (Moscone West)
1:30 PM *P2.1
Electrohydrodynamic Jet Printing for Digital Microfabrication John Rogers, University of Illinois, Urbana, Illinois.

Electrically driven formation of droplets in conducting fluids combined with electrostatic control of droplet trajectory forms the basis of a method that can be used to print fluids with sub-micron resolution. This talk describes basic aspects of this approach, together with its use in printing a variety of fluids, including suspensions of single walled carbon nanotubes, solutions of conducting polymers, and range of dielectric materials. Simple devices, such as organic transistors and light emitting diodes demonstrate some of the patterning capabilities. Advantages and disadvantages compared to conventional thermal or piezeoelectric ink jet printing for these classes of applications will be described.


2:00 PM P2.2
Carbon Nanofiber Forests as the Volume Exclusion Feature in a Cell Mimic Device. Jason Fowlkes^1,2, Scott Retterer², Ben Fletcher^1,2, Mike Simpson^1,2, Kate Klein^1,2, Anatoli Melechko² and Mitch Doktycz²; ¹The University of Tennessee, Knoxville, Tennessee; ²Oak Ridge National Laboratory, Oak Ridge, Tennessee.

A biological cell mimic device has been fabricated using standard integrated circuit fabrication techniques. Carbon nanofibers (CNFs) are an integral component of the cell mimic structure and have been evaluated as the membrane mimic component of the device. The CNFs have successfully demonstrated size-selective transport, are amenable to chemical functionalization, and affect transport at the molecular scale. These properties are integral to effective membrane performance. This paper further explores the use of CNFs in the cell mimic environment to replicate features of the crowded biological cytosol. Macromolecular crowding in the biological cytosol, on the order of 20-40% volume exclusion, affects both the transport and thermodynamic activity of molecular species. The effective concentration of molecules may exceed the real concentration by several orders of magnitude in the biological cell. CNFs were found to mimic specific characteristics of the crowded biological cytosol. For example, molecular diffusion was reduced to levels observed in prokaryotic cells. The integration of CNFs as the crowding feature in the cell mimic device provides a further step towards realizing functional, cell replicas capable of parallel sensing and actuation.


2:15 PM P2.3
Use of Poly(ethylene glycol) (PEG) Photolithography for Integration of Cells and Microdevices He Zhu, Jun Yan and Alexander Revzin; Biomedical Engineering, University of California, Davis, Davis, California.

Seamless integration of biological and electrical/mechanical components is critical for successful development of BioMEMS. In this presentation, we will describe the use of photopatternable biomaterial, poly (ethylene glycol) (PEG), that can be utilized in a fashion similar to photoresist. In this process, PEG prepolymer solution is spin-coated onto a surface and exposed to UV light through a photomask, resulting in formation of cross-linked PEG hydrogel micro-domains. Because the patterning process is similar to traditional “top-down” photoresist lithography, PEG gel microstructures can be registered with pre-existing microfabricated layers. Two applications utilizing PEG photolithography for biological packaging of microdevices will be highlighted. In one application, gold electrodes, packaged in PEG gel, were selectively modified with avidin followed by the attachment of biotynilated antibodies specific to T-lymphocytes. Presence of the avidin and antibodies on the electrodes was verified by immunofluorescent staining. T-lymphocytes were shown to selectively attach onto the antibody-modified electrodes. In the future, this device may be employed for capture, interrogation and electrochemical release of cells. In the second application, we will demonstrate ability to deposit PEG hydrogel microstructures containing active enzymes onto microfabricated gold electrodes. This application will be valuable for constructing miniature enzyme-based electrochemical biosensors for multi-analyte detection.


2:30 PM P2.4
Tools for Manipulation of a Two-Dimensional Fluid Membrane. Bryan Lawrence Jackson¹ and Jay T. Groves^1,2; ¹Chemistry, University of California - Berkeley, Berkeley, California; ²Physical Bioscience and Materials Science Divisions, Lawrence Berkeley National Laboratory, Berkeley, California.

Hybrid systems consisting of biomolecules coupled to inorganic scaffolds show potential for the design of novel biosensors and research tools. As a two dimensional fluid supported lipid membranes add capabilities for dynamic rearrangement not present at other surfaces, but provide additional challenges for spatial and temporal manipulation. Recently, several soft lithography techniques for concurrently patterning proteins and lipids have emerged. However, these techniques suffer from limited resolution and efficiency. Traditional lithographic techniques relying on reactive surface coatings called resists can be much easier to use, but are not compatible with supported bilayers because bilayer formation is very sensitive to the chemical composition of the underlying silica substrate. Here we utilize thin aluminum films as sacrificial layers that prevent surface fouling during lithography. This technique has proven useful for producing sharp patterns of supported lipid bilayers with additional biomolecules like fibronectin and inorganic electrodes for further device capabilities.


2:45 PM P2.5
Improved Neuronal Adhesion to the Surface of Electronic Device by Engulfment of Protruding Micronails Fabricated on the Chip Surface. Micha E Spira¹, Dotan Kamber¹, Ada Dormann¹, Carmen S. Bartic⁴, Gustaaf Borghs⁴, Shlomo Yitzchaik³, Keren Shabtai³ and Joseph Shappir²; ¹Neurobiology, The Hebrew University of Jerusalem, Jerusalem, Israel; ²Engeneering, The Hebrew University of Jerusalem, Jerusalem, Israel; ³Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel; ⁴MCP/ART, Cell Based Sensors & Circuits, IMEC vzw, Leuven, Netherlands.

Integration of neurons with microelectronic devices has been a subject of intense studies over the last decade. One of the major problems in assembling efficient neuro-electronic hybrids systems is the low electrical coupling between the components. This is mainly due to the fundamental property of living cells to secrete an extracellular matrix forming a cleft between the plasma membrane and any substrate to which they adhere. This cleft shunts the current generated by the neuron, or the device, and thus reduces the signal to noise ratio. Increasing the seal resistance formed between the neurons and the gate surface is thus a fundamental challenge which could improve the coupling coefficient. Here we demonstrate a new approach to improve the physical and electrical coupling between neurons and the surface of electronic chip by harnessing a basic property of cells namely, to internalize particles, such as bacteria, by phagocytosis. To that end we fabricated gold micronails that protrude from the transistor gate surface (see R. Huys this meeting). The protruding micronails are constructed from a one micrometer-long stalk with a diameter of 500 nm, and a sub-micrometer head. The stalk and head were functionalized by peptides that facilitate phagocytosis. We found that both cultured Aplysia neurons and human cardiomyocyte readily engulf the micronails forming tight physical contact between the cells plasma membrane and the surface of the device. Individual cells can engulf several micronails thus forming a very tight physical contact with the transistor surface. Calculations based on the dimensions of the cleft formed in-between the plasma membrane and the micronailed-transistor surface, reveal that the seal resistance formed under this condition, and the ensuing coupling coefficient between the excitable cells and the device are significantly improved. This prediction is now being validated by direct measurements.


3:30 PM *P2.6
Sacrificial Layer and Rapid Prototyping Methods for Creating Microfluidic Devices in Various Materials. Adam Woolley, Chemistry and Biochemistry, Brigham Young University, Provo, Utah.

Lab-on-a-chip systems are seeing increased application in biomolecular analysis. However, materials constraints in the fabrication of high-performance microchips still hinder the broader use of microdevices in bioanalysis. Indeed, microfabrication of materials such as poly(dimethylsiloxane) (PDMS) is fairly easy, but separation performance is often less than ideal; in contrast, glass microchips provide quality separations, but device construction is much more involved and expensive. Our efforts have been directed toward the development of novel approaches for creating microfluidic systems to improve biomolecular analysis. Specifically, we have focused on using hard polymeric materials to form multilayer microchips and to rapidly prototype microfluidic devices; in addition, we have applied thin-film sacrificial layers in constructing microfluidic arrays on inorganic substrates. We have devised a phase-changing sacrificial layer fabrication approach that enables straightforward solvent bonding of polymer microfluidic systems.[1] We are now applying this same method in making multilayer microfluidic structures for simplifying multiplex analyses and facilitating on-chip sample labeling. Recently, we developed a solvent imprinting and bonding approach for the rapid prototyping of microfluidic systems in hard polymer substrates. Total device fabrication times are just several minutes, and quality electrophoretic separations can be achieved in these microchips. Our new methods are as easy to carry out as PDMS molding, but offer better device material characteristics and separation performance. Moreover, we have studied thin-film sacrificial layer techniques for constructing microcapillaries.[2] Our thin-film microfluidic devices offer excellent performance in rapid separations of amino acids and peptides, and provide opportunities for the direct integration of optical and electrical capabilities on-chip. These advances in the fabrication of lab-on-a-chip systems broaden the range of materials available for microchip construction and should enable continued improvements in biomolecular analysis.<p> References<p> [1] Kelly, R.T.; Pan, T.; Woolley, A.T. Phase-Changing Sacrificial Materials for Solvent Bonding of High-Performance Polymeric Capillary Electrophoresis Microchips. Anal. Chem. 77, 3536-3541 (2005).<p> [2] Peeni, B.A.; Lee, M.L.; Hawkins, A.R.; Woolley, A.T. Sacrificial Layer Microfluidic Device Fabrication Methods. Electrophoresis in press (2006).


4:00 PM P2.7
Fluorescence Spectroscopy on a Moving Particle Oliver Schmidt, Michael Bassler, Peter Kiesel and Noble Johnson; Palo Alto Research Center Inc. (PARC), Palo Alto, California.

An optical lab-on-a-chip system is presented that enables fluorescence spectroscopy to be performed on moving analytes. The analytes are continuously excited within a novel optical waveguide structure. Fluorescence spectra are recorded as the analyte traverses the detection area of a chip-size spectrometer that is integrated onto a microfluidic platform. To achieve a strong interaction between the excitation light and analyte we use an anti-resonant waveguide: the light is guided within the target-containing medium, thereby enabling a continuous excitation of a large volume. Guiding the excitation light within the lower-refractive-index fluid is achieved by coupling the light into the waveguide at a specific angle of incidence. A compact spectrometer is integrated along the fluidic channel. The spectrometer consists of a detector array that is coated with a linear variable band-pass filter. The filter converts the spectral fluorescence information into a spatially dependent signal that is analyzed by the detector array. Such chip-size spectrometers are especially applicable for characterizing moving analytes. The operational system will be demonstrated by characterizing polystyrene beads coated with a fluorescent dye. The fluorescence was excited with a 533 nm green laser at a flow speed of 2 mm/s and recorded during flow with a spectral resolution of 5 nm. Experiments are in progress to measure native fluorescence spectra of bacteria in solution with UV excitation light.


4:15 PM P2.8
3D Microhorns for Capillary Driven Microdroplet Transport. Chunguang Xia, Andrew Cox and Nicholas Fang; MechSE, UIUC, Urbana, Illinois.

The rapid advancement of two-dimensional microfluidic systems in recent years now enables a multitude of micro total analysis systems to perform complex sorting and processing at the microscale on disposable lab-on-a-chip devices. It is becoming an inevitable thrust of research and development of the micro and nano total analysis systems in both the consumer and military applications to drive to ever higher levels of integration, requiring the exponential growth of material transport and information processing functions such as sensing and manipulation at the shrinking dimensions. However, current planar manufacturing technologies face the challenge of attaining a high degree of integrated functionalities on the same device, as they offer very limited layers in the device architecture and constrain the interconnect density for heterogeneous integration. In this work, we will pursue the design and microfabrication of 3D microchannels using an innovative parallel micro-freeform technology, the projection microstereolithography[1]. Using a data projector as the dynamic mask, our technology defines high resolution micro-objects by solidifying a light curable solution. By playing a movie that incorporates the cross-sectional patterns and stacking the defined patterns on a small and precise elevator, 3D microstructure are fabricated in a layer-by-layer fashion. In our preliminary works, a set of molecularly imprinted methacrylic acid and poly(ethylene glycol) microstructures are demonstrated with resolution better than 1 micron, sufficient to reproduce the intricacy of the smallest capillaries in the microfluidic networks. This technology provides a unique prototyping method of highly complex 3D microstructures at the table top. In addition, this process offers a variety of functional and biocompatible polymers and hydrogels with tunable surface properties and compliance that can be patterned for the heterogeneous integration of the lab-on-a-chip devices and microfactories. To demonstrate the technology, an array of 3D branching microhorns are designed and fabricated. The conical shape of the capillaries functions as a hydraulic machine: by Pascal’s law, the pressure difference at the smaller and larger meniscus generates a force that drives the wetting droplet towards the narrower end. This phenomenon resembles the movement of fluid in the stems of plant to the leaves. The flow rates of various fluids driven by the capillary force gradient in the conical capillaries will be analyzed. Variations of the horn geometry and their effects on fluid flow rates through the microhorns will be reported. Theoretical analysis of the surface forces involved will be also presented. These branching microhorns show great promise as vital components of fully three dimensional microfluidic systems. [1] Sun C., Fang N., Wu D.M., Zhang X., “Projection micro-stereolithography using digital micro-mirror dynamic mask”, Sensors and Actuators A, 121(2005)113-120.


4:30 PM P2.9
Patterned Conducting Polymers for All-Polymer Cell Electroporation Microsystems. Niels B. Larsen¹, Thomas Steen Hansen^2,1, Keld West¹ and Ole Hassager²; ¹Danish Polymer Centre, Risoe National Laboratory, Roskilde, Denmark; ²Department of Chemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.

Cancer immunotherapy shows increasing potential for assisting in fighting cancer, based on reinjection of dendritic cells “trained” in the laboratory to recognize a patient’s cancer cells. The training proceeds via transient formation of nanopores in the cell membranes induced by a pulsed high electrical field. Highly defined electroporation of single cells has been demonstrated by a number of methodologies during the past decade. However, none of these techniques may be easily up-scaled in a cost-effective manner to handle the large number of cells required for immunotherapy (10-100 million trained dendritic cells). Furthermore, the initial generation of high numbers of dendritic cells, by chemically induced differentiation of the patient’s blood monocytes in the laboratory, is a labor intensive and costly procedure. The differentiation process and subsequent electroporation procedure are therefore well suited as targets for direct integration in a lab-on-a-chip configuration. We have set out to produce such an integrated microsystem based exclusively on commodity polymers for microchannel structures combined with micropatterned conductive polymers (CP) as active field generators and active components of the microfluidic pumping system. Our technology platform is based on new methodologies, to be presented here, for integrating and patterning CP layers into the surface of bulk polymers: Integration occurs via solvent-induced blending of a nanoscale thickness CP layer into a thermoplastic polymer surface [1]. This results in mechanically highly stable surfaces retaining the conductivity of the CP layer. Surprisingly, the procedure works equally well for thermoplastic elastomers which may be strained by more than 50% without irreversible change in conductivity [2]. Patterning of the free-standing or integrated CP occurs in a fast parallel micropatterning (<2 micrometer resolution in seconds to minutes) procedure based on spatially selective transfer of an oxidant from a stamp surface relief to CP areas to be deactivated [3]. We have demonstrated the application of these combined methodologies for the fabrication of an all-polymer electroosmotic microfluidic pumping system, suitable for the slow controlled release of agents inducing monocyte differentiation [1]. Furthermore, we have manufactured interdigitated microelectrode arrays for controlled electroporation of large cell numbers and shown their ability to electroporate and transfect live dendritic cells by messenger-RNA coding for enhanced Green Fluorescent Protein. Results of both demonstrations will be presented. [1] T.S. Hansen, K. West, O. Hassager, N.B. Larsen. Synthetic Metals. In press. [2] T.S. Hansen, K. West, O. Hassager, N.B. Larsen. Advanced Materials. Submitted. [3] .S. Hansen, K. West, O. Hassager, N.B. Larsen. Advanced Functional Materials. Submitted.


SESSION P3: Poster Session
Chair: Sonia Grego
Tuesday Evening, April 10, 2007
8:00 PM
Salon Level (Marriott)
P3.1
Nanoporous Silicon Sensor for Biological Applications Gagik Ayvazyan and Vahe Buniatyan; Semiconductor R&D Center, EAA, Yerevan, Armenia.

Nanoporous silicon is a unique and versatile material that has several features that make it especially attractive for biological sensors, including a very high surface area to volume ratio, simple and inexpensive fabrication techniques, and suitability for integration with silicon electronics. Offered patented architecture and method of making biosensors provide a sensitive way to measure small changes in the in electrical properties (capacitance and conductance) of nanoporous silicon that occur when exposed to organic solvents or when biological molecules attach to the internal surfaces. In particular, use of unilateral electrical contact allows a complete exposure of the surface to the sensing species and reduces the generation of ionic currents through the porous matrix. Pore selective distribution (gradient in pore sizes) in matrix increases sensitivity of biosensors. Based on offered sensor engineering prototype of portable, hand-held diagnostic device is developed. Such sensitive label-free devices can be used by consumers worldwide, for example to detect the presence of specific biological agents (viral DNA, proteins, and potentially bacteria) or organic solvents (ethanol, acetone, benzene). Preliminary testing demonstrated the main advantages of the proposed sensors, including miniaturization, portability, high throughput, high signal to noise ratio, and production of on-site and real-time results.


P3.2
Using Tubular Millifluidics as a Versatile Tool Box for The Generation of New Complex Architectures: Some Integrative Chemistry Synthetic Pathways Wilfrid Engl², Cindy Hany², Pascal Panizza² and Renal Backov¹; ¹CNRS-Universite Bordeaux-I, Pessac, France; ²GMCM, UMR CNRS 6626, CNRS-Université de Rennes, Rennes, France.

There is today a crucial need for new complex hierarchical materials possessing diverse functionalities at different length scales. To design such multiscale architectures accompagned with either organic, inorganic or hybrid compounds in nature are assembled within the new concept of Integrative Chemistry.[1] Herein, beyond the first use of Millifluidic toward shaping complex integrated materials, we demonstrate that "Modular Tubular Millifluidic Synthesis" (MTMS) overcomes the limitations of microfluidics synthesis while maintaining its high potentialities. By assembling together elementary modules and integrating their corresponding functions, modular set-ups can be designed “on demand” to engineer newly advanced materials in characteristic sizes ranging from 50 microns up to a few mm. The great versatility of this method is limited only by the number of combinations possible using the modular tool box and one’s imagination. This is illustrated through the formation of double and triple macro-emulsions, non-spherical particles containing several liquid compartments and the encapsulation of solid objects of various shapes in drops.[2] Beyond, and again for the first time we have combined sol-gel chemistry and milli-fluidic to generate silica ceramic engineering their sizes and aspect ratios. The particle sizes are controlled by varying the flow rates of the continuous and disperse phases within the home-made milli-fluidic reactor. Also, the silica particles aspect ratio can be tune by adjusting the constrained geometry of the milli-fluidic devices, leading to the production of rod-like silica ceramics. Both SAXS and Nitrogen physisorption Its potential use as a production tool for industry is also addressed.[3] This new process combined either with polymeric networks or sol-gel chemistry is appearing thus as new Integrative Chemistry pathways. 1- R. Backov, Soft Matter, 2006, 2, 452. 2- W. Engl, C. Hany, P. Panizza, R. Backov, Adv. Mater., Submitted 3- M. Tochibana, W. Engl, P. Panizza, R. backov, Chemical Engineering and Processing, Submitted.


