Symposium Organizers
Mehmet R. Dokmeci Harvard Medical School
Brigham and Women's Hospital
Junji Fukuda University of Tsukuba
Ali Khademhosseini Harvard-MIT Division of Health Sciences and Technology
Hirokazu Kaji Tohoku University
II3: Poster Session
Session Chairs
Tuesday AM, November 29, 2011
Exhibition Hall C (Hynes)
II1: Microfluidics for Cellular Microenvironments I
Session Chairs
Monday PM, November 28, 2011
Room 206 (Hynes)
9:30 AM - **II1.1
Microfluidic Technology for Building and Handling 3D Tissue Structures.
Shoji Takeuchi 1 2
1 , Univ. of Tokyo, Tokyo Japan, 2 , JST ERATO, Tokyo Japan
Show AbstractIn this presentation, I will talk about several approaches for 3D tissue construction based-on MEMS/Microfluidic technology. We demonstrated a 3D tissue structure by stacking the "cellular" beads into a 3D mold. Since this method allows us to fabricate 3D in vivo like tissue structures, it can be applicable in the fields of regenerative medicine and drug development. To prepare the cellular beads, we used an axisymmetric flow focusing device (AFFD) that allows us to encapsulate HepG2 cells within monodisperse collagen beads. We then seeded 3T3 cells on the surface of the collagen beads. Finally HepG2 and 3T3 cells were successfully made contact with each other. Moreover, by putting these capsules in a 3D chamber and incubating them, we successfully established complicated and milli sized 3D structures. We believe that altering the shape can be possible as simple as changing the mold, and will try to combine multiple types of cells to create more complex system that functions as a living organism.
10:00 AM - II1.2
A Polymeric 3D Artificial Compound Eye for Wide-Angle Imaging.
Hansong Zeng 1 , Yi Zhao 1
1 Biomedical Engineering Department, Ohio State University, Columbus, Ohio, United States
Show AbstractThis abstract reports a bio-inspired artificial “compound eye”, which can view optical images with wide field-of-view. The technology is achieved by a smart microfluidic configuration, which uses cost-effective materials and simple fabrication. Because of the unique features, this device can potentially be applied in areas including endoscopic visualization, military surveillance, environment monitoring, consumer electronics and so forth [1].Biological research reveals that mammalian animals and insects have distinct vision mechanisms [2]. The former usually have a pair of camera eyes whose focal length can be adjusted to obtain a high definition image; while the latter usually have compound eyes that have a wide field-of-view but poor resolution. In this work, an engineering solution that integrates features of both mammalian animals’ and insects’ eyes is realized using an opto-microfluidic system.The structure is realized by two layers of microfluidic channel network. In the first layer, an array of circular membranes is deployed at the close end of the channels. The refractive liquid medium is supplied to actuate the membranes and form the lenses. The curvature and hereby the focal length of each lens is adjusted by the pumping pressure. In the second layer, another medium with different optical refractive property is applied to create the hemispherical dome. It also steer the lenses to orient in different directions. The device is fabricated using standard soft lithography process. The pressure is controlled by external pressure sources in this work. The focal length of an individual lens can be tuned from the infinity to less than 1mm with a small pressure change, suggesting that an integration of micro-pumps into the system is possible in our future work. The prototype of a 3D artificial compound eye is fabricated. The images acquired clearly show that each lens behaves as an individual camera eye and the multiple images form a comprehensive view around the optical device.In conclusion, an advanced 3D artificial compound eye with focus-tunable lenses is successfully developed using opto-microfluidic technology. The optical device is able to present images with wide field-of-view and high definition. It mimics the structures of both insects’ compound eyes and mammalian animals’ camera eyes and enjoys the advantages of both.Reference[1]W. Sturzl, et al, "Mimicking honeybee eyes with a 280 degree field of view catadioptric imaging system," Bioinspiration & Biomimetics, vol. 5, 2010.[2]J. W. Duparre et al., "Micro-optical artificial compound eyes," Bioinspiration & Biomimetics, vol. 1, 2006.
10:15 AM - II1.3
Hybrid Silicon MEMS/Biogenic Silica Microfluidics Platform for Separating and Detecting Transport of Ions and Molecules.
Kai-Chun Lin 1 , B. Ramakrishna 1 , Xiaofeng Wang 2 , Michael Goryll 2
1 School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, Arizona, United States, 2 School of Electrical, Computer and Energy Engineering, Arizona State University, Tempe, Arizona, United States
Show AbstractMicro- and nanofluidic technology offers not only the potential to dramatically reduce sample volume and enhance the speed of fluidic separation and (bio-) chemical detection, but also to selectively perturb locations on a micro/nanostructure. Top-down design of nanofluidic devices, however, is still a challenging endeavor. Biogenic silica nanostructures, derived from diatoms, possess highly ordered porous hierarchical nanostructures. The complex 3D arrays of silica pores have very unique properties, making it the ultimate nanofluidic device to select and capture molecules and colloids. In this research, we exploit the unique properties of Coscinodiscus wailesii - a species with a fairly large structure (~200 microns in diameter), that exhibits a narrow size distribution of nanopores on the order of 40 nm in diameter. We will present how a top-down/bottom-up approach can be used to integrate biogenic silica nanostructures with micromachined silicon substrates to create micro/nano hybrid systems and devices. Results from the study of transport phenomena will be presented for ions, such as hydronium, potassium and sodium, as well as polystyrene nanobeads gold nanoparticles and drug molecules of interest. We expect to leverage the understanding of the fluidic transport phenomena through well-characterized hierarchical pore structures towards designing hybrid devices for applications in separation and sensor technologies. Initial results will be presented on addressing single cells that are cultured on the surface of these hybrid structures.
10:30 AM - **II1.4
Neuroscience on a Chip.
Albert Folch 1
1 Bioengineering, University of Washington, Seattle, Washington, United States
Show AbstractCell culture technology is falling behind in the pace of progress. As animal and bacterial genomes and proteomes are being fully probed with DNA chips and a wide array of analytical techniques, a picture of cells with dauntingly complex inner workings is emerging. Yet cell culture methodology has remained basically unchanged for almost a century: it consists essentially of the immersion of a large population of cells in a homogeneous fluid medium. This approach is becoming increasingly expensive to scale up and cannot mimic the rich biochemical and biophysical complexity of the cellular microenvironment.Microtechnology offers the attractive possibility of modulating the microenvironment of single cells and, for the same price, obtain data at high throughput for a small cost. Microfluidic or “Lab on a Chip” devices, in particular, promise to play a key role for several reasons: 1) the dimensions of microchannels can be comparable to or smaller than a single cell; 2) the unique physicochemical behavior of liquids confined to microenvironments enables new strategies for delivering compounds to cells on a subcellular level; 3) the devices consume small quantities of precious/hazardous reagents (thus reducing cost of operation/disposal); and 4) they can be mass-produced in low-cost, portable units. Not surprisingly, in recent years there has been an eruption of microfluidic implementations of a variety of traditional bioanalysis techniques. I will review the latest efforts of our laboratory in the development of cell-based microdevices for neurobiology studies, such as neuromuscular synaptogenesis, axon guidance, and olfaction.
11:30 AM - **II1.5
Multipurpose Microfluidic Probes: Dipoles, Quadrupoles and Electrochemical Sensors for Studies with Cells and Tissue.
David Juncker 1
1 Biomedical Engineering Department, McGill University, Montreal, Quebec, Canada
Show AbstractMicrofluidic probes have been proposed for local perfusion and processing of surfaces, cells and tissues. Here, we will present our recent work in this area and present data on perfusion of organotypic slices using dipolar probes with one injection and one aspiration aperture. Next, we will present theoretical analysis of dipolar and quadrupolar microfluidic probes along with experimental validation. We introduce the concept of floating gradient that is formed at the stagnation point of a microfluidic probe and show that it can be rapidly tuned in space and time yet with minimal shear stress. We will discuss the integration of electrodes into the probes for electrochemical sensing of proteins by the probe. Challenges and future work will conclude this presentation.
12:00 PM - **II1.6
Microfluidic Platforms for Study of 3D Cell Chemotaxis within Biomaterials.
Amir Shamloo 2 , Sarah Heilshorn 1
2 Mechanical Engineering, Stanford University, Stanford, California, United States, 1 Materials Science & Engineering, Stanford University, Stanford, California, United States
Show AbstractChemotaxis, the directed migration of cells in response to a soluble biochemical gradient, is a critical process that directs cell movement during embryonic development, adult tissue remodeling, and cancer progression. Traditional in vitro chemotaxis assays (e.g., Boyden chambers and transwells) have a number of severe limitations, despite being widely used within the cell biology community. These traditional assays are not conducive to direct cell imaging, are generally difficult to quantify theoretically, do not produce stable concentration gradients, and cannot be adapted to test chemotaxis within 3D biomaterials. In response to these limitations, several groups have developed the use of microfluidic gradient generators to study cell chemotaxis. Microfluidics have several advantages for chemotactic studies, including ease in fabrication, low consumption of costly reagents, the capability to perform parallel experiments on a single chip, and the ability to predict and fabricate equilibrium concentration profiles of soluble cues. Building on this work, we have designed a microfluidic chemotactic generator that enables real-time visualization of chemotaxis and collective cell migration within 3D biomaterials. These microfluidic platforms are being used to study the biomechanical and biochemical factors that regulate endothelial cell movement during sprouting morphogenesis. Endothelial cell sprouting is a critical early step in angiogenesis, the formation of new blood vessels from existing conduits. Using this platform, we have identified that the G-protein coupled receptor 124 (GPR124) is a previously unknown regulator of blood vessel development in the brain. Furthermore, we have used these devices to screen various biomaterial formulations for their ability to induce stable endothelial sprouting upon exposure to vascular endothelial growth factor (VEGF) gradients. Intriguingly, our experiments find that endothelial sprouts alter their sensitivity to VEGF depending on the matrix density, suggesting a complex interplay between biochemical and biomechanical factors. As matrix density increases, steeper VEGF gradients and higher VEGF absolute concentrations are required to induce directional sprouting. In lower density matrices, endothelial sprouts were frequently observed to change their direction of growth by turning to reorient parallel to the VEGF gradient, a behavior reminiscent of the path-finding behavior of neuronal axons. In contrast, in higher density matrices this turning phenomenon was only rarely observed. Together, these results suggest new anti-angiogenic strategies for potential cancer treatment as well as pro-angiogenic strategies for regenerative medicine scaffolds.