P3.3
Functionalized Microfluidic Channels And Resistive-Pulse Sensing For Cell-Surface Antigen Detection. Andrea Carbonaro¹, Lydia Sohn¹ and Lucy Godley²; ¹Mechanical Engineering, University of California Berkeley, Berkeley, California; ²Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, Illinois.

The expression of a particular antigen on the cell surface can indicate a pathological condition of the cell. For this reason, the detection of antigens bound to the cell membrane plays a crucial role in disease detection and monitoring. Here, we describe our ability to use resistive-pulse sensing integrated with microfluidics and surface chemistry to sense and separate cells based on their surface antigens. The sensing is done without labeling of the cells, which can subsequently be isolated for further study. In resistive-pulse sensing, a non-conductive particle flowing through a channel blocks the flow of current, thereby leading to a transient increase, or pulse, in the channel’s electrical resistance. Electronic pulses can be characterized in terms of their magnitude and width: the first is strictly related to the size of the particle, and the latter indicates the transit time of the particle as it travels through the channel. In addition, for aspherical particles, such as cells, the shape of a resistive pulse can provide useful information about the shape, orientation, and motion of cells through the channel. Our integrated microfluidic device consists of a 15 μm-wide × 15 μm-high × 800 μm-long channel, which connects two reservoirs, and is embedded in a polydimethylsiloxane (PDMS) slab that is permanently bonded to a glass slide having two sets of lithographically defined Ti/Pt electrodes. The area between the electrodes on the glass substrate is functionalized with antibodies using surface silanization and then derivatization with N-5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), a heterobifunctional cross-linker, which is covalently coupled to the antibodies. We generate a flow by applying a pressure (0.5-2.0 psi) to one of the two reservoirs, and we measure the transit time of cells as the width of the resistive pulses generated by cells as they flow one by one through the channel. When cells flow near a channel wall functionalized with antibodies, the antigen-mediated interaction of the cells to the antibodies functionalized on the wall results in a force, which is function of the antigen-antibody affinity, the antibody and antigen density, and the shear rate of the flow. When there is high affinity between the antigens on the cell surface and the antibodies functionalized on the channel walls, the force of the interaction slows down the cells, leading to longer transit times and correspondingly wider resistive pulses. When the antigen-antibody affinity is low, our data show that the interaction effect is negligible. In addition, the analysis of pulse shape shows a change of cell motion when the channel is functionalized as opposed to the case in which the channel is unfunctionalized. We have used our resistive-pulse technique to develop a number of important assays (e.g. immunophenotyping of leukemia), which we will describe in this talk.


P3.4
Microbioassays Based on Nanoparticle Agglutination and Evaporation Driven Separations inside Droplets on a Chip. Vinayak Rastogi, Suk Tai Chang and Orlin D. Velev; Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina.

We present a new on-chip bioassay technique based on nanoparticle agglutination inside microdroplets floating on the surface of dense fluorinated oil. The floating droplets are captured and transported by alternating electric fields created through addressable arrays of electrodes below the oil. Evaporation on top surface of droplet leads to rapid collection of the particles suspended in the droplets in their top region. Experimental results and theoretical simulations show that this microseparation is a result of series of processes driven by mass and heat transfer during the evaporation. The assays based on agglutination of antibody-conjugated particles in the presence of analyte are read out by the pattern of particle collection on the droplet using the evaporation-driven microseparation. These droplet-based microbioassays require samples of nanoliter volume, which are confined within the droplet and cannot contaminate the walls of the vessel. The experimental results for different assay formats are interpreted by theoretical analysis of the kinetics of particle agglutination and mass transfer processes inside the droplets. The performance of the droplet-based microbioassays will be compared to the one of conventional hand held assays.


P3.5
Abstract Withdrawn


P3.6
Electroseparation in Microfluidic Channels Using Inverse Opal Structure Jau-Ye Shiu and Peilin Chen; Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan.

Gel electrophoresis and capillary gel electrophoresis are widely used for the separation of biomolecules molecules. With increasing demand in the miniaturized devices such as lab-on-a-chip, it is necessary to integrate such separation component into a chip format. Here we describe a simple approach to fabricate robust three-dimensional periodic porous nanostructures in the microchannels for the separation of DNA molecules. In our fabrication procedure, the colloidal crystals were first grown at the desired area inside the PDMS microchannel using evaporation assisted self-assembly process. Then the void space inside the colloidal crystals can be filled with sol-gel or SU-8 photoresist. After solidification, the inverse opal structure in the microchannel can be obtained by removing the nanoparticles with proper solvent. Our results indicated both sol-gel and SU-8 photoresist can be used to construct inverse opal structure inside a 5 mm long microfluidic channel with pore size around 25-30 nm. To demonstrate the capability of separating different size of biomolecules in our device, DNA markers labeled with fluorescence dye was first loaded into reservoir by electrokinetic injection. After applying proper voltage, DNA molecules with different size can be separated within one minute and visualized in a microscope.


P3.7
Surface Enhanced Raman Scattering using Built-in Noble Metal Nanoarrays in Integrated Optofluidic Devices Chul-Joon Heo^1,2, Se Gyu Jang^1,2, Seung-Kon Lee^1,2 and Seung-Man Yang^1,2; ¹Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Chungnam, South Korea; ²National Creative Research Initiative Center for Integrated Optofluidic Systems, KAIST, Daejeon, South Korea.

Molecular specific detection using surface enhanced Raman scattering (SERS) has been widely investigated after its initial discovery. The high-density metal nanoarrays have been used as a SERS substrate because gaps between adjacent metallic nanostructures induce extremely intense local electromagnetic fields, known as “hot spots,” upon optical excitation. Colloidal lithography is a robust method for fabricating regularly ordered nanostructures in a controlled and reproducible way using spontaneous assembly of colloidal particles. SERS enhancement characteristics could be tuned by changing the materials or conditions for fabrication. In this study, embossed nanostructures of polymer thin film with different shapes and spacings were fabricated via colloidal lithography. Then, metallic embossed structures with high density hot spots were created by sputtering noble metals such as gold (Au) and silver (Ag). The enhancement on Raman signal and tunability of afore-mentioned structures were confirmed by the SERS spectra of Rhodamine 6G and other molecules on metal nanoarrays. Finally, SERS active substrates which were fabricated by colloidal lithography were integrated in optofluidic chips to achieve in-situ molecular and multi-channel detection of Rhodamine 6G and other molecules.


P3.8
Modeling of Nanoscale Inorganic-organic Hybrid System for in-situ Molecular Recognition. Sehoon Jeon¹, Ui Seong Kim¹, Chee Burm Shin¹ and Jongheop Yi²; ¹Division of Energy Systems Research, Ajou University, Suwon, South Korea; ²School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea.

Miniaturized devices (e.g. Lab-on-a-chip, MEMS, μ-TAS, etc.), based on a combination of microfabrication technology and life sciences, are widely used in medical field such as in diagnostics, drug-delivery system, etc. For in-situ molecular recognition by using Surface Plasmon Resonance (SPR) in a nanoscale inorganic-organic hybrid system, we constructed a simple micro-flow system to diagnose of protein (superoxide dismutase, SOD) mutation which may cause ALS (Amyotrophic Lateral Sclerosis, Lou Gehrig's Disease). By microfluidic modeling, we can analyze the role of geometry and process conditions on the performance of microfluidic devices, and get better understanding of the complex biochip systems, obtaining of experimentally inaccessible information, and reducing design time by exploring more design options. To find the optimum design of components of microfluidic devices for using on SPR, microfluidic modeling was performed to predict the velocity distribution of the fluids. With this designing guideline by modeling, we made simple flow cells (2×2cm) with polydimethylsiloxane (PDMS) which is widely used for SPR cell. We did capillary flow experiments with various concentrations of salt-water solutions to maximize mixing.


P3.9
Continuous-flow PCR Chip with PDMS / ITO Heater Patterned Glass for DNA Amplification. Seung-Ryong Joung¹, Yung-Jin Choi¹, Chi-jung Kang¹, Jaewan Kim¹ and Yong-Sang Kim^1,2; ¹Nano Science and Engineering, Myongji university, Yong-in, Gyeonggi, South Korea; ²Electrical Engineering, Myongji university, Yong-in, Gyeonggi, South Korea.

A polydimethylsiloxane (PDMS) / indium-tin-oxide (ITO) heater-patterned glass polymerase chain reaction (PCR) chip for continuous-flow DNA amplification was proposed. Continuous-flow PCR chip enables fast thermal cycling and series amplification, which are difficult to achieve in a conventional PCR or a micro-chamber PCR chip. The continuous-flow PCR chip is consisted of a PDMS microchannel layer and an ITO heater patterned glass layer. High repeatability and ease of fabrication of PDMS microchannel can be performed by negative molding method. The fabricated microchannel width and depth are 250 µm and 200 µm, respectively. Also, the total working length of the PDMS microchannel is 1340 mm which is equivalent for 20 cycles of amplification. A 2:2:3 microchannel length ratio for three different temperature zones namely denaturation, annealing, and extension was assigned, respectively. Six indium-tin-oxide (ITO) thin film heaters, two on each temperature zone, were mounted on a glass substrate by photolithography and wet etching techniques. The ITO heating calibration guaranteed the stable and precise liquid heating control in each region. Upon the operation of the fabricated continuous-flow PCR chip, the amplification of plasmid DNA pKS-GFP with 720-DNA base pairs was found successful with a total reaction time of 15 minutes which is four times faster than a conventional PCR machine. The integration of the fabricated continuous-flow PCR chip with other micro-fluidic systems for a micro-total-analysis-system (µ-TAS) or a lab-on-a-chip application is prospected in future works.


P3.10
Cell Behaviour on Nanoscale Hierarchical Patterned Surfaces Bimalraj Rajalingam¹, Myoung-Woon Moon², Ashkan Vaziri² and Ali Khademhosseini^1,3; ¹Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; ²Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts; ³Harvard-MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts.

We report a novel technique for fabricating hierarchical wrinkling patterns on the surface of PDMS in micron and submicron level. Initially stretched PDMS slabs were exposed to plasma treatment and then allowed to relax. The wavelength of the wrinkling patterns can be effectively selected by controlling the oxidation time, while the initial stretch controls the amplitude of wrinkles. Using this technique, wrinkling patterns with wavelength in the range of 50 nm to 5 microns and amplitude in the range of 20 nm to 400 nm were fabricated. We also show that wrinkles in the submicron level can be nested within larger wrinkles using multi step plasma treatment, resulting in hierarchical wrinkling patterns. We used this technique to manufacture wrinkling patterns with wavelength in the range of 50 nm to 5 microns and amplitude in the range of 20 nm to 400 nm, as well as patterns comprising hierarchical wrinkles. We characterized the surface using atomic force microscopy and scanning electron microscopy. These patterns were employed to study the role of surface topology on the behavior of NIH-3T3 cells. Cells aligned and elongated on these patterns depending on the surface topology. Moreover, the cells proliferated on these nanopatterned substrates and gave rise to progeny that was also aligned on these nanopatterned substrates. We systematically studied the role of surface topology on the orientation and the morphology of NIH-3T3 cells as well as their cytoskeletal structure. The double scale wavelength effect on the cell behavior was also explored. We varied the duration of the multi step plasma treatment and the strain of the PDMS samples to arrive at the optimal wavelength and amplitude for controlling the cell orientation. The alignment of the cellular focal adhesions and actin fibres in relation to the variations in the amplitude of the waves was analyzed. The cell orientation and elongation is a function of the wavelength,with wavelengths around 1 to 2 microns showing maximal orientation and elongation. Moreover the cells dynamically changed their orientation over time, with cells after three days of incubation showing better orientation compared with those after 12 hours of incubation. Our results indicate the potential of the developed technique for controlling the cell orientation and morphology in tissue engineering templates and in the fabrication of biomimetic surfaces.


P3.11
A Controlled Release Approach to Generate Microengineered Hydrogels of Controllable Shapes and Sizes made from Fast Gelling Polymeric Precursors Giovanni Talei Franzesi¹, Yibo Ling^1,2, Bin Ni³ and Ali Khademhosseini^1,4; ¹Harvard-MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts; ²Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts; ³Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts; ⁴Center for Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts.

Microscale hydrogels are useful for a variety of applications such as drug delivery, tissue engineering and food sciences. However, some of the most useful and better studied fast gelling ionically and pH-crosslinked hydrogels are not amenable to standard micromolding and micropatterning techniques that are commonly used to generate microscale hydrogels. Here, we present an approach that uses controlled release of the gelling agent to generate molded structures of this class of hydrogels. As model hydrogels we used alginate representing an ionically crosslinked hydrogel and chitosan, a pH dependent hydrogel. To micromold the hydrogels calcium alginate was molded between a plasma-cleaned PDMS mold and a calcium-containing agarose slab and subsequently gelled by the controlled release of calcium ions from the agarose. The slab provides a physical barrier while simultaneously inducing the gelation of the hydrogel precursor, resulting in the formation of both membranes and microparticles of controlled morphology. A similar approach was used to obtain chitosan micropatterns, by using an agarose at high pH. Using this approach, features with lateral dimensions between 5 µm and 2000 µm, and vertical dimensions between 10 µm and 200 µm could be obtained. In addition cells could be embedded in microgels in a range of densities (10E3 - 10E8 cells/ml) and remained >80% viable. Different cell types could be co-cultivated, either in two separate hydrogel phases micromolded on top of each other or one cell type could be encapsulated in a micromolded hydrogel, while the other is seeded on top of the structure. Furthermore, by varying the precursor concentration and gelling conditions the mechanical properties of the micropatterned hydrogels were controlled. These microgels (often as small as 10 µm) remained stable for >2 weeks incubation in cell culture media at 37°C. Thus, we demonstrate that controlled release of the gelling agent can be used to micromold hydrogels not amenable to traditional molding approaches.


P3.12
Fabrication of Superhydrophobic Micro/Nanostructures Donghyun Kim¹, Joonwon Kim¹, Woonbong Hwang¹, Hyun Chul Park¹ and Kun-Hong Lee²; ¹Mechanical Engineering, POSTECH, Pohang, South Korea; ²Chemical Engineering, POSTECH, Pohang, South Korea.

A surface was created with the same superhydrophobic property as the lotus leaf (Lotus Effect) by dipping of sandblasted porous alumina into polytetrafluoroethylene (PTFT, Teflon®: DuPont™) solution. The fabricated artificial lotus leaf had PTFT micro/nanostructures. This fabrication process has several advantages, including low fabrication cost, simplicity and easy coverage of a large area. The sandblasted porous alumina template was fabricated by sand blasting of aluminum sheet and anodization in oxalic acid. To obtain PTFT micro/nanostructures, PTFT replication based on the dipping method was used, with a 0.3 w% PTFT solution. To remove the aluminum and alumina layers, wet etching by chromic and phosphoric acid mixed solution and liquid HgCl2 solution was used. The fabricated surface has a superhydrophobic property whose apparent contact angle of the PTFT micro/nanostructures was approximately 165 degrees and sliding angle is less than one degree.


P3.13
Abstract Withdrawn


P3.14
Dynamic Properties and Flow rates of Novel Piezoelectric Micropump Sang-Jong Kim^1,2, Dae-Yong Jeong¹, Chong-Yun Kang¹, Ji-Won Choi¹, Hyun-Jai Kim¹, Man-Youmg Sung² and Seok-Jin Yoon¹; ¹Thin Film Material Research Center, Korea Institute of Science and Technology, Seoul, South Korea; ²Department of Electrical Engineering, Korea University, Seoul, South Korea.

In recent years, much attention has been given to developing the microfluidic systems based on MEMS(Micro Electro Mechanical System) technologies. Among them, micropump have a large potential for an application to micromechanical analysis system such as LOC(Lab On a Chip) as well as embedded medical devices. Micropumps have been developed using several actuation methods such as a electrostatic, thermopneumatic, electroosmotic, piezoelectric, etc. Most of them require complex structure. But, the piezoelectric actuation has advantages of the relatively simple structure, low power consumption and high pumping performances. In this paper, the characteristics of the novel piezoelectric micropump were studied. We have designed and fabricated 3 types of micropump. The first type consists of single piezoelectric element and single top electrode. The second is the separate two piezoelectric elements which include top electrode, respectively, Final third type is made of single piezoelectric element including two separate top electrodes. The diffuser has been optimized as 1 mm length and 1 to 2 ratio width from the fluidal analysis. The micro diffuser and the chamber were fabricated using bulk micromachining method. The parts of actuator, bottom electrode Ag and piezoelectric thick films were fabricated using screen printing method, respectively. Piezoelectric thick films were sintered by RTA(Rapid Thermal Annealing). Top electrodes Pt were deposited by DC sputtering system and lift-off. We investigated dynamic properties by vibrometer and measured the flow rates.


P3.15
Chitosan for Selective Biofunctionalization of Microsystems. Stephan Koev^1,4, Vlad Badilita^1,4, Hyunmin Yi⁷, William Bentley^3,5,6, Gregory Payne^5,6, Gary Rubloff^2,4,6 and Reza Ghodssi^1,4,6; ¹Department of Electrical and Computer Engineering, University of Maryland, College Park, Maryland; ²Department of Materials Science and Engineering, University of Maryland, College Park, Maryland; ³Fischell Department of Bioengineering, University of Maryland, College Park, Maryland; ⁴Institute for Systems Research, University of Maryland, College Park, Maryland; ⁵University of Maryland Biotechnology Institute, College Park, Maryland; ⁶Bioengineering Graduate Program, University of Maryland, College Park, Maryland; ⁷Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts.