12:30 PM - II1.7
A Microfluidic Assay for Measuring Electrical Conductivity of Gap Junction Channels.
Cedric Bathany 1 , Derek Beahm 2 , Steve Besch 2 , Frederick Sachs 2 , Susan Hua 1 2
1 Dept. of Mechanical & Aerospace Eng. , SUNY Buffalo, Buffalo, New York, United States, 2 Department of Physiology and Biophysics, SUNY-Buffalo, Buffalo, New York, United States
Show AbstractGap Junction Channels are the transmembrane protein structures that connect neighboring cells and responsible for the intercellular exchange of ions and metabolites. Most cells are known to express multiple connexins that form different types of junctions, and unfortunately, there are no known blocking reagents for a specific type of junction channel. We have previously developed a microfluidic based assay capable of measuring gap-junction mediated dye diffusion in cultured cells. In this paper, we present a microfluidic chip that measures the conductance across gap junction channels in real time. The chip contains tri-stream laminar flow across a 2D cell array. Two platinum electrodes are located under each side-stream forming a four-point resistance measurement. The middle stream contains sucrose solution that creates a non-conducting fluid barrier, known classically as the sucrose gap. Thus, when current is passed from one side of the gap to the other, it can only flow through the cells. The microfluidic channel was constructed using SU-8 on Pyrex glass substrate and Platinum electrodes deposited by sputtering deposition. Numerical simulation and empty channel calibration experiments were conducted to characterize the performance of the chip in terms of multiple flow control, interface stability, and fluid exchange time. Using this sensor device we tested the effect of the gap junction blocker, 2-APB, on Cx43 gap junctions in Normal Rat Kidney (NRK) cells. The results show that 2-APB reversibly inhibits Cx43 gap junction channels, and the blockage is dose dependent. The conductance was reduced by 13 kΩ and 63 kΩ in the presence of 100 and 200 µM of 2-APB, respectively, compared with control experiment. The time course of impedance change was fit using Boltzmann equation and it was found that the half saturation of the inhibition and recovery of 2-APB was approximate 51±10 sec. This chip allows us to measure conductance and molecular diffusion across gap junction channels simultaneously. Using the same chip, we have simultaneously measured the diffusion of 5-(and-6)-Carboxyfluorescein Diacetate, AM (5(6)-CFDA) and the conductivity through Cx43 in the presence of 2-APB. The results show 2-APB inhibits both electrical and diffusion coupling of gap junction channels in NRK cells with similar kinetics. The presented work demonstrated that our microfluidic sensor provides a generic platform for screening pharmacological agents, and the kinetics of the inhibitors on electrical conductance and molecular diffusion can be compared.
12:45 PM - II1.8
Method for Efficient Droplet Extraction from Covered Droplet-in-Oil Electrowetting-on-Dielectric Devices.
Haig Norian 1 , Ioannis Kymissis 1 , Kenneth Shepard 1
1 , Columbia University, New York, New York, United States
Show AbstractElectrowetting is the phenomenon in which a polarizable liquid droplet undergoes a reduction in contact angle in the presence of an applied electric field. By selectively assigning voltages to an array of metal electrodes coated with a hydrophobic dielectric, we can facilitate droplet transport down to the picoliter scale, enabling a true lab-on-chip without the use of bulky mechanical pumps common in channel-based microfluidic devices. Due to the rapid evaporation of such small volumes of liquid, the typical electrowetting lab-on-chip configuration consists of the analyte/reagent droplets immersed in an oil, often dodecane, and sealed with a coverslip. Extraction of the droplet from the covered oil region into oil-free uncovered configuration has been analyzed in our laboratory setup. We propose the use copper perfluorooctanoate as a means of preventing excess oil flow over the output electrodes. We gauge the quality of droplet extraction by measuring the dodecane percentage in our extracted droplet. We explore the effects of coverslip height, coverslip material, coverslip edge geometry, and oleophobic region geometry on the droplet extraction from an electrowetting-on-dielectric microfluidic device.
II2: Clinical Diagnostic Devices
Session Chairs
Monday PM, November 28, 2011
Room 206 (Hynes)
2:30 PM - **II2.1
Development of Electrochemiluminescence and Surface Plasmon Resonance-Based Immunosensors with Surface Accumulable Molecules.
Ryoji Kurita 1
1 , National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan
Show AbstractThe electrochemiluminescence (ECL) and surface plasmon resonance (SPR) based immunosensors for measuring a trace level of disease markers are shown. It is well known that thiols form a self-assembled monolayer on a metal surface, and this has been widely used to modify metal surfaces. We employed this characteristic for a highly sensitive immunosensors by obtaining a surface pre-concentration of thiol molecules formed by the enzymatic reaction of labeled antibody.Thiocholine, a kind of thiol, was found to be a very useful coreactant with tris(2,2′-bipyridyl)ruthenium complex for bright ECL emission because thiocholine has the bifunctional properties of ECL acceleration and surface accumulation on a gold surface by the gold-thiol binding. Therefore, we propose an ECL based enzyme-linked immunosorbent assay (ELISA) employing a labeling enzyme that produces thiocholine. This marks the first time that ELISA and ECL detection have been combined. Acetylcholinesterase was used as the labeling enzyme to convert acetylthiocholine to thiocholine. Then the thiocholine was collected on a gold electrode surface by the gold-thiol binding. A bright and distinctive emission was observed at 1150 mV (vs. Ag-AgCl) on the gold electrode with a thiocholine monolayer as a coreactant in the presence of Ru complex. Our new method was successfully employed to realize a high signal to noise ratio immunoassay thus enabling us to measure trace levels of disease marker protein and DNA.We also applied a micro immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable SPR system. Sample BNP solution was introduced into the micro immunosensor after an immuno-reaction with acetylcholine esterase labeled antibody (conjugate) and only unbound conjugate was trapped on the BNP immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multi-step immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device.
3:00 PM - II2.2
Multiplexed, Enzyme-Free Pathogen Detection Using a DNA Nanobarcode Microfluidic Device.
Roanna C. Ruiz 1 , Mark Hartman 2 , Hector Acaron 2 , Thua N. Tran 2 , Shawn Tan 1 , Dan Luo 2
1 Biomedical Engineering, Cornell University, Ithaca, New York, United States, 2 Biological and Environmental Engineering, Cornell University, Ithaca, New York, United States
Show AbstractIdentifying specific pathogens via nucleic acid-based tests is becoming an essential detecting method for a variety of fields including medicine, agriculture, and food safety. Although current assays such as polymerase chain reaction (PCR) have high sensitivity, these methods have certain limitations: they can be time consuming, require specially trained personnel, enzymes, and expensive equipment, and typically only detect one pathogen per test. To overcome these challenges, there is a need for user-friendly point-of-care (POC) devices that perform multiplexed sensing in an automated, efficient, and cost-effective manner. Due to enhanced controllability and diverse design capability, DNA nanotechnology is an ideal approach to interface with nucleic acid materials, offering an attractive alternative to current pathogen detection methods. Towards this end, we have utilized DNA as a structural polymer to design novel branched-DNA-based “fluorescent nanobarcodes” that can identify multiple pathogens simultaneously in a single test using a non-amplified sample. Each DNA nanobarcode carried a unique fluorescent color ratio code that corresponded to a specific pathogen nucleic acid (DNA and/or RNA). Our detection approach was implemented in a microfluidic device that identified pathogens via an addressable array of DNA nanobarcodes. Our microfluidic device provides a novel enzyme-free platform for automated, efficient, and cost-effective multiplexed detection of pathogens.
3:15 PM - II2.3
Label-Free Biomolecule Detection in Nanowall Arrays.
Takao Yasui 1 2 , Noritada Kaji 3 , Yukihiro Okamoto 2 , Manabu Tokeshi 1 2 , Yasuhiro Horiike 4 , Yoshinobu Baba 1 2 5
1 Applied Chemistry, Nagoya University, Nagoya Japan, 2 FIRST Research Center for Innovative Nanobiodevice, Nagoya University, Nagoya Japan, 3 ERATO Higashiyama Live-Holonics Project, Nagoya University, Nagoya Japan, 4 , National Institute for Materials Science, Tsukuba Japan, 5 , National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu Japan
Show AbstractRecently, many researchers have paid attention to lab-on-a-chip (LOC) or micro-total-analysis-systems (µTAS) devices due to an advantage which these devices could integrate through whole laboratory procedures in biotechnology and chemical industry. Despite the fact that the pretreatment system and separation system showed a great progress in nearly a decade, the detection system considerably made a slow progress. During the past decades, numerous nanostructures to separate DNA and protein molecules have been reported, since they have tremendous advantages to realize ultra-fast analysis of biomolecules with ultra small sample consumption, compared with the conventional gel-based separation technologies, including microchip, capillary, or gel electrophoresis. On the other hand, the detection system still relies on the fluorescenct based detection. The reason is that it is difficult to detect the bare biomolecules due to a limitation of microscopic resolution, and therefore, many reports have been forced to use fluorescent molecules to detect separated biomolecules. As for the judgement for whether a particular molecule resides or not, it is no problem to label it with fluorescent molecules, but when it comes to clinical applications, it is of a great importance to utilize the bare biomolecule separated and extracted from samples. The technique to detect biomolecules with no fluorescent molecules has been desired. In this time, we demonstrated a new detection method using nanostructures without any fluorescent molecules. This method we develop uses the diffraction phenomenon which nanostructures have intrinsically. Another benefit using nanostructures is that they have a potential to be a sieving matrix for biomolecules.In this study, an array of nanowall, which is 600 nm wide and 2.7 µm high, was fabricated on a quartz chip by electron beam lithography, photolithography, and plasma etching, as described elsewhere. We precisely controlled the nanowall spacing of 200 nm. We used 532 nm emission line of a diode pumped solid-state laser with output power of 20 mW for the incident laser. A light chopper modulated the incident laser with a modulation frequency at 1 kHz. The laser was focused by 10×/0.30NA objective lens and diffracted by nanowall arrays. The diffracted laser was detected by a photodiode and fed into a lock-in amplifier. The time constant of the lock-in amplifier was set to 100 ms. For the performance assessment of our label-free detection system, we measured the signal shift from air to water, and had a result of S/N ratio over 300. We also detected the difference between water and 3×TBE (267 mM Tris-Borate and 6 mM EDTA) buffer. Finally, we could distinguish the signal from native DNA molecules. Owing to the availability to separate DNA molecules in the nanowall chips, it will be feasible to separate and detect label-free DNA molecules simultaneously with the nanowall chips.