We present chitosan-mediated electrochemical assembly of biomolecules on MEMS biosensors. Chitosan is an amino-polysaccharide with unique properties: (i) pH-dependent solubility transition that allows electric signal-guided assembly onto conductive surfaces and (ii) chemical reactivity that allows covalent conjugation of biomolecules for biosensing surface construction. This biofunctionalization method has significant advantages over printing-based techniques such as soft lithography because non-planar device surfaces can be patterned (e.g. sidewalls) and the deposition is electrically controllable. In this presentation we report two BioMEMS platforms that employ chitosan as the biomolecule assembly scaffold for DNA hybridization sensing: a micromechanical sensor and an in-plane waveguide biophotonic sensor. Our micromechanical sensor is a cantilever that detects the binding of biomolecules by the change in deflection due to surface stress (static mode) or by the change of resonant frequency due to added mass (dynamic mode). The cantilever is fabricated of thin films of Si3N4 (500nm) and Au/Cr (100nm) on a silicon substrate by standard lithographic and etching techniques. Chitosan is electrodeposited on the device and probe DNA is covalently coupled to the chitosan film. The device is exposed to target DNA for hybridization, and then to urea solution for denaturation. At each step, the bending of the cantilever in solution and the resonant frequency in air are measured by an optical interferometer. Both static and dynamic responses demonstrate that sequence-specific biological recognition occurs and is transduced to a large mechanical signal. The biophotonic sensor consists of SU-8 polymer optical waveguides and microfluidic channels defined on a pyrex substrate. The facets of the waveguides have transparent indium tin oxide (ITO) electrodes, on which chitosan is electrodeposited and biomolecules are subsequently assembled. The waveguides deliver excitation light to and collect emitted light from the biomolecules for fluorescence or absorption analysis. The waveguides in turn are coupled to optical fibers, which are connected to an external light source and a spectrum analyzer. The key advantage of this device is that the analyte is immobilized on the waveguide facet, which leads to large collection efficiency of the emitted light compared to more common evanescent coupling techniques. The optical biosensor was tested in response to fluorescent DNA hybridization. The measured sequence-specific response demonstrates the feasibility of optical detection by sidewall biofunctionalization with chitosan. This work shows that chitosan facilitates both optical and mechanical biosensing. It is electrodeposited as a stable film at specific electrode addresses, and it is transparent and mechanically responsive. Our future goal is to combine the chitosan-based optical and mechanical sensors in a single multimodal detection platform.


P3.16
Assessment of Fluidic Channels Produced via Femtosecond Laser Iinduced Delamination of Thermal Oxide Films from Silicon Substrates. Vanita R. Mistry¹, Joel P. McDonald² and Steven M. Yalisove³; ¹Mechanical Engineering, University of Michigan, Ann Arbor, Michigan; ²Applied Physics, University of Michigan, Ann Arbor, Michigan; ³Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan.

Recent femtosecond (fs) laser induced damage morphology studies of Si (100) with thin thermal oxide films (SiO2) demonstrated selective delamination of the thermal oxide film under certain laser conditions. Isolated delaminated regions, or blisters, are connected together to form fluidic channels. The mechanism responsible for the delamination is thought a combination of compressive stress relaxation and the force provided by the laser induced ablation of the substrate. A range of material and laser variables were considered for optimal channel production. The optimal oxide thickness for channels was found to be 1200nm. Optimal laser focusing was achieved with a 35 cm plano-convex lens, yielding a spot size of ~70 micrometers on the sample surface. The average laser power observed to produce the most uniform channels was 5.6 mW (< 1% of the available output power), corresponding to a laser pulse energy 5.6 microjoules, or a laser fluence of 0.46 J/cm2. Single pass channels were produced by scanning the sample through the focused laser beam at a constant velocity of 10 mm/s, producing channels with a typical width of 20 micrometers and a height of 350 nanometers. By laterally overlapping single pass channels, the width of channels was extended to greater than 300 micrometers, while the height of the channels was expected to exceed 15 micrometers. Applications of fluidic channels produced in this fashion are being investigated through the design and fabrication of a simple fluidic device. Macro to micro interfacing will be discussed. Our approach uses a PDMS mold with reservoirs that deliver liquid to the channel entrance. For electrophoresis, gold electrodes are directly sputtered onto the top surface of the thermal oxide within the fluid reservoirs. Carboxylate modified polystyrene spheres (20 nm in diameter) are driven through the channels via electrophoretic flow over a range of bias conditions. Details of electrophoresis will be discussed, and potential applications of such channels will be presented.


P3.17
Microfluidic Love Wave Sensor for Highly Viscous Environments. Vincent Raimbault¹, Dominique Rebiere¹, Corinne Dejous¹, Matthieu Guirardel² and Jean-Luc Lachaud¹; ¹Laboratoire IXL, Talence, France; ²Rhodia - Laboratoire du Futur, Pessac, France.

High speed chemistry, pharmaceutics, cosmetic, environment or health monitoring have special needs for integrated liquid microsensors. In many applications, a viscosity measurement is needed. Conventional methods, i.e. rotational rheometers, needs sample preparation that is time consuming and cannot be perform in-situ. A microsensor device integrated in a manipulation loop is useful in terms of time, sample volume reduction and measurement rates. A counterpart of miniaturization is a loss in sensitivity. Through the microsensor devices that can measure viscosity with high sensitivity, Love wave sensors offers great potential. Devices used for this paper are delay lines where the acoustic wave is generated and received by interdigital transducers deposited on an AT cut quartz piezoelectric substrate. A 4µm thick SiO2 guiding layer confines the energy at the surface which makes these sensors especially sensitive to mass deposition effects and viscosity variations of the surrounding media. The sensor is included in an oscillation loop and the oscillation frequency variation is monitored. The main limitation in liquid is acoustic losses that occur due to viscous coupling at the guiding layer/liquid interface. A PDMS microfluidic chip bonded on the acoustic path has been developed to limit liquid volume, isolate interdigital transducers and thus limit these acoustic losses. Thanks to this microfluidic technique, our microsensor can work in highly viscous solutions (silicon oils up to 30 Pa.s) and can be integrated in classical microfluidic setups. In order to improve knowledge of polymers behavior for high frequency and small displacements, polyethylene glycol and polyethylene oxide are studied. These polymers are widely use for clinical, biological, pharmaceutical or commercial purposes. PEG and PEO viscosity is linked to their chain length. Thanks to their solubility, aqueous solutions can be formulated to cover a wide viscosity range. PEG and PEO with different chain length are compared to PEG and PEO solutions with the same η0 obtained by dilution in deionized water. Liquids are also characterized using a rotational rheometer to measure the low frequency dynamic viscosity (η0 in Pa.s). A comparison between these results and previous experiments made on aqueous glycerol solutions and silicone oils (poly(dimethyl)siloxane) is proceed, bringing to light chain length influence on liquid relaxation times. A temperature study of liquids behavior is performed, thanks to the integration of a screen printed resistance on an alumina substrate in contact with the Love wave delay line. Thanks to the relation between time (i.e. frequency) and temperature for polymer solutions, this solution allows to predict liquid viscosity at smaller frequencies than f0, providing more data in the viscoelastic behavior of polymers. A numerical resolution based on the Transfer Matrix Method is used to modelize microsensor frequency shift as a function of liquid properties.


P3.18
Multiplexed Transport and Detection of Cytokines Using Kinesin-Driven Molecular Shuttles. Lynnette Rios and George D. Bachand; Biomolecular Interfaces & Systems Department, Sandia National Laboratories, Albuquerque, New Mexico.

Multiple analyte immunoassays are a simple way to achieve simultaneous analysis of various analytes in a sample without the need to run separate tests. To date, most multiple analyte assays have focused on array-based systems. Such arrays, however, become problematic when analyzing cocktail samples, or samples that contain multiple analytes, due to a dependence on the signal from a single label and the immobilization of only one type of antibody per analyte. The intrinsic limitations of this approach also prevent miniaturization of such array-based devices. As an alternative, we are currently exploring the use of biomolecular active transport systems as a means of downscaling multiple analyte assays for lab-on-a-chip applications. Recently, the ability to capture and transport a wide range of target analytes including proteins, virus particles, and bacterial spore was demonstrated using kinesin-driven molecular shuttles. The molecular shuttles consisted of microtubule filaments that were functionalized with analyte-specific antibodies, thus facilitating selective target capture and transport. In the present work, we have applied this nanofluidic platform for the simultaneous detection of multiple target analytes. Multiplexing of molecular shuttles was achieved by immobilizing biotinylated antibodies against interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) on biotin microtubules using a streptavidin bridge. ELISA results show detection of TNF-α across a broad range of concentrations using the antibody-functionalized microtubules. To facilitate multiplexed detection in nanofluidic architectures, we have functionalized nanocrystal quantum dots (nQDs) of different sizes and spectral emissions with IL-2 and TNF-α antibodies. Current work is focused on applying both the functionalized microtubules and nQDs for simultaneous detection of IL-2 and TNF-α in kinesin-based motility assays. The results of both the multiplexed microplate and motility assays will be presented and discussed. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.


P3.19
A Planar Electroosmotic Micropump for Lab-on-Microchip Applications. Konstantin Seibel, Lars Schoeler, Heiko Schaefer, Marcus Walder and Markus Boehm; Institute of Microsystem Technologies, University of Siegen, Siegen, Germany.

Fully integrated micro total analysis systems such as a lab-on-microchip require precise transport of reagents and analytes from reservoirs to reaction chambers and separation columns. Mechanical pumps and valves have large size, are too complicated for monolithic integration on microchips and have low long-term reliability because of moving parts. The electrokinetic effect is the most common one used for nonmechanical pumping of aqueous solutions in microchannels. The scope of the paper is to provide a theoretical and experimental treatment allowing to optimize critical design parameters for planar electroosmotic micropumps. In order to increase the pump pressure the dimensions of the pumping channel need to be reduced. This method was implemented successfully for the design of high pressure electroosmotic pumps. The suggested design with a vertical arrangement of multiple narrow polymer pumping microchannels reduces the pump area to 1/10 compared to planar micropumps with widened shallow pumping channels. This design allows the fabrication of the channel system in only one process step and is compatible with post-CMOS processing. A simple analytical model has been developed to characterize the flow rate in a field free pressure-driven section of the channel. It is shown that the micropump with optimized dimensions of rib structures makes possible high pressure low voltage pumping. For high pressure capacity the distance between the ribs must be on the order of 0.5-1 µm with an aspect ratio of 10-20. The main problem of electroosmotic micropumps is water electrolysis and gas bubble generation. Electroosmosis and electrolysis are tightly interrelated effects. Electroosmosis requires an ion current in the fluid and a continuous ion current is only possible if electrolysis takes place at the metal electrodes. Therefore, one option to cope with the bubble generation is to place the electrodes outside the main flow path using gel or liquid bridges as ion conductors. The electroosmotic micropump with suggested design using microchannels of SU-8 and polyacrylamide gel electrodes has been fabricated and tested. The photo-polymerized polyacrylamide gel electrodes were fabricated in the side channels using photolithographic technique. The micropump was tested with deionized water as working fluid. Due to the hydrophilic properties of the channel walls and the reduced contact angle of the glass cap, the microfluidic system is self-priming by capillary forces. The pumping rate is bidirectionally linear and reached 10 nl/min at applied voltage of 40 V in 1 cm long pressure-driven channel. The experimental data are in a good agreement with theoretical considerations.


P3.20
Characterisation of Lab-on-chip Electrophoresis Systems with Integrated Amorphous Silicon based Optical Detectors. Lars Scholer, Marcus Walder, Lars Storsberg, Konstantin Seibel, Heiko Schaefer and Markus Boehm; Institute of microsystem technologies, University of Siegen, Siegen, Germany.

Application specific lab-on-microchips (ALM) making use of the combination of complex microfluidic networks with microelectronic circuits and micro optical components allow the realization of miniaturized application specific biological and chemical processing and analysis devices. Fluorescence sensing is one of the most widely used detection technologies, e.g. for DNA fluorescence labelling in micro CE due to its superior sensitivity and specificity. Unfortunately, commercially available fluorescence sensing systems are physically very large, non portable, expensive and constrain the analysis in portable diagnostic and medical care. Integrated semiconductor optoelectronic devices can provide a portable, parallel and inexpensive solution for on chip fluorescence sensing. Monolithic integration of optical sensors by deposition of hydrogenated amorphous silicon (a-Si:H) as thin films on application specific integrated circuit systems (TFA) has recently gained considerable attention. The fusion of a Si:H sensor technology with microfluidic analytical devices provides a dramatic reduction of sample volume and analysis time, resulting in improvement of speed, reliability and efficiency. Most notably, due to the higher absorption coefficient for visible light and the low dark current, an a-Si-H detector is more suitable for the detection of fluorescence light than a crystalline silicon detector. In particular, most applied labelling dyes used for chemical and biological analysis target this light spectrum. In this paper we combine an a:Si-H photo sensor with a fluidic micro channel to detect the fluorescence of a rhodamine analyte mixture. The analyte mixture was excited by light with a wavelength in the range of λExcitation = 450 - 490 nm. The a-Si:H detector reveals a low dark current density on the order of 10-10 A/cm2 and a sufficient dynamic range of ~100 dB under illumination of ~1000 lx as a function of bias voltage. The measurement shows that the movement of the rhodamine plug in the microchannel causes a significant rise in the pin-diode photo current, which correlates to the evaluated signal of a microscope image detector. The photo current difference for excitation and additional fluorescence amounts to 2.4 µA.


P3.21
Simulation and Experimental Characterization of Plug Distortion in On-chip Capillary Electrophoresis Systems with Hybrid Micro Channels. Lars Storsberg, Markus Walder, Konstantin Seibel, Lars Schoeler, Heiko Schaefer and Markus Boehm; Institute of microsystem technologies, University of Siegen, Siegen, Germany.

In capillary zone electrophoresis systems plug widening must be avoided to ensure high seperation efficiency. This applies particulary to lab on a chip applications because of their short seperation channels. A non uniform velocity of the carrier fluid can cause the widening and the plug will be distorted. The velocity profil is not stamp like if the ζ potentials at the channel walls, are different because of different materials used for manufacturing. The plug distortion for on-chip capillary zone electrophoresis systems with rectangular separation channels has been examined. The channels were manufactured in a hybrid layer system. Using glass layers (Corning 7059 and microscope cover plates) for the horizontal and SU 8 layer (MicroChem Corp., USA) for the vertical channel walls. The plug of the fluorescent dye Rhodamine 6G was observed under a microscope with a video camera. A contour of equal concentration was defined by means of an RGB analysis of the pixel signals for the entire separation process, defining the plug boundaries. The process of plug distortion as function of time indicates, that the diffusion of the dye isn’t negligible. The experiment was reproduced in a 3D-simulation, using the parameters of the experiment and varying the ζ potentials at the side walls. Experimental data and simulation results indicate that plug widening caused by different values of the ζ-potential of the channel walls depends strongly on the aspect ratio of the channel cross section. If the height to width ratio is much greater or much smaller than 1, as is often the case for commonly used labchip architectures, plug widening may be negligible. For an architecture using glass for the top and bottom walls, but SU-8 for the side walls, the difference of the ζ potentials was measured to be on the order of only 2.4 mV for a pH of 9.2, suggesting that such device architectures may be used for on-chip electrophoresis analysis without uniform coating of the channel inside for less demanding applications.


P3.22
Configurational Model for the Elctronic Coupling of Neurons to MOS Transistors. Joseph Shappir¹, Ariel Cohen², Carmen Bartic⁴, Gustaaf Borghs⁴, Shlomo Yitzchaik³ and Micha E Spira²; ¹Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel; ²Neurobiology, The Hebrew University of Jerusalem, Jerusalem, Israel; ³Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel; ⁴MCP/ART, Cell Based Sensors & Circuits, IMEC vzw, Leuven, Belgium.

Culturing Aplysia neurons on Floating Gate Depletion type P-Channel MOS Transistors (FGDP) was used to develop an improved model for the electronic coupling of neurons to transistors. The choice of the FGDP enabled the following advantages: 1. Elimination of the need for DC bias between the biological solution and the silicon chip to reduce electrochemical corrosion and current drifts. 2. Removal of the neuron from the most sensitive part of the transistor. 3. independent optimization of the transistor to neuron coupling area (10 μm diameter octagon floating gate extending from W/L = 50/0.5 μm transistor channel). 4. Lower noise level of the P-channel transistor. Basic electrical considerations lead to the conclusion that no field potential signal could be recorded from an isopotential neuron body. To overcome this postulate, it was commonly accepted that the part of the cell membrane which is facing the transistor has different density of ionic channels as compared to the rest of the cell membrane facing the ionic solution thus breaking the isopotential concept. It will be shown that the neuron composed of soma and axon does not behave as an isopotential entity. Difference in shape and timing of the action potentials generated in these two compartments, result in ionic current flow outside the neuron through the resistive path Rseal of the thin layer of ionic solution between the neuron and the silicon chip. Results of very good fit between experimental data and circuit analysis will be shown and discussed.


P3.23
Tunability of Dielectrophoretic Mobility of Semiconductor Nanowires and Its Implications in Device Design. Grace Xing¹, Gabor Galantai³, Vladimir Protasenko³, Masaru Kuno³, Amol Singh¹, Debdeep Jena¹, Ronghui Zhou² and Hsueh-Chia Chang²; ¹Electrical Engineering Department, University of Notre Dame, Notre Dame, Indiana; ²Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana; ³Chemistry and Biochemistry Department, University of Notre Dame, Notre Dame, Indiana.

Both nanocrystals and nanowires have attracted a lot of attentions as building blocks for nano-opto-electronics or hybrid electronics and systems. Furthermore, nanocrystals and nanowires both offer unique properties stemming from quantum confinement, especially in terms of optical properties. However, to interface with the outer world via electronic connections, nanowires are more advantageous since they allow carrier band transport while nanocrystal-based devices suffer from low carrier mobilities due to variable range hopping conduction. We have investigated semiconductor nanowire assembly using dielectrophoresis (DEP) in solution and discovered strong dependence of nanowire DEP mobility on super bandgap illumination. In this talk, we will present the origin of this phenomenon; as device examples, we will report 1) the polarization sensitivity of optical emission and absorption as well as photocurrent of the assembled semiconductor (CdSe and CdTe) nanowires and 2) carbon nanotube enhanced bacteria trapping. We will also discuss the implications of this tunability of semiconductor nanowire DEP mobility and several novel device designs.