3:30 PM - **II2.4
Plasmonic Metamaterials for Multi-Resonant Spectroscopy and High Resolution Optical Trapping.
Hatice Altug 1 , Arif Cetin 1 , Mustafa Turkmen 1 , Kai Chen 1 , Serap Aksu 1 , Ali Yanik 1 , Alp Artar 1
1 Electrical and Computer, Boston University, Boston, Massachusetts, United States
Show AbstractSensitive and quantitative detection of large variety of biological and chemical compounds is needed for early disease diagnostics, environmental monitoring, and homeland security. In this talk, we will introduce plasmonic bio-chemical sensor platforms enabling ultrasensitive label free detection, resonantly enhanced multi-color vibrational spectroscopy as well as high-resolution optical trapping and manipulation. Within the last decades several innovative optical detection technologies have been investigated for sensitive, label-free, quantitative and multiplexed bio-chemical sensing and spectroscopy. Optical biosensors allow transduction of the biomolecular binding signal remotely from the sensing volume, thereby minimizing sample contamination. Unlike mechanical and electrical sensors, they are also compatible with physiological solutions and are not sensitive to changes in the ionic strengths of the analyte solutions. Among optical sensors, methods exploiting surface plasmon resonance (SPR) are taking the lead. Surface plasmons, electromagnetic excitations propagating at metal/dielectric interfaces associated with the collective oscillations of electrons, can interrogate minute changes within the close vicinity of the interface. In fact, SPR is considered as the gold standard technique for label free-detection. Furthermore, plasmons by enabling extreme concentration of electromagnetic fields much below the diffraction limit, strong light-matter interaction and large optical gradients are being explored for highly sensitive surface enhanced spectroscopy as well as for optical trapping and manipulation of analytes. In this talk, we will introduce several plasmonic sensor. First, we will describe a structure which consists of arrays of gold nanopillars for biosensing, nanospectroscopy and optical trapping at the same time. The structure combines the complementary characteristics of localized and extended surface plasmons. We demonstrate high refractive index sensitivities (~675 nm/RIU, where RIU stands for refractive index unit) with large figure of merits (~110). Such good detection limits are attributed to tight localization of plasmonic excitations in nanopillars structures leading to spectrally narrow resonances with enhanced near-field intensities. Plasmonic hot-spots created at the tips of the nanopillar structures also allow trapping of nanoparticles with optical forces as high as 350 pn/W/um^2 at low power excitation sources. We also show that these strong forces around the hotspots can be controlled by incident light polarization allowing direct manipulation of bio-chemical analytes on-chip. Next, we will describe multi-resonant plasmonic metamaterials supporting high optical fields. We will show that these structures can be used to simultaneously enhance different vibrational modes of bio-chemical compounds. Accessing multiple molecular bands is crucial to specifically and accurately identify unknown biochemical.
4:30 PM - **II2.5
Microfluidics for Cell Sorting and Clinical Applications.
Mehmet Toner 1
1 MassGeneral Hospital, Harvard Medical School, Charlestown, Massachusetts, United States
Show AbstractBodily fluids, especially blood, contain a treasure of information about the functioning of whole body. Consequently, blood sampling and analysis are of prime interest for both clinical and biomedical research applications, and hold a central place in the diagnosis of many physiologic and pathologic conditions, localized or systemic. However, tapping into this wealth of information has been significantly limited with the lack of adequate technologies and the unspecific nature of the information generated from the current approaches. Among the new technologies with an increasingly broader impact in biology, microfluidics is extremely attractive for blood and other bodily fluid analysis. This presentation will focus on our recent efforts to bring microfluidics to clinical medicine in (i) cancer, (ii) burns and trauma, and (iii) global health. While each of these applications has drastically different design and engineering requirements, the capture of specific cells in peripheral blood is achieved through the use of binding of target cells to antibody-coated surfaces in precisely controlled micro-channel flows. In cancer, the use of microfluidics in isolating extremely rare circulating tumor cells (CTCs) from ~5 to 10 mL of whole blood and the development of CTC-chip will be discussed with specific examples for the initial utility of the CTC-chip in various cancers. In burns and trauma, a point-of-care microchip to isolate homogeneous population of inflammatory cells from ~100 to 300 μl of whole blood will be presented to obtain both genomic and proteomic information from blood cells without altering their biology. In global-health application, label-free isolation of CD4+ T-cells for monitoring HIV/AIDS patients in highly resource-limited environments from a fingerprick of whole blood (~5 μl) will be discussed. In all these clinically relevant examples, the importance of using samples from real patients in the development of new technology will be emphasized. The challenges and perils in bridging technology development with clinical medicine will also be discussed in the context of these three distinct applications.
5:00 PM - **II2.6
Immuno-Pillar Chips for Clinical Diagnosis.
Manabu Tokeshi 1 2
1 Department of Applied Chemistry, Nagoya University, Nagoya Japan, 2 FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Nagoya Japan
Show AbstractRecently, a concept of "plasma proteome profiling", which a lot of plasma proteins are measured at the same time, has paid much attention to clinical diagnostics [1,2]. By using data from the plasma proteome profiling, we are expecting that a highly accurate diagnosis becomes possible. However, although changes of plasma protein profiles reflect physiological or pathological conditions associated with many human diseases, only a handful of plasma proteins are currently used in routine clinical diagnostics. One reason for this is that there is no method of measuring a lot of plasma proteins with greatly different concentration range easily and rapidly. Very recently, Heath et al., developed a new device (an integrated blood barcode chip) that can detect a dozen different proteins in whole blood simultaneously [3], although this cannot detect wide dynamic range of proteins. These approaches will bring us big benefits: improvement of diagnostic accuracy, early detection of disease, proper treatment depends upon the stage of the disease, and so forth.We are advancing the research aiming at the development of a plasma profiling device for clinical diagnostics. To realize the device, it is necessary to satisfy all the requirement for the device: i.e., provide rapid analysis with high sensitivity, have wide dynamic range, be easy-to-use, require small volumes of sample and reagents, and be fabricated at low cost. Very recently, we developed a new device called an "immuno-pillar chip [4]", which has the desired features for a plasma profiling device. It has hydrogel pillars (diameter: 200 µm, height: 50 µm) fabricated inside a microchannel, with many antibody molecules immobilized onto 1 µm diameter polystyrene beads. For detection of disease markers, we confirmed the chip provides rapid analysis (total assay time: 4-12 min) with high sensitivity, it is easy-to-use (no special skills are needed), and it uses small volumes of the sample and reagent (0.25 µL each for plasma or 2 µL each for whole blood). Moreover, multiplex assay for three biomarkers was also possible.We are working on the development of a multi-biomarker detection chip for the diagnosis of diabetic nephropathy by using this device in cooperation with the Department of Medicine at Nagoya University now. We believe that the plasma profiling chip for clinical diagnostics can be developed by expanding the function of the immuno-pillar chip.[1]N. L. Anderson, N. G. Anderson, Mol. Cell. Proteomics, 1, 845–867 (2002).[2]P. Mitchell, Nat. Biotechnol., 28, 665 (2010).[3]R. Fan, O. Vermesh, A. Srivastava, B. K. H. Yen, L. Qin, H. Ahmad, G. A. Kwong, C. -C. Liu, J. Gould, L. Hood, J. R. Heath, Nat. Biotechnol., 26, 1373-1378 (2008).[4]M. Ikami, A. Kawakami, M. Kakuta, Y. Okamoto, N. Kaji, M. Tokeshi, Y. Baba, Lab Chip, 10, 3335 (2010).
5:30 PM - II2.7
On-Chip Diagnostic System for Circulating Tumor Cells.
Jaehoon Chung 1 , Huilin Shao 1 , Ralph Weissleder 1 , Hakho Lee 1
1 Center for Systems Biology, Massachusetts General Hospitals, Boston, Massachusetts, United States
Show AbstractWe have developed a novel, low-cost and high-throughput microfluidic device for detection and molecular analysis of circulating tumore cells (CTCs). The device captures CTCs directly from unprocessed whole blood, provides on-chip cell labeling for CTC identification, and allows facile cell-retrieval for further analyses. The device operation is based upon a size-selective cell separation technique, which was implemented by a weir-style physical barrier with a gap in the main fluidic channel; blood cells which are smaller than the gap height move straight through following a laminar flow, whereas larger cancer cells deviate from their original path and move along the physical barrier to be collected in a separate outlet. This new system is a versatile CTC analysis platform with many advantages. First, it supports extremely high throughput operation, since the use of weir structure effectively reduces fluidic resistance and enables flow-through separation.Specifically, we achieved >6000-fold CTC enrichment from whole blood at a high flow rate (10 mL/h). Second, the CTC-chip facilitates clear visual verification and enumeration of captured cells during/after operation. For this purpose, we have implemented microwell-shaped capturing structures on the physical barrier. Cancer cells introduced to the device were individually collected at each capture site, allowing single-cell resolution analyses. Furthermore, the captured cells could be profiled in situ by introducing fluorescent antibodies. The chip thus assumes not only high detection sensitivity but also molecular specificity for CTC identification. Finally, the CTC-chip provides a facile way to retrieve captured CTCs. By reversing the flow direction, the cells can be dislodged from their capture sites and collected for downstream investigation (e.g., cell culture and genetic analyses).
5:45 PM - II2.8
Cancer Cells in 3D Microenvironments: Individual and Collective Migration Behaviors.
Ian Wong 1 2 , Daniel Irimia 1 2 , Mehmet Toner 1 2
1 BioMEMS Resource Center, Massachusetts General Hospital, Charlestown, Massachusetts, United States, 2 , Harvard Medical School, Boston, Massachusetts, United States
Show AbstractThe metastasis of cancer cells from the primary tumor to surrounding tissues in the body is ultimately responsible for 90% of cancer-related deaths. However, the mechanistic details of how malignant cells invade into the tissues and vasculature are poorly understood. Here, we use microfabricated 3D environments with controlled chemoattractant gradients to directly characterize individual and collective migration of cancer cell populations with controlled degrees of malignancy. Using automated image analysis techniques, the behavior of these heterogeneous subpopulations can be distinguished and categorized. Finally, this platform is implemented as a high-throughput screen to quantitatively measure the effects of small molecule anti-metastatic therapies.
II3: Poster Session
Session Chairs
Tuesday AM, November 29, 2011
Exhibition Hall C (Hynes)
9:00 PM - II3.1
Evaluation of Performances of Organic BioMEMS as Resonators.
Georges Dubourg 1 , Isabelle Dufour 1 , Claude Pellet 1 , Cédric Ayela 1
1 , IMS laboratory, Talence France
Show AbstractPolymers are promising materials for MEMS and sensor applications. They are particularly attractive for sensitive biosensing applications due to their low cost, good processability, bio-compatibility and tuning properties, such as Young’s modulus. Actually, an organic free-standing structure is more flexible than a silicon one: the use of an organic microcantilever offers the possibility of characterizing many deformations in dynamic mode that are difficult or impossible to be observed with silicon structures. Thereby, an organic cantilever can operate at high resonant frequencies improving their sensitivity for sensing applications. In the same time, the common fabrication methods are mainly restricted to photosensitive materials. Therefore, the introduction of versatile methods to pattern materials such as thermoplastics and biopolymers which are not affected by the standard photolithography is challenging. In this context, the presented work proposes a new collective microfabrication process of all-organic microcantilever chips made of PMMA material. This method is based on the hierarchical combination of shadow-masking and wafer-bonding processes. The shadow-masking combines deposition and patterning in one step thanks to spray-coating through a polymer microstencil that gives the opportunity of patterning thermo and photo sensitive materials. The resulting organic structures are then transferred onto SU-8 chips by using an SU-8 wafer-bonding process that is well-adapted for the wafer-level fabrication of organic cantilevers.The second part of this study focuses on the dynamical characterization of resulting microstructures as resonators to evaluate the structured material performances. The polymer-based microcantilevers are actuated by the electromagnetic Lorentz force obtained by an external magnet and an alternative electrical current in a conducting path integrated on the structure. For this, the gold path is deposited on the structures by thermal evaporation through polymeric microstencil. With this approach, fifteen out-of-plane resonant modes have been measured, including torsional and flexural ones. Resonant frequencies above 1MHz have been obtained for the fifteenth mode enhancing the organic structure sensitivity of 2 decades compared to the first mode. Thereby, organic cantilevers used as resonator have the potential to serve as highly sensitive devices for biological sensing applications. In addition, the unconventional use of microcantilevers in dynamic mode knows a fast-growing interest. More specifically, the use of cantilevers in the in-plane vibration modes may be of potential interest for detection in liquid media where viscous damping occurs. Currently, a piezoelectric material is mandatory to generate this mode while, in our case, the flexibility of organic cantilevers will allow the direct observation of the first longitudinal resonant mode.
9:00 PM - II3.10
Functional Design of Porous Drug Delivery Systems Based on Laser Assisted Manufactured Nitinol.
Igor Shishkovsky 1
1 Laboratory of Technological lasers, Lebedev Physics Institute of Russian Academy of Sciences, Samara branch, Samara Russian Federation
Show AbstractPrevious our studies have shown the presence of shape memory effect (SME) in the biocompatible porous nitinol (intermetallic phase NiTi), fabricated by the selective laser sintering (SLS) method [1]. In the living tissue under raise of temperature (beginning disease) the size of pore will decrease an account of austenite phase transformation and a pharmaceutical composition will extrude from the pores. And wise versa on the cooling stage (a tissue temperature returns to normal) the intake of drug will stop. Depending on the type of the three dimensional structure of scaffold, determined on the stage of computer aid design, the velocity of penetration is possible to control. Because the scaffolds consist of random pores, we propose to derive the inhomogeneous surface strain distribution numerically by combining micro-compression experiments with Finite Element (FE) model. [1]. Shishkovsky I.V. Laser synthesis of functional mesostructures and 3D parts. Moscow. Fizmatlit Publ.: 2009. ISBN 978-5-9221-1122-5. 424 p.
9:00 PM - II3.11
Study of Double Emulsion Behavior by Optical Force.
Kyungheon Lee 1 , Sang Bok Kim 1 , Kang Soo Lee 1 , Seung Hwan Kim 1 , Sang Youl Yoon 1 , Hyung Jin Sung 1
1 Mechanical Engineering, KAIST, Daejeon Korea (the Republic of)
Show AbstractOptical force has been used in numerous fields as tool to manipulate micro particulate, such as cell, micro/nano particles and bio-molecules. Because of its non-invasive nature, many research fields adapted optical force for manipulating single or multiple micro particulate. Among several micro particulates, single and multiple layered emulsions are used in many research fields. Encapsulation of specific ingredient with emulsion is widely used to chemical science, biological cell encapsulation study, drug delivery, food and synthesis of specific shape of micro particulate. Several parameters determine the size of each emulsion and its frequency such as flow rate of each fluid, geometry of generating device and fluid properties. In order to obtain high quality encapsulation and other synthesis, it is very crucial to manipulate emulsions based on its size and ingredient properties.In the present work, we demonstrate manipulation of double emulsion that depends on differences of refractive index and relative size between inner and outer emulsion by optical force. We derived and calculated the analytic expression of optical force on a pair of concentric spheres with photon stream method and measured the behavior of double emulsion by the optical force. The behavior of double emulsion was also calculated with the optical force distribution and particle motion governing equation. For the experimental measurement, a simple device for generating double emulsion with surface treated co-flowing geometry was employed and laser beam propagate perpendicular to the double emulsion and outer fluid flowing direction. When the double emulsion passed through the laser beam, the scattering force pushed them in the direction of laser beam propagation and the double emulsion move their position in the plane perpendicular to the fluid flow. The shifted distance was controlled by emulsion size ratio, refractive index and other optical parameters of double emulsion. To identify the refractive index of selected fluid mixture, defocusing based micro refractometer measurement was carried and other parameters were also controlled. The analytical results were compared with experimental data and were found in good agreement. This work has potential uses in double emulsion sensing, separation for drug delivery and emulsion related biomedical applications.
9:00 PM - II3.12
Planar Impedance Sensing Device for Cellular Response Studies.
Jinwang Tan 1 , Xin Zhang 1
1 , boston university, Boston, Massachusetts, United States
Show AbstractThis project is aiming to develop a cell impedance sensing and analysis system for cellular response studies. Cellular responses are kept close eye on in the experiments with cell models where a variety of labeling techniques and optical observation are the most common approaches. Despite the impressive achievement attained with these methods, current and future studies strive to provide effective and quantitative detection, capable of achieving real-time monitoring of transient cell responses. Bioelectronics techniques, with the help of advancement in automatic detection, have gradually been utilized by biomedical researchers to study the cellular responses. These techniques, such as electrical cell-substrate impedance sensing (ECIS) and real-time cell electronic sensing (RT-CES), have been developed to determine the cell-substrate or cell-cell adhesion based on the average morphology of a large number of cells. The advancement of shrinking electrode dimension into subcellular level will provide even more undisturbed cell morphology and electrical properties with isolated cells.The planar electrodes have been created with microfabrication processes to carry out the impedance measurement of certain electrode/electrolyte interface.As the first step of this project, we have developed our device for cell sample preparation and chemical delivery.The device consists of the following functional sections: 1) A microfluidic section that delivers cell suspension and chemicals with well designed network; 2) An alternating current electrokinetics (ac-EK) section that positions HEK cells onto the sensing electrodes by dielectrophoresis. The novel design of the electrode array makes it serve as both trapping and sensing electrodes, which significantly reduce the complexity of our design as well as the cost of fabrication. Following steps such as cyclic spectrum measurements of working electrode impedance and automatically data fitting for electrochemical impedance spectroscopy (EIS) will be performed in the future.
9:00 PM - II3.13
Nanostructured Selenium for Preventing Biofilm Formation on Medical Devices.
Thomas Webster 2 , Qi Wang 1
2 School of Engineering, Brown University, Providence, Rhode Island, United States, 1 Department of Chemistry, Brown University, Providence, Rhode Island, United States
Show AbstractIn this study, we coated traditional implants with selenium nanoparticles to impart antibacterial properties directly onto the surface of medical devices. Selenium can kill bacteria by depleting their thiol levels. The nano-scale size of selenium nanoparticles increases the surface area of selenium available to interact with and kill bacteria. For this, selenium nanoparticles were synthesized through a simple reaction between glutathione and sodium selenite (4:1 molar mixture) and at the same time were coated on the surface of various medical devices. These substrates included PVC (polyvinyl chloride), polycarbonate, PU (polyurethane), co-polyesters and silicone. After coating, tape tests and fluid flow assays were used to test the strength of adhesion of the selenium nanoparticles on the substrate surfaces. SEM images of the substrate surfaces were taken before and after the adhesion tests to determine coating strength. We achieved very strong adhesion for some of the substrates, like PVC. We also used many methods, such as plasma treatment, UV light treatment, changing temperature, altering pH and coating time, to optimize the coverage and attachment strength of selenium nanoparticles onto all the substrates. Lastly, experiments with bacteria (specifically, Staphylococcus aureus) were conducted to determine the effectiveness of the selenium coating for killing bacteria or preventing bacteria from attaching. Bacteria colonization decreased significantly when polymers were treated with selenium coated samples and substrates with higher concentrations of selenium attached less bacteria, which indicated that the selenium coating could inhibit bacteria growth and deserves further investigation.