P3.24
Nanofluidic Size Focusing of Functionalized CdSe Quantum Dots Louis J Tribby¹, Youn-Jin Oh¹, Timothy J Boyle², Timothy N Lambert², Cornelius F Ivory³ and Sang M Han¹; ¹Chemical & Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico; ²Inorganic Chemistry and Nanomaterials, Sandia National Laboratories, Albuquerque, New Mexico; ³Chemical Engineering, Washington State University, Pullman, Washington.

We have conducted size-separation of surface-functionalized fluorescent quantum dots (QDs) based upon their electrokinetic mobility in “gate” biased nanochannels and field flow fractionation. We are utilizing lithographically fabricated nanochannels (approximately 10⁴ to 10⁵ parallel channels), where each channel is on the order of 100 nm wide, 500 nm deep, and 16 mm long. In order to acquire extreme size focusing, we systematically manipulate and detect the influence of size, pH, and net surface charge on the electrokinetic mobility and resulting size distribution. We place different charges on the surface of the QDs to tailor the particles’ response to various induced field gradients in the nanochannels. We have synthesized functionalized CdSe QDs that can be easily modified with different charge carriers in aqueous as well as organic solutions. The resulting size distribution is evaluated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and high-efficiency dynamic light scattering sampling. The advancing speed of NCs in the channels is measured by laser scanning confocal fluorescence microscopy (LS-CFM) and multiple internal reflection Fourier transform infrared spectroscopy (MIR-FTIRS). Experimental measurements will be presented in further detail within the context of size-separation. The authors acknowledge generous support from NSF-NIRT (CTS-0404124) and Keck Foundation. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.


P3.25
Integration of Immobilized Polymeric pH, O2 and Glucose Sensors into a Novel Microbioreactor Array for Use in High Throughput Cell Culture Optimization. Denis F Leroux¹, Scott E Miller¹, Brett Bernier¹, Xin Yu Li¹, George J Vella¹, Michelle Muscatello², Stunja E. Lee² and Sanford A. Asher²; ¹BioProcessors, Woburn, Massachusetts; ²University of Pittsburgh, Pittsburgh, Pennsylvania.

Biopharmaceutical process optimization is a bottleneck in drug development. BioProcessors has developed an automated platform for mammalian cell culture that enables high-throughput cell culture bioprocess optimization for the production of recombinant proteins for the therapeutic, vaccine and diagnostics industries. The SimCell™ Automated Management System uses novel disposable microbioreactor arrays to perform hundreds of cell culture experiments in parallel at the sub-milliliter scale. An essential element is the integration of non-invasive optical chemosensors to automatically monitor on-line key control parameters that affect cell growth and protein yield. The main challenge is the incorporation of known sensing chemistries into disposable microbioreactors using low-cost manufacturing techniques. pH sensors were developed by copolymerization of a pH sensitive dye into a hydrogel and integrated into the microbioreactors by using a conventional screen-printing process followed by a polymerization scheme. DO sensors have been integrated onto an oxygen-permeable substrate while avoiding oxygen interference from the environment. Various schemes for the integration of optical glucose sensors using Bragg diffraction will be reviewed. An overview of application-specific challenges will be presented including detection schemes for the integration of Biomass sensing based on Optical Density (OD) measurement, pH sensing based on ratiometric fluorescence measurement, Dissolved Oxygen (DO) sensing based on fluorescence life-time decay and glucose sensing based on Bragg diffraction. Data from cell growth experiments demonstrating the concurrent measurements of biomass, pH, DO and glucose using a SimCell microbioreactor array, will be discussed showing the similarity with those obtained from conventional bench-top bioreactors with offline analysis. Precision of +/- 0.05 pH unit is achieved with pH sensors operating between pH 6 and 8 and control of pH has been demonstrated to +/- 0.1 pH units by periodic fluid additions. Data from DO sensors operating between 0.02 and 0.2 atm with a precision of +/- 0.0004 and +/- 0.01 atm (1SD) respectively, will also be shown. Preliminary data on optical glucose sensing in cell culture will also be presented.


P3.26
Development of A First-Responder Fluorescence Reader for Microplate Cytokine Assay of Human Immune Response to Disease. David B Fenner, D. I Rosen, A. A Ferrante, A. E Stevens, C. E Bigelow and S. J Davis; Physical Sciences Inc, Andover, Massachusetts.

The practical utility of technologies for early detection of disease has been limited in many cases by the absence of instruments suitable for first responders and in scenarios such as the field hospital. Bioanalysis with sandwich microarray is a technique whereby fluorescent labels on antibody-antigen pairs at microarray spots are imaged in a laboratory setting for quantitative assay. New methods and instruments are needed for point-of-care use. Microplate-based schemes provide multiplexed assay of a large number of human cytokines including those that are known to indicate imbalances in the immune system. Analysis of blood is the common method but saliva is more readily available and also able to provide quick indication of immunological challenge. We report development of a highly portable system configuration with microarray cytokine capture set, dye selection, excitation by filtered LED and fluorescence imaging by 16-bit CCD camera. At present, plates have 16 microarray wells, with 12 cytokines per well and quadruplicate spots. Excitation with multiple one-watt amber (~590 nm) LEDs provides efficient, uniform illumination directly over each full microarray well. The fluorescence from an array of spots (100-200 micron diameter) in a well is imaged through a long working-distance objective, long-pass filter and onto a cooled CCD chip. Image data is immediately transferred into a laptop PC for data reduction. Assay with this small, portable battery-powered reader provides cytokine concentrations over about three orders of magnitude and measured to as little as ~5 pg/ml, the same limit as for laboratory instruments. Profiles of selected cytokines in whole, unprocessed saliva are known to be clinically significant as fingerprint indicators of human disease state. Such indications of a generalized disease state can precede the development of clinical symptoms and hence take on extra significance. The cytokine profiles of a small clinical study of healthy and flu-vaccine challenged individuals, assayed in this manner, will be reported. Opportunities to extend this field-use technology for human health assay to include other biomarkers is discussed. Work supported by Wright-Patterson AFRL-HE through SBIR contract FA8650-06-C-6647.


P3.27
Real-Time Detection of Micron-Sized Magnetic Beads using a Highly Sensitive Spin-Valve Sensor for a Chip-cytometer. Jong Wook Roh¹, Sun Gu Yi¹, Kyoung Il Lee¹, Oh-Taek Son², Hyo-Il Jung² and Wooyoung Lee¹; ¹Department of Materials Science and Engineering, Yonsei Univ., Seoul, South Korea; ²School of Mechanical Engineering, Yonsei Univ., Seoul, South Korea.

The development of a chip-cytometer detecting magnetic beads using a spin-valve sensor has recently attracted great interest since it is capable of realizing both cell-separation and cell-counting on a chip. In order to implement a chip-cytometer, the real-time detection of magnetic beads moving in a microfluidic channel is essentially required for cell-counting. For this reason, sensors with high sensitivity and large signal to noise ratio, i.e. GMR (giant magnetoresistance) spin-valve sensors, are prerequisite to detect moving magnetic beads in a microfluidic channel. In this work, we report on the real-time detection of moving magnetic beads using a highly sensitive spin-valve sensor integrated in a microfluidic channel. The generic structure of a spin-valve was Co₈₄Fe₁₆(20)/NOL/Ni₈₁Fe₁₉(25)/Co₈₄Fe₁₆(10)/Cu(17)/Co₈₄Fe₁₆(20)/Ir₂₂Mn₇₈(75)/Ta(or Au)(50) (Å). In this work, NOLs (nano-oxide layers) were employed in order to enhance the sensitivity and to enlarge the range of a magnetic field resolved by a spin-valve sensor. The spin-valve sensor was observed to exhibit about 10 % MR (magnetoresistance). A combination of electron beam lithography and a lift-off process has been utilized to fabricate a spin-valve structure (w = 4 μm, l = 20 μm). A PDMS (polydimethylsiloxane) microfluidic channel with a height of 90 μm and a width of 100 μm was fabricated in order to transport superparamagnetic beads with d = 8.8 μm (SPHERO™ SVM-80-5) toward an active area of the spin-valve structure. Magnetic beads dispersed in deionized water were funneled into the microfluidic channel using a syringe pump. In order to generate a magnetic dipole field of magnetic beads, a DC magnetic field of 34 Oe was applied to the longitudinal direction of the spin-valve structure during the direct measurement. The real time detection of a single-bead was observed by the direct measurement of a magnetic dipole filed from a moving magnetic bead using a spin-valve sensor. It was found that the real-time signal voltage of 0.3 μV sharply dropped when a magnetic bead approached the active area of the spin-valve sensor. The signal voltage output recovered the initial voltage as the magnetic bead completely passed over the active area. This signal voltage drop is attributed to a fringe field of the magnetic bead, which partially cancels the applied field in the free layer of the spin-valve structure. The optimization of the flowing rate of the magnetic beads was performed in order to stabilize the real-time signal voltage. We extend our study to the real-time detection of animal cells coated with magnetic beads for the biological application. Our results demonstrate the possibility of implementing a chip-cytometer for biological applications using high-sensitive spin-valve sensor integrated with a microfluidic device.


P3.28
Nanolithography Studies of Chemical and Topographical Signaling on Osteoblast Cells. Somjai Sangyuenyongpipat¹, Ananda Sagari Arcot¹, Sergey Gorelick¹, Mikko Laitinen¹, Timo Sajavaara¹, Paavo Rahkila², Sulin Cheng², Matti Putkonen³ and Harry J Whitlow¹; ¹Physics, University of Jyvaskyla, Jyvaskyla, Finland; ²Sport and Health Sciences, University of Jyvaskyla, Jyvaskyla, Finland; ³Beneq Oy, Ensimmäinen savu, Vantaa, Finland.

Understanding the dynamical processes in bone development at the cellular and sub-cellular level will be an important key for understanding and developing effective treatments for bone formation disorders. In order to study living bone cell function, a method using nanometer-scale materials process technology to create an artificial environment to study bone cells under the microscope is being developed. In this study murine pre-osteoblast cells have been grown on lithographically produced substrates. The Si and glass substrates were first coated with a thin layer of hydroxyapatite-like material by sputtering or atomic layer deposition. Subsequently, 2D patterns were written using electron beam lithography while 3D patterns were produced using the high aspect ratio writing capability of MeV proton beam writing. After culturing the cells they were fixed and stained with a fluorescent stain to image the cytoskeleton under the confocal fluorescence microscope. Cell-growth substrates where a hydroxyapatite-like surface is exposed or covered in nanometer-scale geometric patterns are used to investigate how this signal effects pre-osteoblast cell proliferation and function. In a related investigation focused MeV ion beam lithography has been used to fabricate cell growth substrates with 3D patterns and controlled feature size to investigate how topography on a nanometer and micrometer scale influence cytoskeleton organization in pre-osteoblast cells.


SESSION P4: Biochemical analysis and Biosensors I
Chair: A. Woolley
Wednesday Morning, April 11, 2007
Room 2005 (Moscone West)
8:30 AM *P4.1
Abstract Withdrawn


9:00 AM P4.2
Dielectrophoresis based On-chip Particle Concentrator Using Carbon Nanofiber Electrode Arrays. Prabhu U Arumugam^1,2, Hua Chen¹, Jessica E Koehne¹, Alan M Cassell^1,2 and Jun Li¹; ¹NASA Ames Research Center, Moffett Field, California; ²UARC/UCSC, Moffett Field, California.

We report the use of carbon nanofiber nanoelectrode arrays (CNF-NEA) based dielectrophoresis (DEP) to develop an on-chip sample concentrator with E.coli as a model species. Sample preparation is one of the key functions in detection of biologically important organisms. Traditionally, it is performed through separate, standalone centrifugation and filtration systems and then transferred to a detector. The main disadvantages are cross-contamination, slow processing times, high cost and the need for skilled personnel. Recent efforts are geared towards the development of lab-on-a-chip systems (LOAC) with integrated sample processors and detection capabilities, which is yet to be fully realized. The reason for the slow progress is due to lack of design methodologies, tools and standards owing to application-specific requirements, complexity in integration of multifunctional components, and the difficulty in processing large sample volumes. We need new technologies to seamlessly integrate sample processors and develop a compact, rapid, fully automated, real-time bio-monitoring system for health care, environmental and homeland security applications. DEP is defined as the translational motion of neutral matter in an electric field gradient. The particles are separated based on their electrical properties such as permittivity and conductivity. The gradient is generated by applying an AC potential between the nanoelectrode array located on the bottom of the microchannel and a conducting electrode on the top. The advantage of using a NEA compared to existing interdigitated microelectrode arrays (MEA) is that it can concentrate particles more efficiently from high speed microflows (10-100’s of mm/s) due to high electric field gradients. This is a precondition for applications where the entire analysis needs to be performed in few minutes. We discuss in detail (i) design guidelines for on-chip concentrator through multi-physics modeling and simulations, (ii) the merits of using NEA vs. MEA in terms of collection efficiency and stopping distance, (iii) systematic study of various parameters such as microchannel dimensions, NEA array size, density and spacing, fluid velocity, particle size, electrical properties of the particle and medium, amplitude and frequency of AC voltage, and (iv) microfabrication and packaging issues.


9:15 AM P4.3
On-chip Collection and Concentration of Live Cells and DNA Based on a Combination of Dielectrophoresis and AC ElectroHydrodynamics Sonia Grego¹, Ketan H. Bhatt², Clifford T. Tse², Jonathan A. Black¹ and Orlin D. Velev²; ¹RTI International, Research Triangle Park, North Carolina; ²Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina.

We show how the use of alternating electric fields can be exploited for on-chip collection and manipulation of particles, small organisms and large biomolecules by a combination of dielectrophoresis (DEP) and AC electrohydrodynamics (AC-EHD). First, we will present a microfluidic chip that collects and concentrates colloidal particles from bulk liquid medium to a specific region of the surface. Alternating fields are applied to dilute suspensions of microspheres enclosed between a patterned silicon wafer and an ITO-coated glass slide. The latex particles entrained by the liquid flow are collected in the center of conductive "corral" patterns. The leading effect in the particle collection process is AC-EHD. We discuss how the electrohydrodynamic flows emerge from the spatially non-uniform field and interpret the experimental results by means of electrostatic and hydrodynamic simulations. On-chip collection of latex particles, yeast cells and microbes will be demonstrated. Second, we will discuss how AC-EHD methods can be combined with DEP above interdigitated electrode patterns. DEP capture of lambda-phage DNA fragments at a typical field intensity of 100 V/cm as a function of different gap sizes is investigated. The frequency is the most sensitive parameter affecting the performance of these devices as it allows “tuning” of the interactions from AC-EHD to DEP. Positive DEP (pDEP) combined with AC-EHD is observed at low frequencies, so particles are moved and captured in the areas of the highest field intensity, which are the edges of the bottom electrodes. Negative DEP (nDEP) is observed at higher frequencies, where the particles are pushed to the area of minimum electric field, tenths of microns above the electrode surface, where they can be transported by AC-EHD. The potential of these electrode designs in devices for biological analysis will be discussed.


9:30 AM P4.4
Label-Free Investigation of Enzymatic Catalysis on Surface-Bound Substrates: How Secondary Structure and Accessibility Affect Enzymatic Proteolysis of a Surface-Bound Polypeptide. Jasper Hardesty¹, Luis Cascao², Jim Kellis², Channing Robertson¹; ¹Chemical Engineering, Stanford University, Stanford, California; ²Genencor International, Palo Alto, California.

In this work, I study the interactions of enzymes with model substrate surfaces under varied environments and label-free conditions. My model system is based on serine proteases - a class of enzymes that digest proteins - and surface-bound polypeptide substrates. Previous studies have shown that single point mutations made at the surface of the enzyme, but distant from the active site, have little effect on solution activity but can have profound effects on activity with surface-bound substrates. So how do single point mutations affect the interplay between the surface processes of adsorption, reaction, and surface diffusion? With regard to substrate, how might secondary structure and film density influence surface reactivity? Furthermore, other studies have shown that labels can alter biological interactions, and given the sensitivity of our enzyme-substrate systems, it is desirable to avoid labels. Ultimately, it is hoped that elucidating the primary factors that drive overall surface reactivity will lead to rational engineering of enzymes to meet specific needs. In addition, development of label-free techniques to interrogate surface biocatalysis can lead to improved tools to researchers in many science disciplines.


9:45 AM P4.5
Electroosmotic Flow and Electromigration of DNA Molecules in a Microfluidic Device Investigated by Surface Vibration Spectroscopy. Tomoyuki Miyoshi, Ayumi Hirano, Ryotaro Yamaguchi, Ko-ichiro Miyamoto, Yasuo Kimura and Michio Niwano; Tohoku University, Sendai, Japan.

Microchip electrophoresis is attracting much attention as a high-throughput technique for separating trace amount of biological samples such as DNA and proteins. It is important for the fabrication of high-performance microchips to characterize the migration properties of biological samples and electroosmotic flow (EOF) in microfluidic channels. In this study, we have investigated the electrokinetic properties of DNA molecules in microfabricated fluidic devices using infrared absorption spectroscopy in the multiple internal reflection geometry (MIR-IRAS). Microchannels were fabricated on Si MIR-prism surfaces by using thermal oxidation, photolithographic patterning, wet chemical etching and bonding with hydrofluoric acid. Sample solutions of 10, 30 and 50-based oligonucleotide (dA10, dA30, dA50) in heavy water (D2O) were added to a sample reservoir, while a microchannel and a waste reservoir were filled with pure D2O. The electrokinetic properties of oligonucleotides in the microchannels were monitored by MIR-IRAS under a constant applied potential between the sample and waste reservoirs. We first measured the rate of EOF in the microchannels by changing a sample solution from D2O to H2O. The O-H stretching and bending modes of H2O appeared around 3400 and at 1640 cm-1, respectively. On the contrary, the O-D stretching mode of D2O around 2500 cm-1 decreased in intensity. When the sample solution was switched from H2O to D2O, the peak due to D2O appeared, while the peaks due to H2O decreased in intensity. The time courses of both experiments were in agreement with each other. The EOF rate was found to decrease with increase of the NaCl concentration. The electrophoresis rate of oligonucleotide at +50 V in D2O was slower than the EOF rate, giving rise to a peak at 1627 cm-1. This peak is due to the C=N and C=C stretching vibration modes of adenine. When the concentration of NaCl was increased to 500 mM, no noticeable peaks were observed around 1630 cm-1 throughout the experiment (1000 s), suggesting that the electrophoresis rate of oligonucleotides was faster than the EOF rate. In summary, we proposed a label-free method for monitoring the EOF and electromigration of DNA in microfluidic channels by MIR-IRAS. The major advantage of our method is that we can avoid undesirable perturbations that we often encounter in the conventional fluorometric method.