9:00 PM - II3.14
Facile Technique for Cell Patterning and Multiple Cell Types Co-Culturing.
Alexander Efremov 1 2 , Eliana Stanganello 1 , Steffen Scholpp 1 , Pavel Levkin 1 2
1 Department of Toxicology, Karlsruhe Institute of Technology, Karlsruhe, Baden-Württemberg, Germany, 2 Department of Applied Physical Chemistry, University of Heidelberg, Heidelberg, Baden-Württemberg, Germany
Show AbstractThe ability to control spatial arrangement of different cell types is crucial for in vitro cell function studies, for designing of tissue constructs that mimic the organization of in vivo cell compartmentalization and variety bioassays [1]. Although the existing cell patterning technologies allow co-culturing of different cell types, they are usually limited to relatively simple geometries. On the contrary, methods used for obtaining complex geometries are usually applicable for patterning of only one cell type. We have developed a facile method enabling conjoint culturing of more than one cell types in areas with virtually unlimited geometrical complexity.Our method is based on the formation of highly hydrophilic (HH) areas (17.4°±0.5°, static water contact angle) surrounded by superhydrophobic (SH) borders (146.1°±2.3°, advancing water contact angle). The HH/SH patterned surface is fabricated by first formation of a nanoporous HH poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) polymer layer on a glass plate followed by UV-initiated fluorination of its surface through a photomask. The surface modification method we use is based on grafting of poly(2,2,3,3,3-pentafluoropropyl methacrylate) brushes on the surface of HEMA-EDMA matrix using photografting[2]. The very high difference in wettability of hydrophilic areas and the surrounding SH border allows us to enclose aqueous solutions (e.g. cell suspensions) inside the hydrophilic areas. Thus, the cell patterning is carried out by filling separated hydrophilic reservoirs with suspensions of different cell lines followed by the co-culturing of adhered cells in the same medium.We characterized the patterned surfaces by scanning electron microscopy, X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. The nanoporous structure of the polymer film assures transparency of sample that permits monitoring of cells by means of optical microscopy. HH/SH surfaces with different geometries of patterned area were utilized to control spatial arrangement of different cell lines on the substrates. We also performed patterning of primary embryonic zebrafish cells to mimic formation of Sonic Hedgehog gradients in vitro. Our preliminary results confirmed good adhesiveness and viability of the tested cell lines. The smallest obtained distance between hydrophilic reservoirs divided by a SH gap was 30 μm that corresponded to the average diameter of a eukaryotic adhered cell. Close proximity of patterned areas, flexibility in pattern geometry, transparency of the polymer film, good cell adhesiveness and viability as well as versatility of the method for the preparation of patterned substrates make the technique an excellent approach for cell patterning, mimicking natural cell arrangements in vivo, studying cell-cell communications and application in a variety of bioassays.[1] Kaji et al. Biochimica et Biophysica Acta, 2011[2] Zahner et al. Adv. Mater., 2011
9:00 PM - II3.17
Metal Assisted Plasma Etching (MAPE).
Teena James 1 , David Gracias 1
1 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractWe describe a novel phenomenon of metal assisted plasma etching of silicon (Si) substrates. Etching is accelerated in the vicinity of noble metal patterns resulting in the single-step formation of novel structures for biomedical applications. These structures include nanoparticle coated microfluidic channels that have been used for sensing. We also describe the creation of nanoporous silicon membranes (with conical pores) and describe their applications in biomolecular separations and ion rectification.
9:00 PM - II3.18
Development of a Microscale Uni-Axial Loading Device for Intercellular Mechanotransduction Study.
Qian Wang 1 , Yi Zhao 1
1 Biomedical Engineering Department, Ohio State University, Columbus, Ohio, United States
Show AbstractThis paper reports development of a microdevice that can deliver controllable uni-axial stress to live cells, where controllable tensile or compressive stress can be loaded on selected cells while keeping other cells unloaded. The propagation of mechanical signals regulated by cell-cell communication can thus be quantitatively studied.Cells in tissues in vivo are constantly subjected to mechanical loads. These mechanical signals are essential for maintaining cellular functions. Current loading devices often expose all cells in culture to one loading condition at the same time. Mechanical loading to selected cells is yet to come. In this work, a device that can apply controllable compressive/tensile uni-axial loads to selected cells is demonstrated. The device consists of two polydimethylsiloxane (PDMS) substrates. The top substrate consists of an array of rectangular membranes. Each membrane is 7500μm in length, 500μm in width, and 50μm in thickness. The bottom substrate contains a microfluidic network. Upon loading, the fluid in the microfluidic channel deforms the membrane and applies stress to cells cultured on the top surface of the membrane. Given the large length-to-width ratio, uni-axial loads can be applied. In this design, each membrane is independently deformable to deliver desired levels of strain to cells in different regions.To examine the actual strain, a microdots array (5μm in diameter and single spaced) is patterned on the membrane of the top substrate. Displacements of the dots upon membrane deformation are optically determined, where the three-dimensional profile of the membrane can be reconstructed. In–plane strain field is then derived from the displacement map of the microdots array using classic large strain/displacement equations with the Lagrangian strain operator. The result shows that the uni-axial strain at the membrane center ranges from about 5% compressive to about 25% tensile, validating the capacity of the device in applying both tensile and compressive loads. A wider range can be achieved by adjusting the membrane geometries and the pumping parameters.Cell testing is performed using murine skeletal myoblast cell line C2C12. After culturing C2C12s in the device and exposing them to 0.5Hz cyclic strain (-5% to 10%) for 4 days, the cells align along the length direction. The myoblasts are then allowed to differentiate into myotubes. The result shows that not only the myotubes on the loading membranes exhibit linear alignment, but also the unloaded myotubes between the loading membranes align in the same direction. The alignment efficacy is dependent on the distance between the loading sites. Such effect is believed due to cell-cell communication. This work validates that the reported microdevice is capable of investigating cell-cell communication by selectively loading certain cells in culture while leaving other unloaded, which holds a promise for intercellular mechanotransduction studies.
9:00 PM - II3.19
Adhesion and Cohesion in Structures Containing Suspended Microscopic Polymeric Films.
Wanliang Shan 1 2 3 , Jing Du 1 2 , Emily Hampp 1 2 , Hannah Li 3 , George Papandreou 3 , Cynthia Maryanoff 3 , Wole Soboyejo 1 2
1 Mechanical and Aerospace Engineering, Princeton University, Princeton, New Jersey, United States, 2 , Princeton Institute for the Science and Technology of Materials, Princeton, New Jersey, United States, 3 , Cordis Corporation, a Johnson and Johnson Compay, Spring House, Pennsylvania, United States
Show AbstractThis paper presents a novel technique for the characterization of adhesion and cohesion in suspended micro-scale polymeric films, which is based on a combination of experiments and computational models. On such films, load is applied using probes that were fabricated by focused ion beam (FIB) techniques. The underlying stresses associated with the different probe tip sizes were computed using a finite element model (FEM). The critical force for failure of the film substrate interface is used to evaluate adhesion, while the critical force for the penetration of the film evaluates cohesion. When testing a standard material, polycarbonate, a shear strength of approximately 70 MPa was calculated using Mohr and Coulomb’s theory; this value is in agreement with literature results. The technique was applied to the measurement of adhesion and cohesion in a model drug-eluting stent called NEVOTM Sirolimus Eluting Coronary Stent (SES), which contains suspended polymeric films in metallic Co-Cr alloy reservoirs. The cohesive strength of the formulation was found to be comparable to that of plastics.
9:00 PM - II3.20
Visualization of NIR Propagation in Quasi-Zero Index Photonic Crystals Using Upconverting Nanoparticles.
Jingyu Zhang 1 , Daniel Gargas 1 , Teresa Pick 1 , Scott Dhuey 1 , Emory Chan 1 , Alexis Ostrowski 1 , Brett Helms 1 , James Schuck 1 , Deirdre Olynick 1 , Stefano Cabrini 1
1 , Lawrence Berkeley National Lab, Berkeley, California, United States
Show AbstractLanthanide-doped upconverting nanoparticles have interesting properties for bioimaging. Here, we present the use of upconversion nanoparticles (NaYF4: Er3+) to image near infrared (NIR) light propagation in photonic crystal (PC) waveguide with conventional optical microscopy. The PC structure is composed of a subwavelength negative index (n=-1) PC slab and a positive index (n=1) air slab in periodic arrays, which show quasi-zero refractive index (QZRI) and collimate 1.55µm wavelength light propagation beyond the diffraction limitation over millimeters. Er3+ doped NaYF4 nanoparticles were used to convert 1.55 µm to visible light through a multi-photon absorption based on sequential energy transfers involving real metastable-excited states. Nanoparticle-assisted NIR light mapping has distinct advantages over other methods such as typical NIR setups which are limited by the NIR wavelength, or the expensive near field scanning optical microscope (NSOM) which is limited by extremely shallow depth of field and long scanning times. In our technique, nanoparticles on PC waveguides are illuminated by a continuous wave laser to generate upconverted luminescence with high sensitivity to local field intensity at optical wavelength resolution. The QZRI PCs on SOI wafer were prepared by electron-beam lithography and cryo-Si plasma etching. We optimized the synthesis, surface treatment, concentration, and deposition methods of the nanoparticles solution in order to produce a layer of 10-20 nm Er3+ doped NaYF4 nanoparticles uniformly distributed on the surface of the PC holes over a large area (2 x 2 mm). Such a layer does not significantly change the PC refractive index. On such structures, the photoluminescence (PL) intensity (530/550 nm) dependence of pumping intensity (1550 nm) has been measured and used to quantify the enhanced local field intensity confined in photonic crystals.
9:00 PM - II3.4
Apatite Coating on Porous Silicone for BioMEMS.