10:30 AM *P4.6
Materials Processing Methods and Issues in the Development of Nanofluidic Systems for Biomolecular Analysis. Gabriel P. Lopez¹, S. R.J. Brueck¹, Sang M. Han¹, Cornelius F. Ivory¹, Dimiter N. Petsev² and Scott S. Sibbett¹; ¹University of New Mexico, Albuquerque, New Mexico; ²Chemical Engineering, Washington State University, Pullman, Washington.

This talk will present an overview of materials processing methods developed at the University of New Mexico in the development of integrated micro- and nanofluidic systems for biomolecular analysis. Methods for rapid prototyping, high resolution lithography and facile fabrication of enclosed and porous nanofluidic channel arrays will be presented. These methods allow the creation of fluidic systems that allow dynamic control of electrokinetic molecular transport through specific localized regions of micro and nanofluidic channels. Methods for achieving molecular focusing and separations include field gradient focusing, isoelectric focusing, nanoelectrosmosis and electrokinetic analogues of field effect transistors. In addition, the molecular-scale cross-sectional dimensions of these channels may permit entirely new separations that are difficult or impossible to perform in larger-scale channels. Materials-dependent system properties, materials processing, and materials related performance issues will be emphasized.


11:00 AM P4.7
Automated Formation of Lipid bilayer Membranes within a Microfluidic Device for Channel Protein-based Sensing. Noah Malmstadt, Jason Poulos and Jacob Schmidt; Department of Bioengineering, UCLA, Los Angeles, California.

Membrane channel proteins are major pharmaceutical targets and recent work has also shown their potential as single molecule sensors. The membranes into which these proteins must be incorporated for measurement can be problematic to form and are extremely fragile, limiting channel protein-based sensing technology. In response to these shortcomings, we have created a microfluidic device capable of directing the self-assembly of lipid bilayer membranes within it. The microfluidic channels are molded in PDMS, and the solvent absorptive properties of this elastomer are used to mediate solvent extraction from a droplet of lipid-containing organic solvent. The lipid is left behind, eventually forming a lipid bilayer membrane, into which single channel proteins can be incorporated and measured. This new method of membrane formation lends itself very readily to further miniaturization and in an array format. We show the formation and measurement of these membranes and single molecule transport measurements of the proteins incorporated therein. We report on the development of a second generation of this device in which membrane arrays for automated high-throughput measurements of channel proteins are created. This technology has potential applications for drug discovery and screening as well as small molecule sensing. “Automated Formation of Lipid-Bilayer Membranes in a Microfluidic Device” Noah Malmstadt, Michael A. Nash, Robert F. Purnell, and Jacob J. Schmidt, Nano Lett. 6(9), 1961-1965 (2006)


11:15 AM P4.8
Parallel Gene Synthesis in a Microfluidic Device. David Sun Kong¹, Peter Carr¹, Lu Chen² and Joseph Jacobson¹; ¹MIT Media Lab, Center for Bits and Atoms, MIT, Cambridge, Massachusetts; ²Chemical Engineering, MIT, Cambridge, Massachusetts.

It has long been recognized that the capacity to design and synthesize genes and longer DNA constructs can be enabling to a broad cross section of applications within molecular biology including the design of genetic circuitry, the engineering of entire metabolic pathways for target molecule manufacture, and even the construction and re-engineering of viral and bacterial genomes. The core technology for custom DNA synthesis centers on the assembly of pools of oligonucleotides (oligos), typically less than 50 nucleotides in length, into increasingly larger DNA molecules. The most widely reported methods for building long DNA molecules involve variations of the polymerase-mediated assembly technique collectively termed Polymerase Construction and Amplification (PCA). Here, much like in the more conventional Polymerase Chain Reaction (PCR), three temperature steps are employed to denature, anneal, and elongate the various overlapping oligos until, after multiple rounds of thermocycling, the desired full length DNA construct is obtained. Using such polymerase-mediated techniques, researchers have successfully synthesized DNA constructs as large as tens of kb. Despite these promising results significant challenges remain, most significantly the cost and time of synthesizing long constructs. Currently, the cost for custom gene synthesis services is significant, on the order of $1.00-$1.60 dollars per base pair, with the major expenditure components for such long syntheses being attributable to reagent and sample handling. Microfluidic technology provides an elegant means to overcome these limitations. By scaling reactions down to volumes of less than a microliter, reagent costs can be substantially reduced. Furthermore, microfluidic technology enables highly parallelized synthesis along with the potential for automated sample handling and process integration. In this paper we report the synthesis and amplification of various genes in a poly(dimethylsiloxane)-based microfluidic device. Genes such as GFP (933 bp), dsRed (733 bp), a Holliday junction cleavase (hjc) gene from the bacteriophage SIRV-1 (390 bp), and a variant alba gene from S. solfataricus (327 bp) were synthesized. In other reports oligos for gene synthesis were synthesized in situ in a microarray, cleaved from a glass substrate and subsequently assembled in macroscopic (≥ 5 μl) reactions. In contrast, we have synthesized these DNA constructs in parallel within four 500 nanoliter reactors of a microfluidic device. Furthermore, the minute oligo concentrations utilized (10-25 nM each oligo) are significantly lower than concentrations expected to be attainable (without amplification) from high density oligonucleotide microarrays. Thus, such a microfluidic approach should be compatible with DNA microarray-derived oligonucleotides, further reducing the cost of this crucial reagent.


11:30 AM P4.9
Chitosan-mediated Enzyme Assembly toward Rebuilding a Metabolic Pathway in the Microfluidic Environment. Xiaolong Luo^1,5, Jung J. Park⁸, Hyunmin Yi⁷, Angela T. Lewandowski^3,6, William E. Bentley^1,6, Gregory F. Payne⁶, Reza Ghodssi^4,5 and Gary W. Rubloff^2,5; ¹Fischell Department of Bioengineering, University of Maryland, College Park, Maryland; ²Department of Materials Science and Engineering, University of Maryland, College Park, Maryland; ³Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland; ⁴Department of Electrical and Computer Engineering, University of Maryland, College Park, Maryland; ⁵Institute for Systems Research (ISR), University of Maryland, College Park, Maryland; ⁶University of Maryland Biotechnology Institute (UMBI), University of Maryland, College Park, Maryland; ⁷Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts; ⁸Polymers Division, Materials Science and Engineering Laboratory, NIST, Gaithersburg, Maryland.

We demonstrate enzyme assembly at readily addressable sites in the microfluidic environment by utilizing two unique properties of the amino-polysaccharide chitosan. The pH responsive solubility of chitosan enables site-selective electrodeposition onto conductive inorganic surfaces within microfluidic channels. Secondly, the abundant primary amine groups on chitosan allow covalent assembly of biomolecules while preserving biological activity. We have utilized chitosan as the biointerface for facile in situ biomolecule assembly in microfluidic environment. The site-selective assembly of proteins enables multiple-step, multiple-site bioreactions in metabolic engineering and many applications. For example, we can harvest an important cell signal molecule Autoinducer 2 (AI-2) if the precursor SAH is introduced over immobilized enzymes Pfs and LuxS. Here we present the unique features of our microfluidic device for biomolecule assembly, the enzyme assembly procedures and the first enzymatic reaction toward rebuilding a metabolic pathway of producing AI-2 in the microfluidic environment. The microfluidic wafer is fabricated of Au/Cr as electrodes and SU-8 polymer as microfluidic channels on a Pyrex wafer with standard lithographic and etching technique. Then the wafer is leak-tightly sealed by a top sealing PDMS layer, and the SU-8/PDMS junction is compressed with two packaging Plexiglas plates by pressure-adjustable compression bolts. Fluidic connectors are assembled and connected to external pressure-driven aqueous transport, and electric Pogo pins are assembled and connected to electrical signal to guide the biomolecule assembly onto selective sites. To assemble Pfs enzyme within microfluidic channel, a tyrosine tagged enzyme Pfs is first conjugated to chitosan. Then the Pfs-chitosan conjugate is introduced into a microfluidic channel, which is already treated with buffer to minimize nonspecific binding. Current signal is applied to the cathode to electrodeposit the Pfs-chitosan conjugate onto the readily addressable assembly site, and the electrodeposited film is then neutralized by buffer. After the enzyme is immobilized, the substrate SAH solution is continuously introduced into microchannel. The reaction product is collected and analyzed using high performance liquid chromatography (HPLC) to determine the efficiency of the enzymatic reaction. The HPLC analysis results show that the assembled Pfs efficiently convert 100% of SAH into intermediate product SRH and by-product adenine, while the negative control (no electric signal when Pfs-chitosan solution is in) shows only 20% conversion rate. Currently we are working on assembling the second enzyme LuxS to convert SRH into AI-2. Our end goal is to achieve a complete bio-microfactory by integrating a cell-based sensor to identify the final product AI-2.


11:45 AM P4.10
On-chip Detection of Chemiluminescent Biomolecules Using an Integrated Thin Film Silicon Photodiode. Ana Teresa Pereira^1,2, A. Pimentel¹, V. Chu¹, D. M. Prazeres² and J. P. Conde^1,3; ¹INESC Microsistemas e Nanotecnologias, Lisbon, Portugal; ²Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisbon, Portugal; ³Dept. of Chemical and Biological Engineering, Instituto Superior Técnico, Lisbon, Portugal.

Current biochip data acquisition is based primarily on the use of fluorescence microscope image capture of the emission from a fluorescent marker. Although these optical systems have high sensitivity, they require the use of complex image acquisition and processing systems. On-chip electronic data acquisition could improve both the speed and the reliability of the biochip pattern analysis. Previous work has demonstrated integrated photodetectors for fluorescently-tagged biomolecule detection. However, these systems require an integrated filter system to cut the excitation light. The use of chemiluminescence instead of fluorescence for on-chip detection has the advantage of not requiring either these filters or the use of an external light source. This would allow a simple integrated platform for on-chip electronic data acquisition. In a chemiluminescence analysis system, an enzymatic label such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) is used to tag the biomolecule. In the presence of the appropriate reactants, these enzymes can catalyze a light emitting reaction that can be detected by a photodiode. Two different device configurations have been developed for the successful detection of HRP in solution by capturing the light emitted by a chemiluminescent reaction using an integrated thin film amorphous silicon p-i-n photodiode. Amorphous silicon photodiodes show high photosensitivity, low dark current, and can be deposited on glass, plastic and steel substrates. In the first configuration (A), the bottom electrode of the photodiode is a transparent conductive oxide (ITO) deposited on a glass substrate. By means of a flip-chip technique the glass substrate faces the reaction chamber. In the second configuration (B), the top electrode is transparent and is passivated by silicon dioxide. In configuration B, the solution is in direct contact with the passivation layer of the photodiode. A set of a-Si:H n-i-p photodiodes with lateral dimensions of 200 x 200 microns were fabricated on a glass substrate and tested. In both device configurations it is possible to detect in real time the presence of HRP in solution. The sensitivities so far obtained are in the range of nanomole of HRP per liter of solution. Efforts are under way to increase the sensitivity of the devices. In addition, device B is being tested for detection of surface immobilized antibodies labelled with HRP, which simulations suggest should be possible. If these tests are successful, multiple parallel immunoassays could be performed using on-chip detection with integrated photodetectors.


SESSION P5: Biochemical Analysis and Biosensors II
Chair: Orlin Velev
Wednesday Afternoon, April 11, 2007
Room 2005 (Moscone West)
1:30 PM *P5.1
Encapsulation of Yeast Cells in Alginate Hydrogels Generated from Monodisperse Double Emulsion Drops. Carlos Martinez^1,2, Jin Woong Kim¹, Manuel Marquez^1,2,3 and David Weitz¹; ¹DEAS, Harvard, Cambridge, Massachusetts; ²INEST, PMUSA, Richmond, Virginia; ³Center for Computational Nanoscience, NIST, Gaithersburg, Maryland.

We have developed a technique to encapsulate yeast cells in alginate hydrogels from monodispersed water/oil/water double emulsion drops made using a capillary microfluidic device. The capillary microfluidic device consists of two tapered cylindrical glass capillaries with different tip diameters (dsmall = 20 μm to 40 μm and dlarge = 40 μm to 120 μm), that are aligned facing each other and nested within a square capillary tube. In this device the innermost fluid is pumped through the smaller tapered capillary tube, while the middle and outer fluids are pumped in opposite directions through the outer coaxial region. The three fluids are forced through the larger tapered round capillary resulting in the hydrodynamic focusing of the coaxial flow. The inner and middle fluids then break into drops, forming the double emulsions. The inner drop contains an aqueous 2% alginate solution plus yeast cells (Ncells ~ 106/mL), while the outer drop consists of mineral oil with 0.4% by wt. SPAN 80. Such a low percentage of surfactant provides just enough stability for these drops to be collected in a 500 mM CaCl2 solution. Hydrogels are formed when the aqueous alginate inner drop breaks from the mineral oil shell and comes in contact with the Ca2+ ions in solution. The hydrogels were left in the CaCl2 solution for 15 minutes to ensure full crosslinking and to minimize cell damage by the Ca2+ ions. Excess CaCl2 and mineral oil were then removed via several short centrifuging steps. Hydrogels with diameters ranging from 30 µm to 200 µm were obtained by varying the capillaries tip diameters and the flow rates of the inner and middle fluids. There were on average 0.2 cells per hydrogels and their occurrence followed a Poisson distribution. Cells were found to be viable for several days after encapsulation (using a live/dead stain). This technique provides an effective and biocompatible way to encapsulate cells in alginate hydrogels.


2:00 PM P5.2
Templated Self-assembly of Magnetic Particles for Microfluidic Cell Sorting. Antoine-Emmanuel Saliba¹, Eleni Psychari¹, Laure Saias¹, Vincent Studer² and Jean-Louis Viovy¹; ¹Curie Institute, Paris, France; ²ESPCI, Paris, France.

In the present paper we present a new method for achieving cell separation based on the self-assembly of magnetic beads guided by a ferrofluid pattern. Superparamagnetic beads are known to self-organize in columns under an external magnetic field forming an hexagonal 3D array on the first order and glass like elsewhere. These arrays have been used for separating long DNA [1]. However the pore size achievable by natural self-organization is typically smaller than 10 µm [2] and thus poorly adapted for mammalian cells sorting. In addition, arrays prepared this way cannot withstand the viscous drag imposed by cells in a flow. We propose here to impose a predefined order and pore size to the array by patterning a paramagnetic template at the bottom of a microfluidic channel. This is achieved by stamping ferrofluid using « micro-contact printing » (µCP) [3]. Under an external magnetic field, the template becomes magnetized creating a local magnetic gradient around each spot of ferrofluid. This guides the self-organization of the magnetic beads and leads to the formation of an array typically made of 10μm-diameter columns with a pore size of 40μm. In order to form an affinity column, magnetic beads are prealably grafted with an antibody mAb directed against a cell type surface antigen. A cell mixture is then flowed through the formed array and cells bearing the antigen are specifically recognized by the mAb and retained on the column. In the conference, we shall demonstrate the ability of the ferrofluid patterned surface to fix magnetic columns. The flow resistance is improved by several orders of magnitude as compared to spontaneous, non-templated arrays. We finally quantify a specific depletion of lymphocytes B from a mixture of lymphcytes B and T one the base of the differential expression of the specific lymphocytes B antigen CD19. This technique combines a high specificity and a high yield. In addition in contrast with the situation occurring in conventional cell sorting, the cells are still viable after capture, and directly available for high resolution microscopic observation. references 1. P. Doyle, J. Bibette, A. Bancaud and J. L. Viovy, Science, 2002, 295, 2237. 2. N. Minc, C. Futterer, K. D. Dorfman, A. Bancaud, C. Gosse, C. Goubault and J.-L. Viovy, Anal. Chem., 2004, 76, 13, 3770 - 3776. 3. Y. Xia and G. M. Whitesides, Annu. Rev. Mater. Sci., 1998, 28, 153-84


2:15 PM P5.3
A Controllable Microfluidic Gradient Device for Studying Neuronal Polarization. Ning Ma¹, Mu-Ming Poo² and Lydia L. Sohn¹; ¹Department of Mechanical Engineering, University of California at Berkeley, Berkeley, California; ²Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California.

We have developed a microfluidics-based device that generates a stable soluble guidance cue concentration gradient for investigating axonal chemotaxis. The device, consisting of a series of alternating cell-culture chambers and reagent reservoirs that are interconnected via microchannels, establishes and maintains steady concentration gradients within static cell-culture chambers. Concentrations of both small molecules (pharmaceutical agents and second messengers) and macromolecules (neurotrophins and other proteins) are easily achieved and quantified in this device. Thus, this device is ideally suited for quantitative studies of neuronal polarization and axon pathfinding of shear-sensitive primary neurons in response to micro-environmental cues. The device is designed to have three cell-culture chambers of size 20,000 μm × 1000 μm × 100 μm, and four reagent reservoirs. The volume of the reagent reservoirs was designed per the different diffusivities of the guidance cues used, and ranges from 2 ml for the guidance cues with lower diffusivities (e.g. BDNF, D ~ 5x10-7 cm2/s) to 6 ml for those with higher diffusivities (e.g. membrane permeable cAMP, D ~ 3x10-6 cm2/s). We use standard photolithography and soft-lithography techniques to fabricate the device. In more detail, we lithographically create a negative master on a silicon wafer, which is subsequently cast into a poly(dimethylsiloxane) (PDMS) slab, a well-known biocompatible material that has been used for a variety of cell-culture applications. The PDMS slab is then cleaned and reversibly sealed to a Poly-L-Lysine coated microscope slide. Specific guidance cues with different concentrations, e.g. 100 μg/mL, 10 µg/mL, etc., are loaded into different reagent reservoirs. They then diffuse through the interconnecting microchannels and establish specific concentration gradients in each cell-culture chamber. We have successfully cultured embryonic rat hippocampal neurons in our devices and have observed good cell viability over seven days of culture, a critical time period during which neurons mature and polarize. The rat hippocampal neurons in our devices undergo normal polarization and develop an axonal process that sprouts from the cell body. In this presentation, we will show these results and preliminary data that demonstrate our ability to measure the minimum concentration gradient required to guide the path-finding of the axon growth-cone in response to known guidance cues. Overall, our device is truly a platform technology, as it is also capable of identifying novel guidance cues that regulate neuronal development and guide axonal movement. As well, it enables the simultaneous testing of multiple environmental cues to discern potential signaling crosstalk. Our future studies include evaluating opposing and reinforcing gradients of guidance cues to identify combinations that optimize the distance over which the axon can be guided.


2:30 PM P5.4
A Microfluidic Array with Micro Cell Sieves for Cell Cytotoxicity Screening Zhanhui Wang¹, Min-Cheol Kim¹, Manuel Marquez² and Todd Thorsen¹; ¹Massachusetts Institute of Technology, Cambridge, Massachusetts; ²Research Center, Philip Morris USA, Richmond, Virginia.

There is currently great interest in cell arrays for cell-based studies to improve experimental throughput. Microfluidics is an inherently scalable technology, offering an exciting new alternative to fabricate cell arrays for cell studies in a parallel fashion. In recent years, this method has been applied to several cell-based biological studies including mammalian cell patterning in an enclosed array, cellular responses to chemical gradients, investigation of celluar differentiation, and observation of dynamic gene expression. However, current microfluidic cell arrays are unable to screen multiple cytotoxins with multiple living mammalian cells. In addition, there are some difficulties to fabricate microfluidic live cell array for toxin screening. Uniform cell loading and distribution are main challenges in a large array format, because cells are suspended in medium and very small perturbations to fluid flow will significantly disturb cell positions in culture chamber with nanoliter volume. Inhomogeneous cell loading and distribution can affect cell viability evaluation after toxins exposure, leading to false toxins screening result. In this report, we developed a microfluidic array platform with lithography molding technology for high-throughput cell cytotoxicity screening. The channels in this platform were individually addressable in both directions (column and row), enabling parallel loading of various cell lines in one direction and introducing of different toxins in the other direction. The channels for cells seeding were orthogonal to channels for toxins exposure, and each region at channel intersection was a circular chamber which was compartmentalized by array valves. Several micro cell sieves were built in each culture chamber to form several low flow velocity regions, and cells can be trapped and immobilized within cell sieves. Cell number and distribution in chambers can be conveniently controlled by the adjustment of cell sieve number, distribution and size.


2:45 PM P5.5
Development of a Lab-on-a-Chip for the Characterization of Human Cells. Peter Ertl, Lukas Richter, Christoph Stepper, Hubert Brueckl, Rudolf Heer and Michael Kast; ARC-Seibersdorf research, Nano-Systems-Technologies, Vienn, Austria.

Over the past decade, the miniaturization of analytical techniques by means of MEMS technology has become a dominant trend in research. The creation of microanalytical systems, such as biochips have demonstrated the ability to provide quantitative data in real-time and with high sensitivity. Microfluidic biochips or lab-on-a-chip systems are vital for biological analysis because they allow spatial and temporal control of growth conditions. Monitoring cell behavior under varying conditions and understanding genotype-phenotype interactions in the context of a living cell is expected to have a considerable impact on medicine. The principle behind cell analysis is that a cellular phenotype represents the expression of a genotype, thus revealing gene function and its interaction with the environment. However, to gain a deeper biological understanding of cells, it is necessary to first make progress in experimental devices, as well as computational and analytical methods. The objective of the developed biochip is to monitor real-time cellular phenotype dynamics under varying conditions. The lab-on-a-chip is designed to continuously assess cell viability, reproduction and metabolic activity over long periods of time using different sensors on a common chip platform. The integrated fluidic and heating systems allow controlled manipulation of living cells adhered to modified/activated chip surfaces that are comparable to biological niches. Furthermore, the chip contains an integrated reference arm providing a low-noise detection environment by eliminating background signals and interferences. The presented work addresses aspects of chip design, fluidic flow profiles, sensor characterization and on-chip cultivation of HeLa cell growth. Additionally, sensor performance will be shown using various microbial strains of known differences in cell morphologies. Furthermore, chemometric analysis from data obtained with cellular dielectric spectroscopy and pattern recognition results will be presented.


3:30 PM *P5.6
Strategy and Method for Construction of Micro-Nano Chemical Process on Microchip. Takehiko Kitamori, Department of Applied Chemistry, The University of Tokyo, Tokyo, Japan.

TBD


4:00 PM *P5.7
Lab-On-A-Chip Devices for Protein Analysis & Clinical Diagnostics. Anup Singh, Sandia National Labs, Livermore, California.

Lab-on-a-chip or microfluidic devices are attracting significant attention in the area of biochemical analysis because of their portability, speed of analysis, potential for multiplexing and high-throughput, and ability to analyze minute sample volumes. In this talk, I would present a few examples of application of microfluidic chips for protein separation and clinical diagnostics. The two most commonly used techniques for protein separation are chromatography (e.g., HPLC) and gel electrophoresis. Their miniaturization holds substantial promise for analysis of complex biological samples as microchip-based separation offers faster analysis (minutes), better sensitivity, and ability to analyze minute amounts of sample. Microchip-based chromatography and gel electrophoresis were developed using a photopolymerization technique to controllably and reproducibly place porous polymer matrices in the channels of a chip. The polymer matrices can be cast in situ in less than 10 minutes and are robust and reproducible with respect to separation characteristics. Microchips containing photopatterned acrylate were used for chromatography of peptides and amino acids and yielded separations that were fast (6 peptides in 45 sec), efficient (up to 600,000 plates/m) and reproducible (run-to-run variability <3%). SDS-PAGE-in-a-chip was developed by using photopolymerized crosslinked polyacrylamide and led to separation of 6 proteins of molecular weight from 20 to 200 kD in less than 30 seconds using a 1 mm-long channel. An integrated on-chip concentrator enabled detection of proteins at concentration as low as 100fM. Taking advantage of the small size of the chips and the rapid analysis they offer, we have also developed a point-of-care device for detection of disease biomarkers in saliva. Saliva offers many advantages over other bodily fluids because it is easy to collect using noninvasive methods in both clinical and non-clinical settings. The device performs rapid microfluidic chip-based immunoassays (< 3-10 minutes) with low sample volume requirements (10 µL) and appreciable sensitivity (nM-pM). Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The chip has been integrated with miniaturized electronics, optical elements, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device is being tested by detecting biomarkers in saliva samples from patients diagnosed with periodontal disease.


4:30 PM P5.8
Biosensing using Thin Film Microresonators. Joao Pedro Conde^1,2, Teresa Adrega¹, Guandong Zhang¹, Ana Teresa Pereira^1,3, Samadhan Bhaulal Patil¹, Duarte Miguel F. Prazeres^2,3 and Virginia Chu¹; ¹INESC MN, Lisbon, Portugal; ²Deptartment of Chemical and Biological Engineering, Instituto Superior Tecnico, Lisbon, Portugal; ³Center for Biological and Chemical Engineering, Instituto Superior Tecnico, Lisbon, Portugal.

There has been growing interest in using microelectromechanical systems (MEMS) as biological sensors. Microresonators, in particular, have been proposed as sensors to detect and quantify the presence of specific biological compounds. Thin-film silicon and polymer-based MEMS have recently been developed to benefit from the advantages of thin-film technology. Thin film materials are of great interest for electronic devices and MEMS applications due to their relative low cost and simple processing. Thin-film silicon and all-polymer suspended microbridges incorporating a conductive polymer are fabricated on glass substrates using surface micromachining. The use of low temperature processing (< 110°C) allows the use of substrates such as glass, plastic and stainless steel. In addition, thin-film MEMS are CMOS compatible enabling the monolithic integration of MEMS with its control electronics. This work presents DNA and protein sensors based on thin-film microresonators. The sensor works by the measurement of the resonance frequency shift induced by a specific biological reaction on a resonating microbridge. Hybridization of DNA oligonucleotides and antibody-antigen interactions are the biological models used. The thin-film silicon sensor is fabricated using surface micromachining and consists of a n⁺-a-Si:H/ aluminum bilayer microbridge with SiO₂ patterned on the top. An aluminum gate underneath the bridge allows the electrostatic excitation of the microbridge. The resonance frequency of the microbridges is in the 1-10 MHz range. Quality factors in vacuum are of the order of 1000. For the polymer-based microbridges, a blended conductive polymer material of polymethyl methacrylate (PMMA) and Baytron P (a waterborne dispersion of the polymer complex PEDOT and PSS) is used as the structural layer. In the fabrication process, a Cr gate is first deposited and patterned on a glass substrate. Then, an Al film is deposited and patterned on the Cr gate to function as the sacrificial layer. The resonance frequency of the polymer bridge is in the MHz range and the quality factor in vacuum is of the order of 100. Covalent and electrostatic immobilization of DNA oligonucleotide probes, and subsequent hybridization of target DNA, as well as covalent immobilization and adsorption of probe proteins, and their subsequent interaction with target proteins, will be described for thin-film silicon microresonators. In these devices, resonance frequency shifts are below 1% in vacuum and are controlled by the mass loading of the microstructure. These results will be compared to the adsorption of proteins and DNA on polymer microresonators. Larger frequency shifts (above 1% in vacuum) are observed in the polymer MEMS biosensors, and these shifts are attributed to a higher sensitivity of the polymer microresonator to the stress induced by the immobilization of the biomolecules, because of the significantly lower rigidity of the polymeric microbridges.


4:45 PM P5.9
Chip-Scale Affinity Microcolumn Biosensors for Toxic Agents Mangesh T. Bore¹, Aurelio Evangelista¹, Linnea Ista¹, Steven R.J. Brueck² and Gabriel P. Lopez¹; ¹Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico; ²Center for High Technology Materials, University of New Mexico, Albuquerque, New Mexico.

This presentation will describe a microscale, multi-threat agent detection system (including unknown agents) using toxin receptor binding and electrokinetic separations in microchannel. Most current threat detection systems rely on precise identification of the biological or chemical toxin. While this approach has its uses, it is ineffective against either newly developed or modified threats that, by novelty or design, can evade precise recognition elements. In our strategy, the potential physiological effect is key, and the exact identity of the threat agent is secondary. Because the detector is the target of the threat, or one of the targets of the threat, either novel threats, or those deliberately designed to thwart current detection schemes, will be quickly detected. The technology is based on two essential components: binding of potential toxins to receptors, enzymes and other biomolecules known to be affected by toxic agents (including chemical, biochemical and biological agents) followed by detection of binding events by altered electrokinetic mobility. We have developed a rapid prototyping method for forming packed microcolumns at the intersection of a cross microfluidic design containing sample, separation and waste streams. This simple cross design forms the prototype component for all of the chip designs envisioned. We have conducted proof-of-concept experiments that demonstrate that the microfluidic design for achieving the new biosensing approach envisions works. Specifically methods for microcolumn packing, sample introduction, pumping, analyte capture, analyte release, separation of receptor and receptor/ analyte complex, and finally detection of receptor and receptor analyte complex (receptor/toxin pair is Ganglioside GM1/Cholera Toxin).


SESSION P6: Assembly and Synthesis of Micro/Nanostructures
Chair: Joao Conde
Thursday Morning, April 12, 2007
Room 2005 (Moscone West)
8:30 AM *P6.1
Magnetic Nanocrystals as Building Blocks for Biotags and Sensors. Christopher Bruce Murray, Chemistry & Materials Science, University of Pennsylvania, Philadelphia, Pennsylvania; Nanoscale Materials & Devices, IBM T. J. Watson Research Center, Yorktown Heights, New York.

The synthesis of colloidal nanocrystals provides a rich family of nanoscale magnetic building blocks with which to assemble new biological tags and from which to assemble novel devices. This talk will briefly outline some of the current “best practices” in preparation, isolation and characterization of magnetic nanoparticles (Co, Ni, Fe alloys and FePt, CoPt3 intermetallics and a range of iron oxides an transition metal ferrites). I will share some of our effort to make the magnetic materials water soluble and to functionallize these with DNA as a well model biotin-avidin labels. Magneto-resistance in magnetic nanocrystal films. These transport properties are extremely sensitive to the nature and dimension of the organic ligands surrounding the particle and thus could be the basis for potential sensing technologies. The potential to design new nanoparticles tags expands dramatically with the creation multifunctional nanoparticle systems. I will share progress in the design of multicomponent systems. The potential to exploit biological interaction to direct these assembly processes is particularly exciting. Although work to date has exploit simple chemical and electrostatic interactions the prospect of harnessing more specific biochemical interactions could open and entirely new front in this research.


9:00 AM *P6.2
Continuous Microfluidic Reactors for Polymer Colloids. Eugenia Kumacheva, Zhihong Nie, Minseok Seo, Shengqing Xu, Ethan Tumarkin, Hong Zhang and Patrick Lewis; Chemistry, University of Toronto, Toronto, Ontario, Canada.

This presentation reviews our progress in the continuous production of polymer colloids by microfluidic methods. The method includes the generation of highly monodisperse monomer droplets by microfluidic emulsification and in-situ (on chip) solidification of these droplets by means of photopolymerization or gelation. The strategy has the following features: (i) it produces particles with an exceptionally narrow size distribution; (ii) it offers control over microbead shapes; (iii) it produces particles with a precise control over their internal structure; (iv) it is applicable to a variety of materials, including: gels, polymers and copolymers, and polymers doped with functional additives; (v) it can be used to carry our successive reactions to generate hybrid microbeads.


9:30 AM P6.3
Controllable Fluidic Assembly of Nanostructures by Chaotic Advection. David A Zumbrunnen, Mechanical Engineering, Clemson University, Clemson, South Carolina.

Research in the author’s laboratory has demonstrated that nano-scale materials can be controllably assembled in the melt by instilling chaotic advection [1]. Smart blending devices have resulted that are currently being adopted. Chaotic advection refers to chaotic motions in fluid markers that can arise even in response to simple flow fields [2,3]. Chaotic advection has two defining and related characteristics of significance to in situ structure development. In one characteristic, the region of space enclosing an initial minor component body becomes stretched and folded. Stretching and folding can occur recursively until nano-scale dimensions are attained. In another characteristic, the positions of individual nano-particles diverge exponentially fast over time. Networks of particles can emerge with features such as alignment or interconnections that can be tailored. Unlike mixing, material components become organized on progressively smaller length scales and nano-structured materials with hierarchical features can arise. In addition to the nano-scale dimensions in materials produced, property enhancements are derived from the structural arrangement of nano-solid additives or nano-scale shapes formed in melt-processable materials. Moreover, it has been found that a multi-layer morphology is a parent to other morphologies. Multi-layers, can for example, transform to dual phase continuous, sponge-like structures. Nano-sponges can thereby be produced [4]. Processing can be done continuously and in either small or large capacities. Miniature devices operate much as do larger devices for large-volume production. Examples of nanocomposites that have been produced include extruded polymeric multi-layer films having thousands of discrete layers and where individual layers have thicknesses of only a few nanometers, multi-layer polymeric films having alternate layers with aligned nano-platelets, composites with aligned nanotubes, and composites with conducting networks. Where molecules are synthesized to self-assemble, results suggest that methods may yield expansive functional supramolecular structures. References [1] Zumbrunnen, D. A., Nano Letters, 2: 1143( 2002). [2] Aref, H., J. Fluid Mech., 143: 1 (1984). [3] Aref, H., Phys. Fluids, 14, 1315 (2002). [4] Joshi, A.S. and Zumbrunnen, D. A., Chem. Eng. Comm., 193: 765 (2006).


9:45 AM P6.4
Microfluidic Electrospinning of Hollow and Core/Sheath Nanofibers. Yasmin Naveen Srivastava^1,2, Manuel Marquez^2,3,4 and Todd Thorsen¹; ¹Mechanical Engineering, MIT, Cambridge, Massachusetts; ²INEST Group Postgraduate Program, Philip Morris USA, Richmond, Virginia; ³NIST Center for Theoretical and Computational Nanosciences, NIST, Gaithersburg, Maryland; ⁴Harrington Department Bioengineering, Arizona State University, Tempe, Arizona.

The versatile technology of electrospinning for the preparation of polymer nanofibers has been recognized as an efficient technique to generate sub-micron scale nanofibers [1,2]. In the conventional electrospinning method, a syringe with a fixed inner diameter of 0.3-1 mm is used as an electrospinning source which, in many cases, limits the process by single jet and single component spinning. Recently, co-axial electrospinning has been extensively exploited as a simple technique to generate hollow and core/sheath nanofibers [3,4,5,6,7]. However, the process of making coaxial-spinnerets is labor-intensive and, to date, has utilized a single spinneret source for generating hollow nanofibers. In this communication, we describe the development of a multichannel microfluidic device for the parallel electrospinning of single, composite, hollow and core/sheath nanofibers. Advantages of this technology over conventional syringe-based methods include the ability to dynamically combine multiple components into a single nanofiber, rapid prototyping and the ability to spin multiple fibers in parallel through arrays of individual microchannels. Nanofibers of poly (vinylpyrrolidone) (PVP) and a conducting composite (PVP+Polypyrrole (PPy)) were successfully fabricated using this microfluidic integration with electrospinning. Fourier transform infrared spectroscopy and conductivity measurements reveal the polymerization of pyrrole in the matrix of PVP. Also, this microfluidic methodology was successfully used to fabricate hollow PVP + titania (TiO2) and core/sheath PPy/PVP nanofibers of the order of 100 nm and 250 nm respectively. The design utilized two layers of microchannels to flow PVP solution as sheath material and heavy mineral oil or pyrrole as the core phase through an array of spinners. Two layers of non-intersecting, stacked 100µm (w) × 100µm (h) microchannels are arranged in a branching tree pattern to provide constant pressure to each of eight outlet spinnerets. Hollow composite nanofibers of PVP + TiO2 were synthesized from the PVP + TiO2 / heavy mineral oil nanofibers by extracting the mineral oil core with octane. Fiber characterization was subsequently carried out using a combination of Scanning electron microscopy, Transmission electron microscopy and Fourier transform infrared spectroscopy. References 1. Formhals, A. US 1,975, 504, 1934. 2. Reneker, D. H.; Chun, I. Nanotechnology, 1996, 7, 216-223. 3. Loscertales, I.G.; Barrero, A.; Marquez, M.; et al. J. Am. Chem. Soc. 2004, 126, 5376-5377. 4. Li, D.; Xia, Y.N. Nano letters 2004, 4, 933-938. 5. Li, D.; McCann, J.T.; Xia Y.N. Small 2005, 1, 83-86. 6. McCann, J.T.; Li, D.; Xia, Y.N. J. Mater Chem. 2005, 15, 735-738. 7. Sun, Z. C.; Zussman, E.; Yarin, A. L.; Wendorff, J. H.; Greiner, A. Adv Mater. 2003, 15, 1929-1932.


10:30 AM *P6.5
Droplet-Based Microfluidics for High Throughput BioAssays. David A. Weitz, Department of Physics and Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts.