Luci Cristina Vercik 1 , Thiago Antonio Menezes 1 , Leticia Baptista 1 , Andres Vercik 1
1 Basic Sciences Department - FZEA, Universidade de Sao Paulo, Pirassununga - SP Brazil
Show AbstractThe Bio-Electro-Mechanical Systems are built using the well-known fabrication processes of microelectronic industry and incorporate a biological component into its structure. These devices are widely used as biosensors and different kinds of actuators with applications in several areas of technology such as pharmaceutical and food industry, biomedical and environmental monitoring. Lab-on-a-chip systems and micro-total analysis systems (micro-TAS) using cantilever structures are used for detection of DNA hybridization and could be used to detect virus, proteins, microorganisms and small molecules of biotechnological interest. Despite the biocompatibility of BioMEMS, when used as an implantable device, is mainly concerned with the preservation of the tissue function, the lack of this property can also affect de device performance and functionality. To overcome this drawback, several surface modifications have been proposed to enhance the biocompatibility and to avoid biofouling. An alternative is coating the surface with a biocompatible material such as an apatite layer. In this work the coating of porous silicon (PSi) with hydroxyapatite was addressed. The PSi is a material often used in MEMS fabrication and has excellent optical properties, which make it suitable for optical biosensors. Porous silicon was obteined by electrochemical etching of crystalline <100> p-type silicon of microelectronic quality with resistivity between 4–40 Ωcm, wafer thickness of 525μm, previously cleaned with RCA standard processes, using an electrolyte of HF:Ethanol:H2O (1:1:5) and a current of 10mA for 10 minutes. After the porosification of silicon, the samples were treated with 0.5mol/L NaOH solutions for 120 minutes and then immersed in a Simulated Body Fluid (SBF) solution at 37°C for 7 days whereas other samples were coated without NaOH treatment. MEV, FTIR and DRX allowed observing that the samples without pretreatment in NaOH solution were not coated, as expected, whereas the pretreated sample exhibited a uniform apatite coating. FTIR showed absorption bands near 1415 and 1465 cm-1 attributed to vibrations of the CO32- group. This bands overlapped with the P-O(H) vibration of the HPO42- group, which is characteristic of the octacalcium phosphate. The characteristic band of the A-type substitution was observed at 1547cm-1 as well as the band due to the stretching vibrational mode of the PO43- group. These results indicate the formation of a carbonated hydroxyapatite on the PSi surface.
9:00 PM - II3.6
Engineering Hydrid Cunductive Polymer Microelectrode for Improving Biotic/Abiotic Interface.
Takeo Miyake 1 2 , Yuichi Ido 1 , Daisuke Takahashi 1 , Syuhei Yoshino 1 , Kuniaki Nagamine 1 2 , Matsuhiko Nishizawa 1 2
1 , tohoku.univ, Sendai Japan, 2 , CREST, Tokyo Japan
Show AbstractConducting polymers such as poly (3,4-ethylendioxythiophene) (PEDOT) and polypyrrole (PPy) are attractive electrode materials, having the advantages of biocompatibility, high capacitance, and flexibility. They have been utilized in biomedical devices, including implanted electronics and in-vitro devices for culturing cells. We report herein the micropatterning of PEDOT on a hydrogel, such as agarose and collagen, to provide a fully-organic, moist, and flexible electrode [1, 2]. The PEDOT/hydrogel electrodes are prepared through two electrochemical processes: the electropolymerization of PEDOT into the hydrogel and the electrochemical actuation-assisted peeling. The method is versatile and can be used to make micropatterns of PEDOT on other or curvilinear hydrogels.We then demonstrated that the PEDOT/agarose electrode could be used for electrical stimulation of the contractile fibers that make up muscle tissue (myotubes). A film of contractile myotubes within a fibrin matrix was laid on top of the electrode and stimulated with periodic voltage pulses. The electrode induced contraction of the myotubes, and the electrode itself was observed to contract in unison with the myotubes.[1] Sekine, S.; Ido, Y.; Miyake, T.; Nagamine, K.; Nishizawa, M., J. Am. Chem. Soc. 2010, 132, 13174–13175.[2] Nature Asia Materials, doi:10.1038/asiamat.2010.173
9:00 PM - II3.7
Complex Modulus Study of PDMS by Dynamic Nanoindentation.
Ping Du 1 , Chen Cheng 2 , Hongbing Lu 2 , Xin Zhang 1
1 Mechanical Engineering, Boston University, Boston, Massachusetts, United States, 2 Mechanical Engineering, University of Texas at Dallas, Richardson, Texas, United States
Show AbstractA key issue in using Polydimethylsiloxane (PDMS) based micropillars as cellular force transducers is obtaining an accurate characterization of mechanical properties. The Young’s modulus of PDMS has been extended from the ideal elastic constant to the time-dependent viscoelastic function in our previous work. However, the frequency domain information is of more practical interest in interpreting the complex cell contraction behavior. In this work, we investigated the complex modulus of PDMS by using the dynamic nanoindentation technique (DNT). The effects of curing condition and storage time on the modulus were evaluated. The PDMS samples were in a thin-film format with a thickness of ~3 mm. The samples were prepared by mixing the prepolymer Sylgard 184 (Dow Corning) with a curing agent at a volume ratio of 10:1, degassing and then thermal curing in oven. One of the major factors which will affect the mechanical properties of the PDMS samples is the curing condition. Therefore in this work we adopted two schemes: 65 °C for 90 min and 80 °C for 120 min. For the first scheme, three samples prepared and stored for different times were selected to study the effect of storage time on the mechanical properties. The complex modulus was obtained by using the DNT. The DNT tests were conducted by a G200 Nanoindenter system (Agilent) with a sapphire flat-ended cylindrical punch tip (Micro Star Tech.) with a diameter of 2.01 mm. The indenter tip was pre-compressed into the film by a certain depth to assure a full contact, and then a sinusoidal displacement was applied with amplitude of ~50 nm. The indenter tip was vibrated at a discrete number of frequencies in the range of 1-45 Hz, and the corresponding load was measured. After that the complex modulus and loss factor as functions of frequency were obtained from the nanoindenter software package.Both the storage modulus (E’) and loss modulus (E”) clearly show the frequency-dependent behavior: they generally increase as the frequency increases. For the first three samples with the same curing condition, the moduli increase with longer storage times. However, for the sample 4 with higher curing temperature, longer curing time but the least storage time, the moduli are much higher than sample 1 with the longest storage time. Therefore the curing condition has a much profound effect on the mechanical properties of PDMS than the storage time.In summary, we measured the complex modulus of PDMS by DNT, and investigated the effects of curing condition and storage time. We believe this work will help evaluating of the cellular force calculation with more information in the frequency domain.
9:00 PM - II3.8
Solvent-Less Planar Lipid Bilayers Formed in Microfabricated Silicon Chips.
Azusa Oshima 1 , Ayumi Hirano-Iwata 1 2 , Tomohiro Nasu 1 , Yasuo Kimura 1 , Michio Niwano 1
1 , Tohoku University, Sendai Japan, 2 , Japan Science and Technology Agency (JST), Saitama Japan
Show AbstractArtificial planar bilayer lipid membranes (BLMs) have been used for electrophysiological studies of ion channel proteins. By incorporating ion channels into the BLMs, functional properties of the channel proteins can be analyzed under chemically controlled conditions. However, BLMs prepared by the conventional methods (painting, monolayer folding and tip-dip methods) suffered from instability and lack of reproducibility. Although a variety of approaches for the formation of BLMs in microfabricated apertures and microfluidic channels have been reported, most of the devices were combined with the painting method, leading to the formation of BLMs containing organic solvent. Since organic solvent is likely to denature proteins, there is still a great demand for microfabrication-based methods for preparation of BLMs containing less or no amount of organic solvent. In the present study, we propose a new method for preparation of solvent-less BLMs in a microfablicated silicon chip using monolayer folding method. The basis of the device was a silicon wafer covered with a silicon nitride layer. Microapertures were fabricated in the silicon nitride layer using photolithographic patterning and wet chemical etching. Some of the device was further coated with thermal oxide and Teflon-AF. After treated with a silane-coupling reagent to make the surface hydrophobic, the silicon chip was placed vertically in a Teflon chamber. Artificial BLMs were prepared in the microaperture by folding up two phospholipid monolayers spread at the air/water interface. The BLMs prepared in the microapertures showed the resistance of several tens Gohm and the current noise level was 1-2 pA in peak-to-peak after low-pass filtered at 1 kHz. The BLMs were resistant to applied voltage of ±1 V and the lifetime of the membranes was 15-43 h with and without incorporated gramicidin channels. BLMs containing gramicidin channel were tolerant to repetitive solution exchanges. Such mechanically stable BLMs will open up a variety of applications including high-throughput analysis of ion-channel proteins.
Symposium Organizers
Mehmet R. Dokmeci Harvard Medical School
Brigham and Women's Hospital
Junji Fukuda University of Tsukuba
Ali Khademhosseini Harvard-MIT Division of Health Sciences and Technology
Hirokazu Kaji Tohoku University
II4: Microfluidics for Cellular Microenvironments II
Session Chairs
Tuesday AM, November 29, 2011
Room 206 (Hynes)
9:30 AM - **II4.1
Computer Assisted Designing and Biofabrication of 3D Hydrogel Structures towards Thick 3D Tissue Engineering.