This talk will describe the use of microfluidic devices to precisely control independent droplets of water in an inert carrier oil. Each droplet contains several picoliters to femtoliters of fluid, and each can be controlled and manipulated with a high degree of precision. This allows these droplets to be used as minute microreactors for high throughput screening purposes. This talk will describe the microfluidic devices and some potential uses.


11:00 AM P6.6
Programmable Manufacturing of Anisotropic Particle Assemblies by Fluidic Processing. Kyung Eun Sung¹, Deshpremy Mukhija¹, Siva A. Vanapalli¹, Hugh McKay², Joanna Mirecki-Millunchick², Michael J. Solomon¹ and Mark A. Burns^1,3; ¹Chemical Engnieering, University of Michigan, Ann Arbor, Michigan; ²Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan; ³Biomedical Engineering, University of Michigan, Ann Arbor, Michigan.

We present a method for continuous manufacturing of anisotropic particles by fluidic processing using precursor particles that are conveyed and fused in distinct sequences. Controlled manufacture of microparticles with programmable anisotropy and sequence may be applied to emergent applications such as chemical sensors, barcoding, optical materials, and microelectronics. To date, very few techniques have been developed to synthesize non-spherical anisotropic particles, especially those with complex interaction sequences. Because the method uses rigid microparticles, it can make use of solid colloidal precursors that possess a wide range of functionality. In the process, particles are transported by pressure control into the reaction zone of a microfabricated device where physical or chemical fusing occurs. The sequence of particles is maintained by matching the channel and particle dimensions. The particles are held in this zone by means of a constriction in the flow geometry. For physical fusing, a microfabricated on-chip heater is used to pulse device temperatures to the softening temperature of polymer microparticles. Thermal fusing can be performed in less than 300ms, implying current manufacturing rates of >10⁴ particles/hour in a single channel. This pressure controlled manufacturing is fully programmable, allowing complex sequences and configurations of synthesized particles to be obtained. Examples of bonded particle assemblies with sequence and shape anisotropy are presented in addition to channel design heuristics that may be applied to produce general classes of particle anisotropy.


11:15 AM P6.7
Microfluidic Assembly of Granular Shells and Janus Colloidal Granules Robert F Shepherd¹, Jacinta C. Conrad^1,3, Summer K. Rhodes¹, Darren R. Link², Manuel Marquez³, David Weitz⁴ and Jennifer A. Lewis¹; ¹Materials Science, University of Illinois, U-C, Urbana, Illinois; ²RainDance Technologies, Inc., Guilford, Connecticut; ³Phillip Morris USA, Richmond, Virginia; ⁴Physics, Harvard University, Cambridge, Massachusetts.

The microfluidic assembly of colloid-filled hydrogel drops and dried granules of varying shape and composition is investigated. Drops are formed by shearing a concentrated colloidal microsphere-acrylamide suspension in a continuous oil phase using a sheath-flow or a double emulsion capillary device. Silica microspheres are synthesized with different fluorescent cores to allow direct visualization of the process. Homogenous and Janus (hemispherically distinct) spheres and disks are produced by confining the assembled drops in microchannels of desired geometry while granular shells of micron size colloids are produced in a double emulsion micro-capillary device. To preserve their drop structure, photopolymerization of an acrylamide-based hydrogel solution is carried out immediately after drop-breakup. Representative drops and dried granules are imaged using fluorescence and scanning electron microscopy to probe their structural evolution during assembly and drying while micro-CT is used to probe packing of the disk and sphere geometries. Our approach offers a facile route for assembling colloid-filled hydrogel drops and dried granules with controlled morphology and composition.


11:30 AM P6.8
Materials Strategies for Advanced NanoTechnology. Kyung M. Choi, Bell Labs, Lucent Technologies, Murray Hill, New Jersey.

Since we have sought new advances in nanotechnology, developments of new materials and new synthesis techniques have been pursued to meet our growing demands in miniaturization. In this talk, we will present design of new materials by fabricating microfluidic reactors specifically designed for microfluidic synthesis, taking advantage of micro-scale mixing and of the use of quenching sequences for greater reaction selectivity. The use of microfluidics offers a number of potential advantages over existing technology. Chemical mixings and reactions run in microfluidic devices have high thermal and mass transfer rates with an opportunity to use more aggressive reaction conditions allowing for improved product yield. Moreover, high chemical homogeneity can be achieved by complex mixing. The microreactors may also be coupled to additional processing steps (i.e. multistep synthesis) and in some cases the product is transported directly for integration into an application device or as part of an assay. The overall goal is to carry out all operations normally performed in a chemical laboratory including synthesis, processing, mixing, purification and analysis on one microfluidic reactors efficiently and economically using minute amounts of solvents and reagents.


11:45 AM P6.9
Selective Enrichment of Phosphorylated Peptides by Magnetic Nanoparticles and Mesoporous Magnetic Sub-micron Particles. Yi Huang¹, Chia-Kuang Tsung², Qihui Shi², Pengyuan Yang¹, Galen D Stucky² and Xian Chen^1,3; ¹Institutes of Biomedical Sciences, Fudan University, Shanghai, China; ²Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, California; ³Department of Biochemistry and Biophysics, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina.

Protein phosphorylation is a one of most critical posttranslational modifications (PTMs) that regulate various biological processes including signal transduction, cellular regulations, etc. Mass spectrometry (MS) has been emerging as the most precise tool for identifying phosphorylated proteins and mapping out those biologically relevant phosphorylation sites. However, due to the low ionization efficiency of phosphopeptides the signals from phosphopeptides can always be suppressed by their nonphosphorylated counterparts. Pre-enrichment and isolation of phosphorylated peptides from proteolytic peptide mixtures resulting from enzymatic digestions of proteins becomes a critical step for tandem MS/MS-based site determination. This procedure eliminates nonphosphopeptide interferences and specifically enhances the signal from phosphopeptides. The common enrichment strategy is the immobilized metal ion affinity chromatography (IMAC), which uses Fe3+, Ga3+, or other metal ions to capture phosphopeptides. Recently, metal-containing nanoparticles have shown a high efficiency as well as a high extraction capacity for phosphopeptide enrichments because of their large surface area-to-volume ratio. Here we demonstrate a series of our newly developed magnetic nanoparticles (Fe3O4) and submicron mesoporous magnetic particles (TiO2) that can selectively enrich phosphorylated peptides from peptide mixtures in solution. Beta-casein and casein conatining the phosphorylated sites were analyzed by our new nanoparicle enrichers, meanwhile, other four nonphosphorylated proteins (bovine serum albumin, hemoglobin, myoglobin and cytochrome c) were used as the controls of non-phosphorylated peptides. The enrichment efficiency was evaluated by matrix assisted laser desorption and ionization time-of flight mass spectrometry.


SESSION P7: Fluid Transport and Modeling
Chair: E. Kumacheva
Thursday Afternoon, April 12, 2007
Room 2005 (Moscone West)
1:30 PM *P7.1
Multiphase Flow in Small Devices: From Colloidal Shells to Numerical Simulations for Drop Breakup and Gas-liquid Microreactors. Howard A. Stone, Engineering & Applied Sciences, Harvard University, Cambridge, Massachusetts.

Rapid advances have occurred in the use and control of two-phase flows in small devices. Many uses of small drops have been identified and tested. In one variant of this idea, a fluid-fluid interface serves as the site for assembly of colloidal particles, which is one route to synthesizing two-dimensional structured materials. We describe a microfluidic method that allows direct visualization and understanding of the dynamics of the growth of colloidal crystals on a curved interface. We show how this approach allows control over composition and size of the colloidal armor, including making janus shells. In addition, we summarize some of the progress we have made with numerical simulations for two-phase flows in confined systems. In particular, we describe three-dimensional simulations for breakup at a T-junction and, time allowing, will summarize our study of mass transfer in segmented gas-liquid systems.


2:00 PM P7.2
Diffusion Effects on Fractionation by Deterministic Lateral Displacement in a Microfluidic Device. John A Davis¹, David W Inglis¹, Suberr L Chi¹, Robert H Austin² and James C Sturm¹; ¹Electrical Engineering, Princeton University, Princeton, New Jersey; ²Physics, Princeton University, Princeton, New Jersey.

The continuous-flow microfluidic technique of fractionation by deterministic lateral displacement has been used to fractionate sub-micron size polystyrene beads(1) with 1% resolution and to separate red blood cells, white blood cells, and platelets from blood plasma(2). The method device takes advantage of the asymmetric bifurcation of laminar flow around an array of obstacles. This asymmetry creates a size-dependent deterministic path through the device. All components of a given size ideally follow equivalent migration paths which are independent of speed, leading to high-resolution separation at high speeds. In this abstract, we identify two different diffusion mechanisms which can degrade the resolution of the device. Each is quantitatively analyzed and compared to experimental data with good agreement. The model differentiates between “unconstrained” diffusion far from obstacles and “constrained” diffusion, which occurs when the particles are near an obstacle and can only diffuse laterally in only one direction. The unconstrained diffusion causes a general broadening, which includes displacing separated particles away from the ideal deterministic path, causing a non-ideal separation. Constrained diffusion causes sub-threshold non-separated particles to bifurcate, an effect which previously was not understood. The model has no adjustable parameters, and results agree quantitatively with measured data for particle separations with particle sizes down to 200 nm. The results emphasize the improved performance at increased speeds. As the particle size becomes smaller, higher fluidic velocities are necessary to overcome non-idealities caused by diffusion in our deterministic separation device. (1) Science vol. 304 no.14 (2004) 987-990 (2) PNAS vol. 103 no. 40 (2006) 14779-14784


2:15 PM P7.3
Tuning Surface Dynamics for Transport in Microchanels. Lyderic Bocquet, LPMCN, université de Lyon and CNRS, Lyon, France.

I will present various strategies to tune and take benefit of surface dynamics for microfluidic purposes. Surface do strongly affect flows in small chanels due to the no-slip boundary condition for the hydrodynamic velocity at the boundaries. I will first show how to bypass this constraining condition by tuning the surface properties. To this end, we have developped patterned surfaces made of superhydrophobic carbon nanotube carpets, embeded moreover in a microchanel. Measurements of water flows close to these surfaces using micro-PIV demonstrate a large slippage at the boundaries [1], in full quantitative agreement with theoretical predictions for flows on patterned surfaces. In a second step, I will discuss how interfacially driven transport (such as electro-, diffusio-, or thermo- osmosis-) can be strongly enhanced by tuning surface properties [2,3]. Applications of these effects for microfluidic purposes (flow transport, mixing, etc.) will be discussed . [1] « Slippage of water past superhydrophobic carbon nanotube carpets in microchanels », P. Joseph, C. Cottin, J.-M. Benoit, C. Ybert, C. Journet, P. Tabeling, L. Bocquet, Physical Review Letters 97 156104 (2006) [2] « Hydrodynamics within the electric double layer on slipping surfaces », Laurent Joly, Christophe Ybert, Emmanuel Trizac, Lydéric Bocquet, Physical Review Letters 93 257805 (2004). [3] « Giant amplification of interfacially driven transport by hydrodynamic slip : Diffusio- osmosis and beyond », Armand Ajdari, L. Bocquet, Physical Review Letters 96 186102(2006)


2:30 PM P7.4
General Continuum Boundary Conditions for Miscible Binary Fluids from Molecular Dynamics Simulations. Colin Denniston¹ and Mark O. Robbins²; ¹Applied Mathematics, University of Western Ontario, London, Ontario, Canada; ²Physics and Astronomy, Johns Hopkins Univ., Baltimore, Maryland.

Molecular dynamics simulations are used to explore the flow behavior and diffusion of miscible fluids near solid surfaces. The solid produces deviations from bulk fluid behavior that decay over a distance of order the fluid correlation length. Atomistic results are mapped onto two types of continuum model: Mesoscopic models that follow this decay, and conventional sharp interface boundary conditions for the stress and velocity. The atomistic results, and mesoscopic models derived from them, are consistent with the conventional Marangoni stress boundary condition. However there are deviations from the conventional Navier boundary condition that states the slip velocity between wall and fluid is proportional to strain rate. A general slip boundary condition is derived from the mesoscopic model that contains additional terms associated with the Marangoni stress and diffusion, and is shown to describe the atomistic simulations. The additional terms lead to strong flows (~m/s) when there is a concentration gradient and wetting. The potential for using this effect to make a nanomotor or pump is evaluated.


2:45 PM P7.5
Study of FET Flow Control of Proteins and pH Changes in Nanochannels Using Scanning Laser Confocal Fluorescence Microscopy and Multiple Internal Reflection Fourier Transform Infrared Spectroscopy Youn-Jin Oh¹, Danny Bottenus³, Dimiter N Petsev¹, Steven R J Brueck², Lopez P Gabriel¹, Cornelius F Ivory³ and Sang M Han^1,2; ¹Chemical & Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico; ²Center for High Technology Materials, University of New Mexico, Albuquerque, New Mexico; ³Chemical Engineering, Washington State University, Pullman, Washington.

We have studied the field-effect-transistor (FET) flow control and separation of proteins in a parallel array of nanochannels (100 nm W ×500 nm D), using scanning laser confocal fluorescence microscopy (SL-CFM) and multiple internal reflection Fourier transform infrared spectroscopy (MIR-FTIRS). For fluidic FET, a DC potential is applied to the gate surrounding an isolated mid-section of the channels. The gate potential controls the surface charge on SiO₂ channel walls and therefore the zeta-potential. Depending the polarity and magnitude, the gate potential can accelerate, decelerate, or reverse the flow. From our experiments, we observe that the electroosmotic flow of protein molecules is successfully controlled by the gate potential. We also detect a pH shift in the nanochannels according to the surface charge modulation, using SNARF-1 and Fluorescein as pH indicators. For instance, using MIR infrared waveguide, into which nanochannels are integrated, we observe that Fluorescein dye molecules are hydrogenated and dehydrogenated in response to the gate bias and subsequent pH shift. We demonstrate that the pH shift affects the FET flow control with SL-CFM analysis. A nanochannel device containing multiple gates is used to improve the controllability of protein flow and to introduce a pH gradient along the channels for isoelectric focusing. A different potential is applied to each gate to differentially control the surface charge on the SiO₂ channel walls and to create a pH gradient along the channels. The control and separation of proteins, the pH gradient in the nanochannels as a function of gate bias, and the molecular orientation will be further discussed in this presentation.


3:30 PM *P7.6
Microevaporators for Phase Behaviour Studies and Materials Formulation. Jacques Leng^1,2, Mathieu Joanicot² and Armand Ajdari¹; ¹UMR Gulliver 7083, CNRS-ESPCI, Paris, France; ²Lab Of the Future, Rhodia/CNRS/Université Bordeaux 1, Pessac, France.

We illustrate the potential of microevaporators that we have recently introduced for the study of the equilibrium phase diagram of aqueous mixtures, as well as some of their kinetic properties. We demonstrate good control, parallelization, use of minute quantities, and applicability to a broad variety of systems (electrloyte solutions, surfactants, colloidal dispersions, ..). A detailed description of the case of AOT/water mixtures is proposed, for which four different phases are observable. Some perspectives for controlled generation of composite materials are also presented.


4:00 PM P7.7
Distributed Microfluidic Pumping, Mixing and Separations Using Remotely Powered Miniature Diodes. Suk Tai Chang¹, Vesselin N. Paunov², Dimiter N. Petsev³ and Orlin D. Velev¹; ¹Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina; ²Surfactant and Colloid Group, Department of Chemistry, University of Hull, Hull, United Kingdom; ³Department of Chemical and Nuclear Engineering and Center for Biomedical Engineering, University of New Mexico, Albuquerque, New Mexico.

We demonstrate how miniature diodes embedded into microfluidic channel walls can provide locally distributed pumping or mixing functions powered by a global external field. The millimeter-sized diodes attached to the walls rectify the voltage induced between their electrodes from external alternating electric field. The resulting electroosmotic flux localized on the surface of diodes pumps fluids in the microfluidic channel in the direction of either the cathode or the anode depending on their surface charge. The flow velocity linearly increases with applied voltage, but does not depend on the frequency of the applied field. This could eliminate intrinsic problem with vortices in areas of non-uniform field that occur in conventional AC electrohydrodynamic pumps. The localized electroosmotic flow between diodes could also be used to construct microfluidic mixers. Theoretical analysis and numerical simulations of the microfluidic pumping and mixing provided by the diodes on a chip were in excellent agreement with the experimental data. The combined application of AC and DC fields in our microfluidic chips allows decoupling the velocity of the particles and the liquid. By precisely and independently adjusting the magnitude of the AC and DC fields particles with small differences in their charges or sizes could be efficiently separated. The technique can be used in "smart", dynamically reconfigurable, microfluidic chips.


4:15 PM P7.8
Microfluidic Devices to Investigate Kinetics and Thermodynamics of Crystallization Processes. Philippe Laval, Jean-Baptiste Salmon, Galder Cristobal, Jacques Leng and Mathieu Joanicot; LOF, CNRS-Rhodia-Bordeaux 1, Pessac, France.

Thermodynamics of crystallization (solubility, polymorphism), and kinetics of nucleation are crucial information for proteomics, pharmacology and chemical engineering. Moreover, one needs today to obtain rapidly reliable data for a large number of conditions (high-throughput screening). Indeed, temperature, solvent, and salts are known to play a relevant role for the crystallization in solution, and one has to vary many parameters with only a small quantity of liquid. Microfluidics, offering tools to manipulate flows at the micron scale, is a promising tool for such experiments. To perform such measurements, we have developed two original microdevices, using the classical PDMS technology. These microfluidic tools allow us to create droplets (of about 100 nL), playing the role of microreactors containing a given concentration of solute. In the first chip, we are able to store droplets of different concentrations using automated valves, in different microchannels. We also apply temperature gradients using Peltier modules and thermocouples measuring locally the temperatures. We thus apply on a same chip, both a concentration and a temperature gradient on hundreds of stored drops. This allows us to measure directly the solubility curve (concentration vs. temperature) with a small quantity of solution (20 µL). In the second chip, we apply large temperature quenches (up to 50°C in a few seconds) to droplets flowing in microchannels, by controlling temperature gradients on the microfluidic chip and with specific channel geometries. By detecting drops containing crystals after the cooling, we measure the nucleation kinetics at different supersaturations. We have also automated the devices (injection of liquids, temperature control, valves), to carry out rapid screening of these measurements. Such a control of the temperatures and of the concentrations of each droplet enables us to perform statistical measurements of the nucleation rate, and simple estimations of the solubility. Moreover, the use of a large number of small drops is essential to obtain reliable data for mainly three reasons. First, impurities are isolated in a finite number of droplets; second, all the crystallization events are independent; and eventually all the crystals grow from a unique nucleation event. Such features enable us to obtain measurements of the nucleation rate which are close to homogeneous nucleation conditions, and therefore to test the classical nucleation theory. For instance, we show for a model system (an electrolyte solution), that the measurement of the kinetic prefactor of the nucleation rate is not in good agreement with the theoretical estimations. More important, we reveal, for the first time, the presence of a polymorph for that system, and measure its solubility. Such polymorphism can only be detected using the developed microdevices for the reasons discussed above, and could not be observed using macroscopic classical tools.