Makoto Nakamura 1 , Ken-ichi Arai 1 , Hideki Toda 1 , Shintaroh Iwanaga 1 , Kozo Ito 1 , Genci Capi 1 , Toshio Nikaido 2
1 Graduate school of Science and Engineering for research, University of Toyama, Toyama, Toyama, Japan, 2 Graduate school of medicine and pharmaceutical science for research, university of toyama, toyama Japan
Show AbstractTo break several present limitations in tissue engineering, we have addressed to develop an innovative approach. Biofabrication is defined as the fabricationtechnology focused to produce biological products using living cells and/or biological materials. In the present tissue engineering, engineering of thick functional tissues has been one of the big issues for a long time. To realize to produce such tissues, we need the technologies to fabricate thick and complicated 3D structures composed of multi-cell types. Then, we have developed a custom-made 3D bioprinter using inkjet technology and have achieved to construct several 3D structures directly with hydrogel and living cells together.In this paper, firstly, the recent developments in our 3D bioprinter are reported. We added to our 3D bioprinter an additional printing mode where image based 3D laminating printing is possible. We also added active Z-axis control mode, too. Using the versionupped 3D bioprinter, more complicated structures than before were tried. As a result, we recognized the fabrication performance of the new version printer was significantly developed both in the fabrication of complicated structures and in the fabrication of arbitrary designed structures. We reconfirmed the feasibility of computer-assisted designing and direct 3D fabrication for tissue engineering.Owing to such developments, a next issue has been emerged, that is “What 3D structures should be designed and fabricated for effective incubation?”, because such fabricated bio-products must be kept incubated to advance the following process to develop to physiological bio-structures. Here, we report our recent challenges andachievements. Based on our previous experiments, we recognized that cells can survive only within 100micrometers areas from the surface of the structure by usual procedure of cell culture where oxygen and nutrients can be delivered by passive diffusion. Then, we designed the thick 3D structures with significant perfusion routes. At first, multiple bitmap images of the sequential 2D sections of 3D products are imagined and designed in computer. Next, 3D structures in computer, 3D structures were printed out by our 3D Bioprinter using sodium alginate solution as an ink.As a result, such designed 3D structures could be fabricated successfully. As our 3D Bioprinter can fabricate 3D hydrogel structures also together with living cells, we confirmed the promising feasibility of direct 3D fabrication with living cells. And as those designs were all human arbitrary designs, this results also indicated the possibility of creation of the artificial tissues or artificial bio-devices systems, too. This approach of computer-assisted biofabrication will contribute to further innovative advancement of tissue engineering.
10:00 AM - II4.2
Microfluidic Production of Micro-Assemblies with Multiple Geometries and Functionalities.
Kunqiang Jiang 1 , Don DeVoe 2 , Srinivasa Raghavan 3 1
1 Department of Chemistry and Biochemistry, University of Maryland-College Park, College Park, Maryland, United States, 2 Department of Mechanical Engineering, University of Maryland, College Park, Maryland, United States, 3 Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, United States
Show AbstractThe concept of “bottom-up” micro-assembly involves precise positioning and robust connecting of individual microsized subunits into more complex, higher-order structures with desired geometries and properties. Specifically, several issues need to be considered in advance before constructing micro-assemblies, including finding suitable subunits, choosing appropriate linking methods, and developing workable spatial templates. It is notable that microfluidics offers an excellent solution to address all of these various issues. Monodisperse subunits can be generated through unique droplet production mechanism, and microfluidic channels can be used as spatial templates to anchor and assemble subunits into complex patterns. Moreover, robust and stable intraparticle linkage can be achieved by adding external physical impulses or by inducing additional chemical crosslinkers, while desired functionalities can be realized by incorporating various encapsulants into the dispersed phase.As a proof-of -concept demonstration, we have successfully utilized microfluidics to produce uniform chitosan microcapsules as subunits (building blocks), and assembled them into various microstructures with facile control over their geometries and properties. The building blocks are microcapsules of the biopolymer chitosan, which are created by dispersing an aqueous solution of chitosan at a microfluidic T-junction with another stream of an immiscible oil phase. In the process, monodisperse chitosan droplets are continuously generated, and these are subsequently crosslinked by a downstream solution of glutaraldehyde (GA). The functional properties of these chitosan capsule properties can be easily varied by introducing various payloads into the disperse phase, such as magnetic nanoparticles and/or fluorescent dyes. We then demonstrate the assembly and linkage of individual capsules into complex structures, again using GA as the chemical “glue”. We have first created linear microchains with tunable flexibility by adjusting the crosslinking conditions: in the case of magnetic chains, both rigid chains that can be rotated by an external magnetic field as well as semiflexible chains that show a beating motion have been produced. The arrangement of capsules within a chain can also be precisely controlled, e.g., to generate linear chains with alternating fluorescent and non-fluorescent capsules. Besides, other complex structures can also be created, including Zig-Zag chains and Y-shape assemblies, by simply altering the spatial geometries of microchannel templates. In general we have developed a robust microfluidic platform for the construction of complex microassemblies with multiple geometries functionalities, which can serve as futuristic methods of “bottom-up” assembly and should be of interest in various fields like microfabrication, microfluidics, and biomimics.
10:15 AM - II4.3
BioMEMS for Growth of Endothelial Cells.
Susmi Das 1 2 3 , Fatima Merchant 3 , Wanda Zagozdzon-Wosik 1
1 Electrical and Computer Eng., University of Houston, Houston, Texas, United States, 2 Texas Center for Superconductivity, University of Houston, Houston, Texas, United States, 3 College of Technology, University of Houston, Houston, Texas, United States
Show AbstractThe in-vitro growth of endothelial cells (ECs) to form blood vessels is increasingly important in tissue engineering, but the mechanisms governing vasculogenesis are still poorly understood. Results from previously reported studies which have evaluated the influence of substrate material properties such as mechanical, chemical and electrical, topology and topography, and various environmental cues using 2D, 3D as well as sheet based configurations, are difficult to reconcile due to inconsistencies originating from the varied but specific experimental conditions implemented. We have designed a BIOMEMS device for controlled EC growth, which provides mechanical support and specific patterns that facilitate vascular network formation. We utilized various materials fabricated both as 2D and 3D structures in matrices of different geometries, to determine factors that affect cell growth and proliferation. For fabrication, we adapted Si technology and implemented layers of silicon oxide, nitride, and borides. Layers were either grown (thermal silicon oxide) or deposited using Chemical Vapor Deposition (nitride) and e-beam evaporation (borides). Detailed material characterization was done for all layers. Pattering was by optical lithography (Futurrex photoresists) followed by etching using wet and/or dry processes. Lines were patterned in sizes ranging from 5 to 60 µm shapes to mimic vascular networks including capillaries. The geometry and proximity of the patterns as well as their material dependent layer properties and surface passivation, facilitated inter-cellular interaction during the process of adhesion, spreading and locomotion. Next, to observe the contact guiding effect on cell growth in 3D structures, we fabricated grooves in Si using the same 2D patterns where these effects were identified. We used etching in KOH to form V- and trapezoidal grooves 5μm to 50μm wide and 3.5μm to 25μm deep. In selected experiments, grooves were gelatin coated to direct and promote cell alignment and proliferation. Human umbilical vein endothelial cells (HUVEC) were cultured to confluence, split and seeded on the cleaned and sterilized substrates for culturing. Cell cultures were maintained at 37°C with 5% carbon dioxide. The cultured cells were observed via transmitted light, and fluorescence confocal microscopy using viability dyes (5mM acridine orange). HUVEC showed selective contact guidance dependent on material properties, pattern geometry, and surface preparation. Adhesion, elongation, and growth of ECs and their proliferation were obtained on Si3N4 and borides but not on SiO2 or Si. Cellular interaction and formation of a monolayer of vascular networks on the substrate was observed. On the Si substrate with grooves, the cells aligned and were mechanically interlocked in the grooves; and exhibited elongation and growth.
10:30 AM - **II4.4
Chemical Engineering-Based Multiscale Optimization of 3D Cellular Organization and Oxygen Supply In Vitro.
Yasuyuki Sakai 1
1 Institute of Industrial Science, University of Tokyo, Tokyo, Tokyo, Japan
Show AbstractOur main concern is the 3D organization of cultured organ-derived cells such as liver cells in various scales for regenerative medicine and cell-based assay for drug or chemical screenings. In our body, 1) Cells are hierarchically organized at a very high cell density, but 2) The vascular system consistently supplies nutrients and removes waste/metabolites, thus attaining very high per-volume-based functionality. However, arrangement of such functional vascular systems in vitro is still a very difficult issue and it thus becomes a serious problem to simultaneously optimize 3D high-density cellular organization and to secure good mass transfer between the cells and culture medium. Chemical engineering-based analyses, design of tissues, and integration of suitable technologies are very helpful in addressing the problem in various scales. When we really intend to organize large tissue equivalents for implantation therapy, the tissue should at least be arranged with a 3D branching/joining flow channel network as an in vivo vasculature and the channels should be perfused with suitable culture medium containing oxygen carriers. We proposed a design criteria based on oxygen diffusion-consumption around a flow channel in macroporous 3D scaffolds, fabricated them, and evaluated their efficacy and limitation in perfusion culture of liver cells. Also, we checked the feasibility and problems of existing hemoglobin-based oxygen carriers. When we intend to make a small tissue for cell-based assays, we probably do not need to arrange vasculature, but we have to organize the cells to a certain extent and create an in vivo-mimicking micro-environment. We again need to pay a special attention to mass transfers between the organized cells and the culture medium. As one of the solutions, we proposed direct oxygenation through highly oxygen-permeable polydimethylsiloxane (PDMS) membranes to solve completely the limitation of oxygen supply to liver-derived cells in static culture. In particular, we are stressing that meeting the cellular oxygen demand at appropriate physiological concentrations enables highly-efficient aerobic respiration of the cells with less oxidative stresses, leading to spontaneous 3D cellular organizations that have never been observed before in vitro. As such, focusing on oxygen supply to the cells should give a firm basis for the design of culture systems in various scales for various applications.
11:30 AM - **II4.5
Fabrication of Complex Hydrogel Materials by Utilizing Microfluidics and Micromolding.