4:30 PM P7.9
Design & Optimisation of a Micromixer by FLIM and CFD David A Mendels¹, Francois Mendels³, Steven Magennis², Emmelyn Graham² and Anita Jones²; ¹Division of Engineering and Process Control, National Physical Laboratory, Teddington, Surrey, United Kingdom; ²Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre, University of Edinburgh, Edinburgh, Scotland, United Kingdom; ³Cognoscens, Lyon, France.

Over the past ten years, the application of micro-manufacturing technologies to producing fluidic devices has led the rapid development of now widely accepted lab-on-a-chip and point-of-care devices. One of the bottlenecks in micro-fluidics remains mixing, because of the practical impossibility to create turbulence in small channels, emphasised by the low Reynolds numbers of the flows at play - generally smaller than 10. Several alternatives to turbulent mixing have been developed. Two types of mixers are commonly used: passive mixers, which involve the exclusive use of non-actuated elements, and active mixers, which include moving elements such as micro-pumps or dynamically changing the surface interactions. The former have the obvious advantage of being made of one single material, hence being much cheaper and easier to manufacture and operate than their counterpart, and will be the subject of this presentation. In this work, a refined fluorescence lifetime imaging microscopy (FLIM) technique was introduced, where a specific image treatment enhances the contrast of an ANS fluorescent dye, and allows direct comparison to numerical results obtained using ANSYS CFX. Having validated the model of mixing water and methanol in a T-joint, we embarked on the optimisation of the channel geometry. To do so, a number of topological grooves and herringbone structures were simulated and the best compromise in terms of mixing efficiency for this couple of fluids and one inlet flow rate was determined. The micro-channels were further manufactured in glass and quartz, and tested by the same FLIM technique. The mixing efficiency, higher than 90%, demonstrated the usefulness of the approach, while the mixing time was decreased by a factor 3.


4:45 PM P7.10
Electrorheologically-controlled Microfluidic Chips. Weijia Wen, Dept. of Physics, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong.

We report the successful design and fabrication of electrorheological (ER) fluid-based microfluidic chips. As an example for an ER fluid actuated microfluidic mixer, such active chaotic mixer’s main channel flows are stirred by orthogonal side channel flows, driven by hydrodynamic pulsating pumps. Each pulsating pump consists of a chamber with diaphragm plus two out-of-phase ER valves operating in a push-pull mode with a maximum working frequency of greater than 100 Hz. All the valves, pumps and mixing channels are integrated in one PDMS chip. In such a mixer, large perturbations to the main channel flow can be actively controlled by the strength and frequency of external electric fields applied directly on the ER fluid, through parallel PDMS-based electrodes. Experimental results indicate that chaotic mixing can be fully achieved in a distance of 1.8 mm, much shorter than that for any mixer reported to date. Our microfluidic mixer chip is bio-compatible and easy to fabricate. It can be readily integrated with valve arrays and other components, making bio-lab-on-a-chip applications possible. In addition, other ER fluid-based microfluidic components will also be introduced.


SESSION P8: Micromanipulation of droplets, molecules, particles
Chair: Sonia Grego
Friday Morning, April 13, 2007
Room 2005 (Moscone West)
8:30 AM *P8.1
Manipulation and Orientation of Molecules and Particles in Nanofluidic Systems Harold Craighead, Applied Physics, Cornell University, Ithaca, New York.

We have used simple small-scale fluid systems to control the orientation of polymer molecules and particles using a combination of electric fields and flow dynamics. This includes controlling the confirmation and position of individual double-stranded DNA molecules using entropic forces in engineered fluidic systems. We have studied the electrically driven motion and relaxation of these molecules in narrow channels and at interfaces. This manipulation enables approaches to molecular separation by size and to presenting straightened and extended molecules for analysis. In other systems we have incorporated nanoparticles and molecules of interest in polymer solutions and deposited composite nanofibers on device structures. Electrical, optical and mechanical devices have been formed in this way.


9:00 AM *P8.2
Directed Assembly of Colloids, Drops and Molecules by Dielectrophoresis Hsueh-Chia Chang, Chemical Engineering, University of Notre Dame, Notre Dame, Indiana.

Dielectrophoresis (DEP) pertains to the use of non-uniform AC electric fields, imposed by fabricated micro or nano-electrodes, to rapidly manipulate and assemble colloids, drops, membranes and molecules at the micron and sub-micron length scales. As the manipulating force results from an induced particle dipole, the surface charge of the particle is unimportant. Like charged particles can hence be packed below the Debye screening length. Opposite charged particles can be assembled in a regimented fashion in layers or stripes without precipitating fractal aggregates. The induced particle dipole is also sensitive to the particle conductivity/permittivity and dimension/shape, such that particle sorting, preferential concentration and textured patterning can be achieved during the assembly with properly designed electric fields. If the particle polarization is due to double-layer charging by space charges, the surface force includes a tangential stress that is unavailable from dielectric polarization. As such, membrane, contact-line and free-surface stretching can be achieved to produce drops, vesicles or cylinders of desired dimensions. We review such DEP-directed assembly work in our group that has produced composite membranes, nano and micro capsules, micro-screws, giant vesicles, patterned DNA quilts etc. The synthesis is achieved within minutes, which is much faster than chemical or physical self-assembly without a directing electric field, and is often done continuously in a chip.


9:30 AM P8.3
The Application of Insulator-Based Dielectrophoresis Polymer Microfluidic Devices as Particle Separators and the Impact of Dynamic Surface Coatings on Performance. Blake Simmons, Rafael Davalos, Alfredo Morales, Karen Krafcik, Pierre Ponce and Kevin Luongo; Sandia National Laboratories, Livermore, California.

Efficient and reliable cellular separation and enrichment techniques are needed to support a range of analytical functions including pathogen detection, sample preparation, high-throughput particle sorting, and biomedical diagnostics. We have demonstrated simultaneous particle enrichment and separation via insulator-based dielectrophoresis (iDEP) using structures produced in polymer-based microdevices. The polymer chips we have evaluated are fabricated through an injection molding process of the commercially available cyclic olefin copolymer Zeonor®. We demonstrate that the polymer devices achieve the same performance metrics as glass devices. One powerful advantage of iDEP is the capability to separate viable from non-viable organisms, and we will present data that indicates we can differentiate between deactivated and viable endospores. We will also show that the nonionic block copolymer surfactant Pluronic F127 has a strong interaction with the cyclic olefin copolymer at very low concentrations. The presence of these dynamic surfactant coatings positively impacts performance by decreasing the magnitude of the applied electric field necessary to achieve particle trapping.


9:45 AM P8.4
Dielectrophorestic Microfluidic Switching for Lab on a Chip Applications. Lisen Wang and Abraham Lee; Biomedical Engineering, University of California , Irvine, Irvine, California.

A novel design for lateral DEP manipulation of particulates has been demonstrated by placing vertical electrodes in the microchannel side walls of microfluidic devices. With appropriate electrode design, lateral DEP force can be generated so that one can position particulates along the width of the channel. Interdigited electrodes have been designed and studied for the generation of non-uniform electric field. Counter DEP force from another set of electrodes facing the first set can be adjusted by the voltage and frequency applied. The particles can be focused in the middle of the channel, trapped or deflected to the side wall electrodes, or positioned at any equilibrium point along the width of the channel direction with appropriate tuning of the magnitude or frequency of the electrical fields. A DEP microfluidic switch has been demonstrated by the lateral positioning design.This switching can be used to sorting particles or cells into multiple outlets (N>3). The effect of the geometry and flow rate on the performance of the lateral positioning was studied and an analytical solution for the optimal design and operation of DEP electrode arrays has been derived from the proposed model.


10:30 AM *P8.5
Integrated Digital Microfluidic Functions for Chemical and Biological Applications. Richard B. Fair, ECE Dept., Duke University, Durham, North Carolina.

The advent of electrowetting-based digital microfluidic lab-on-a-chip (LoC) technology offers a platform for developing diagnostic applications with the advantages of portability, reduction of the volumes of the sample and reagents, faster analysis times, increased automation, low power consumption, compatibility with mass manufacturing, and high throughput. In addition, digital microfluidics is being applied in other areas such as airborne chemical detection, DNA sequencing by synthesis, and tissue engineering. In most diagnostic and chemical detection applications, a key challenge is the preparation of the analyte for presentation to the on-chip detection system. Thus in diagnostics, raw physiological samples must be introduced onto the chip and then further processed by lysing blood cells and extracting DNA. On-chip separation of DNA has not been reported for electrowetting chips, but preliminary results for the integration of electrowetting and electrokinetic flow separation are described. For massively parallel DNA sequencing, sample preparation can be performed off chip, but the synthesis steps must be performed in a sequential on-chip format by automated control of buffers and nucleotides to extend the read lengths of DNA fragments. A key problem is attaching the DNA to be sequenced to an electrowetting chip to allow multiple-droplet processing. In airborne particulate sampling applications, the sample collection from an air stream must be integrated into the LoC analytical component, which requires that a collection droplet scan an exposed impacted surface followed by its introduction into a closed analytical section. On-chip sample analysis can then be performed using chemical assays or capillary electrophoresis and detection. Advances in integrating optical detection will be described. Finally, in tissue engineering applications, the challenge for LoC technology is to build high resolution (less than 10 µm) 3-D tissue constructs with embedded cells and growth factors by manipulating and maintaining live cells in the chip platform. These applications are discussed and their implementation on electrowetting-based digital microfluidics LoC platforms is described. Open design issues for each application are discussed.


11:00 AM P8.6
Actuation of Discrete Water Droplets Driven by Organic Transistor Based Circuits. Suvid Vikas Nadkarni, Byungwook Yoo and Ananth Dodabalapur; Electrical Engineering, University of Texas at Austin, Austin, Texas.

Most present-day microfluidic system implementations employ the continuous-flow approach. These systems have complex fabrication methods, complicated controls and typically use very high voltages and pressures. Discrete droplet based microfluidic systems tend to be efficient and fairly easy to fabricate. There have been a number of approaches to discrete droplet based systems, many employing the mechanism of electrowetting for the actuation of discrete droplets.[1] An important part of microfluidic devices is the drive circuitry responsible for actuation of discrete unit-sized droplets of liquids. The drive elements for a number of implementations have utilized computer controlled voltage sources and other bulky systems such as waveform generators. It would be ideal if there were to be an ‘on-chip’ drive element generating the desired voltages for the electrowetting-based actuation of discrete droplets. Voltages of the order of 100 volts are typically required for the actuation of discrete droplets and the response time is of the order of milliseconds. The voltages and speeds achievable with organic electronics are well matched to the needs of microfluidic devices. We report the implementation of an organic transistor based complementary metal-oxide semiconductor (CMOS) inverter that is employed for driving a simple microfluidic device that does not use a cover slip or a top grounding plate and has a simple open structure. The planar microfluidic device was fabricated on a glass slide. An array of independently addressable electrodes was e-beam evaporated using shadow masking. 1000 Å of gold was deposited and the pitch of the electrodes was 800 µm. 50 Å of Titanium was deposited before the gold for good adhesion. Cytop, an amorphous fluorocarbon manufactured by Asahi Glass Company and distributed by Bellex International Corp., was spin coated to a thickness of 4000 Å. Cytop served as the dielectric layer and also provided a highly hydrophobic surface. A top-contact CMOS inverter that acted as the driving mechanism for the actuation of discrete water droplets was fabricated using Pentacene and N, N’- bis (n-octyl) dicyanoperylene-3, 4:9, 10-bis (dicarboxyimide) (PDI-8CN2.) which is an air stable n-type semiconductor.[2] Other n-type materials such as copper hexadecaofluoro-phthalocyanine (F16CuPc) have also been used by our group for fabrication of complementary organic circuits. Recent work on organic CMOS circuits reported by Yoo et al. show promising results for various organic circuits. [2],[3] It is envisioned that complex organic CMOS circuits such as decoders will be integrated with discrete microfluidic systems for actuating discrete droplets over long length scales, performing multiple operations on a lab-on-a-chip type system. [1] M. G. Pollack,et al., Lab on a Chip, 2002, 2, 96 (2002). [2] B. Yoo,et al., Applied Physics Letters , 88, 082104 (2006). [3] B. Yoo,et al., IEEE Electron Device Letters, 27, 9, 737 (2006).


11:15 AM P8.7
Engineering of a Genetically Modified Motor Protein for Cargo-Specific Transport. Amanda Carroll-Portillo and George D. Bachand; Biomolecular Interfaces and Systems, Sandia National Laboratories, Albuquerque, New Mexico.

Transport of vesicles, proteins, and signaling molecules within eukaryotic cells is a complex process involving ATP-driven, bidirectional movement of cargo-carrying motor proteins along the three dimensional cytoskeleton. The ability to harness the functions of motor proteins and their associated cytoskeletal filaments (i.e., microtubules and actin filaments) in a manner that mimics eukaryotic intracellular transport has allowed for the synthesis of materials and devices with transport, assembly, and sensory capabilities. Specifically, utilization of kinesin and microtubules in a device format has enabled the capture and transport of synthetic and biological materials through the conversion of ATP into mechanical work. Such devices enable transport at the nanoscale without the physical constraints associated with other types of micro- and nano-fluidic devices. The kinesin and/or microtubules within these devices often require chemical modification prior to system integration in order to impart cargo specificity, a process that can be a time-consuming. In order to maintain this specificity without requiring chemical modification, we have designed a conventional kinesin hybrid with a single chain variable fragment (kinesin-ScFv) incorporated onto the tail end. This two-component protein, easily expressed and purified from bacteria, is designed to capture an antigen and transport it along microtubules affixed to a surface. Experimental analyses show the kinesin-ScFv maintains ATP hydrolysis activity as well as its ability to move bound microtubules in a manner similar to the non-hybrid protein. In addition, the ScFv fragment successfully captures target antigens when tested in vitro (e.g., ELISA), confirming the preservation of the antigenic properties of the ScFv. Overall, these results demonstrate the ability to genetically engineer cargo specificity into kinesin motor proteins without adversely affecting motor function. Attachment of genetically engineered peptides, antigen-specific variable fragments, or phage display sequences to the kinesin tail offers a new strategy for cargo-specific transport that can be applied to a variety of applications including active nanomaterials synthesis and bio-analytical and sensor devices. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000


11:30 AM P8.8
Autonomously Moving Local Nano Probes in Heterogeneous Magnetic Fields. Prajnaparamita Dhar¹, Yanyan Cao², Timothy Kline², Thomas Fischer¹, Tom Mallouk², Ayushman Sen² and Tom Johansen³; ¹Chemistry and Biochemistry, Florida State University, Tallahassee, Florida; ²Chemistry, Pennsylvania State University, University Park, Pennsylvania; ³Physics, University of Oslo, Blindern, Norway.

We try to direct the autonomous motion of nano-shuttles on top of an open lab on chip, i.e. fluid on a chip without material walls. Confinement of the transported material is achieved by magnetic field gradients acting on paramagnetic and ferromagnetic nano-navigators. These gradients arise from magnetic domain walls of uniaxial magnetic garnet films that can be manipulated by homogeneous magnetic fields. Surprisingly, guidance of the navigators is best achieved if they are paramagnetic, and there is a synergy between the autonomous motion and the field guidance. If the magnetic energy is larger than the propulsion energy, ferromagnetic navigators are either trapped by the domain walls or the propulsion power of the rod engine overcomes the magnetic energy landscape easily. In the later case there is only a mild correlation between the propulsion and the magnetic field direction. Adjusting the ratio of the propulsion versus the magnetic energy, one can switch from a roving to a guided shuttle. Varying the propulsion power allows a switching between guided and roving motion simply by varying the amount of propellant, at the same time keeping both the magnetic as well as the propulsion energy well above the thermal energy. One can also use a paramagnetic to ferromagnetic transition to control the different modes of navigation by changing the temperature or in some cases by photoexcitation. This enables magnetic nano-shuttles to be guided to a specific target, and then dispersed at will. Such navigators have the potential to find widespread application in micro and nanofluidics such as drug delivery and distribution.


11:45 AM P8.9
Fabrication and Characterization of Electrowetting on the flexible substrate. Jin-Young Kim¹, Hyun-woo Lim², Min-soo Cho², Chang-hyun Bann², Jin-Goo Park¹, Young Huh³, Seong-chae Jeon³ and Seung-oh Jin³; ¹HanYang Univ., Ansan, South Korea; ²Microbiochipcenter, Ansan, South Korea; ³Korea Electrotechnology Research Institute, Ansan, South Korea.

Electrowetting refers to an electrostatically induced reduction in the contact angle of an electrically conductive liquid droplet on a surface. This Electrowetting is an good method in a microfluidic system without the need for any mechnical component. Electrowetting has become one of the most widely tools manipulating tiny amounts of liquids on surface. It is useful technology that is applied to various applications, such as lap-on-a-chip devices, adjustable lenses, and new kinds of electronic display. In this paper, we fabricate electrowetting devices on flexible substrates for the flexible displays and devices. The electrowetting devices are fabricated on a 0.1mm-thick PET(Poly Ethylene Terephthalate) substrates and a 1mm-thick PMMA(Polymethylmethacrylate) substrates. The electrodes using Pt and ITO(indium tin oxide) are implemented. In this process, the wet-etching step to minimize damage to poly flexible substrates is optimized. Then unlike the typical process using the Teflon, the dielectric layer is deposited as thin as possible using hydrophobic FC(fluorine carbon) thin film on silicon dioxide. The performance of the electrowetting on PET and PMMA is characterized based on the driving voltage and contact angle as a function of FC film thickness. The droplet performance is also characterized, such as the droplet cutting, merging, transporting. Keywords : electrowetting, flexible display, flexible substrate, PET, PMMA, wet-etching, FC film, driving voltage, contact angle, the droplet performance.