Masumi Yamada 1 , Yoji Naganuma 1 , Emi Yamada 1 , Shunta Kakegawa 1 , Sari Sugaya 1 , Minoru Seki 1
1 Applied Chem. & Biotechnol., Grad. Sch. of Eng., Chiba University, Chiba, -, Japan
Show AbstractMicrofluidic and microfabrication techniques are being developed for producing functional biomaterials for tissue engineering applications. We have proposed microfluidic systems to produce hydrogel scaffolds for cell culture, having various shapes including particles, fibers, sheets, thin patterns, and microfabricated plates. For example, we have demonstrated the continuous and rapid production of calcium alginate gel fibers with diameters of 1-200 micrometer, by using a microfluidic device, and the produced fibers were strong enough to roll around longer than 100 m. Several researchers have reported on the synthesis of Ca-alginate or chitosan hydrogel fibers either by using a micro-nozzle or a double capillary. However, it was impossible to obtain complex hydrogel fibers composed of different components in the cross section when conventional methods are employed. We have proposed a microfluidic system for synthesizing Ca-alginate hydrogel fibers composed of hard and soft regions, which enables the guided cell growth along the fiber direction. By introducing sodium alginate solution, buffer solution, and the gelling solution of CaCl2 into a co-flowing microchannel, and by controlling the flow rates and the microchannel geometries, we successfully fabricated anisotropic microfibers with diameter from 5 to 200 micrometer. Also, we employed propylene glycol alginate (PGA) to make a soft core sandwiched by solid shells, and tried to guide the direction of cell growth. As a result, 3T3 or HeLa cells, initially located in the soft core, grew along the soft region and formed linear colonies after several days of cultivation. In addition, we successfully controlled the direction of neurite elongation, and formed cell-cell networks between multiple PC12 cells. The hydrogel fibers with the anisotropic cross-sectional morphologies are promising as a material for enabling guided growth of various kinds of cells and can be used as functional parts for 2D/3D cell assembly for tissue regeneration or transplantation.
12:00 PM - **II4.6
3D Cell Co-Culture System on Hydrogel Micro-Patterned Surface.
Keitaro Yoshimoto 1
1 , The University of Tokyo, Tokyo Japan
Show Abstract The recent progress in the combination of cell culture and microfabrication technologies has stimulated the research on the development of new methods of cell culturing on chips for medical purposes. Especially, the high-performance cell culture to control cell’s functions, such as retention of viability, activity, differentiation and proliferation, is more required in the field of regenerative medicine. In order to realize controllable cell culture for regenerative medicine, it is also important that the in vivo-like culture is fabricated. So we focus on a spheroid co-culture system for hepatic cells and primary hepatocytes. The method for constructing multicellular spheroid plays many important roles in various metabolic pathways and express in vivo-like function, respectively. The micropatterned PEG-gel chip was prepared on glass surface by photolithography technique. In this system, in order to construct feeder-cell micropatterned surface before fabrication of spheroid formation, bovine aorta endothelial cells (BAECs) and non-parenchymal cells (NPCs) were seeded on the constructed PEG-gel patterned surface as single layer. Among the hepatic cells, fetal mouse liver cells (FMLCs) have been studied as a new material for growing artificial livers, liver-cell implantation, and served as a model of undifferentiated hepatocytes in the adult liver. FMLCs are regarded as a suitable cell source for implantation and regeneration due to their genetic normality and potentially proliferative activity in vitro. In this study, we tried to fabricate FMLCs spheroids arrays on micropatterned PEG-gel surface chip and evaluate the activity of the FMLCs and the efficiency of the differentiation induction. FMLCs spheroid were cultivated by seeding FMLCs with various cell concentrations onto the constructed BAECs and NPCs micropatterned surfaces. 10 ng/mL of oncostatin M (OSM), which is differentiation induction for matured liver cell, was added to the incompletely formed spheroid array on day 1 after seeding FMLCs. To assess the liver function of the cultured FMLCs spheroids, albumin secretion was quantified by a sandwich enzyme linked immune sorbent assay as activity of hepatocyte, and CYP450 1A2 activity as differentiation marker of matured hepatocyte was measured using chemiluminescence intensity from luciferin. As results, we succeeded in constructing a two-dimensional array of FMLC spheroids on a micropatterned PEG-gel surface chip. Interestingly, the spheroids did not only show the long viability and high albumin secretion, but also high degree of differentiation induction. This novel co-culture system based on cell-chip technology could provide an interesting new approach for cell culture system for primary, cancer, and stem cells.
12:30 PM - II4.7
Electrohydrodynamic Jet Printing for Hydrogel Cell Culture Substrates.
Michael Poellmann 1 , Kira Barton 2 , Amy Wagoner Johnson 3
1 Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 2 Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States, 3 Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States
Show AbstractChemical and physical factors in the cellular microenvironment strongly influence, if not direct, many aspects of cell behavior. The development of in vitro microenvironments with precise control over adhesion ligands, geometry, and mechanical properties is critical to understanding this regulation. In this work, we introduce a method to pattern polyacrylamide substrates with Electrohydrodynamic Jet (E-Jet) printing. E-Jet printing is a method for patterning features at the micro- and nanoscale. Ink is pushed through a glass capillary tube with applied back pressure, then pulled into a conical meniscus by an electric field. Droplets that jet from the tip of this Taylor cone-shaped meniscus result in spot sizes significantly smaller than inkjet printing. This work represents the first application of E-Jet printing on a soft substrate, in this case, polyacrylamide-co-acrylic acid functionalized with N-hydroxysuccinimide. These hydrogels are architecturally and mechanically similar to soft tissue, and are designed to efficiently form covalent bonds to printed proteins. Non-printed regions are deactivated in subsequent rinsing steps. Substrates are patterned with an easily-detectible protein, IgG, and an extracellular matrix protein, fibronectin. Patterns are printed using drop-on-demand mode to create arrays of equally-spaced spots, continuous jet mode for straight lines, and pulsed mode for high-speed printing. We demonstrate patterns with spot diameters smaller than 5 µm, which compares favorably to microcontact printing. Compared to stamping methods, E-Jet offers much higher flexibility, with the ability to change patterns at the point of printing. Fibronectin-patterned substrates with Young’s moduli ranging from 2 to 65 kPa are shown to support the adhesion, proliferation, and differentiation of mesenchymal stem cells (MCSs), a cell type separately known to be sensitive to substrate stiffness and to adhesive geometry. Such substrates are being used to study how adhesion geometry, chemistry, and substrate stiffness interact to influence the differentiation of MSCs into osteoblasts.
12:45 PM - II4.8
Toward a Lithographically Patterned Bio-Artificial Pancreas.
Jaehyun Park 1 , Yevgeniy Kalinin 1 , Christina Randall 1 , David Gracias 1
1 , Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractWe describe the use of lithographic processes to precisely structure a three dimensional bio-artificial pancreas from the nano to the macro scale. This precision is combined with the utilization of novel self-assembly and molecular surface modification methods to create a device that facilitates adequate diffusion to transplanted islet cells while also enabling immunoisolation. Using both simulations and experiments, we investigate (a) architectural constraints that minimize dead or hypoxic zones; (b) the influence of nanopores in enabling size-exclusion based immunoisolation; and (c) insulin release from devices with encapsulated islet cells.
II5: BioMEMS Tools for Cell Mechanics
Session Chairs
Tuesday PM, November 29, 2011
Room 206 (Hynes)
2:30 PM - **II5.1
Implementation of BioMEMS for Determining Mechanical Properties of Biological Cells.
Svetlana Tatic-Lucic 1 , Markus Gnerlich 1
1 ECE, Lehigh University, Bethlehem, Pennsylvania, United States
Show AbstractEven though microelectromechanical systems (MEMS) have been researched and developed for several decades now, only relatively recently has their full potential in the fields of medicine and biology been recognized and exploited. Within that framework, their applications are particularly attractive in cell biology, because of the nearly perfect compatibility of their sizes, as well as the simultaneous flourishing of both miniaturization science and bioengineering.Determining mechanical properties of biological cells, which was a challenge to do because of the lack of techniques that would be capable of executing this task accurately and efficiently, and on more than one individual cell at the time, proved to be a very fruitful BioMEMS targeted application. In this paper we are reporting on our recent work to determine the mechanical properties of biological cells using a BioMEM system based on an electrostatic actuator with predetermined step-wise deflection, piezoresistive force sensor, temperature sensor for measuring and heater for regulating the temperature of the cell medium, as well as a dielectrophoretic trap for positioning of the cells. There is a number of challenges associated with this system: 1) all of its elements need to be functional in a conductive liquid such as cell medium, 2) the force sensor needs to be extremely sensitive (forces that need to be measured are below 100nN), 3) temperature of the cell medium has to be maintained close to 37degC, and not be elevated during the actuating sequence. We will discuss the material issues, design and characterization details of this system. We will also discuss the results of initial cell mechanics experiments.
3:00 PM - II5.2
The Use of Controlled Surface Topography and Flow-Induced Shear Stress to Influence Renal Epithelial Cell Function.
Else Frohlich 1 2 , Xin Zhang 2 , Joseph Charest 1
1 Bioengineering, Draper Laboratory, Cambridge, Massachusetts, United States, 2 Mechanical Engineering, Boston University, Boston, Massachusetts, United States
Show AbstractPhysiologically-representative and well-controlled in vitro models of human tissue provide a means to safely, accurately, and rapidly develop therapies for disease. Applying mechanical cues, such as sub-micron substrate topography and flow-induced shear stress (FSS), can control cell functions such as alignment, migration, differentiation and phenotypic expression of cells [1, 2]. Leveraging these effects, we combined and independently controlled topography and FSS in a cell culture device to control cell function resulting in a physiologically-representative in vitro model of human tissue. The microscale tissue modeling device (MTMD) coupled an embossed topographical substrate with a molded microfluidic chamber to control both topography and FSS independently. The topographical substrate possessed surface features consisting of 750 nm wide ridges and grooves, generated using a hot-embossing mold that was fabricated via a unique optical lithography, etch, and electroforming process. As a renal cell model, cells from the human renal proximal tubule cell line HK-2 were cultured in the MTMD and exposed to user-defined topography and various FSS levels for two hours. Tests were conducted using both blank and topographical substrates, allowing the effects of FSS and surface topography to be studied independently and simultaneously. Results show that topography and FSS work in concert to elicit cell alignment and influence tight junction (TJ) formation. Cells aligned when presented with both topography and FSS, with alignment levels increasing further as FSS levels were increased. Formation of robust TJs, as measured by ZO-1 intensity and continuity around cell perimeters, increased for cells on topographic substrates. FSS further enhanced the robust TJ formation of cells on topographic substrates. As these alignment and TJ formation changes occurred more rapidly than in previous studies, the topographic patterns may have enhanced or accelerated renal proximal tubule response to FSS, demonstrating the value of the combination of these two mechanical cues. The MTMD provides a more realistic in vitro model of human kideny tissue by administering independently-controlled mechanical cues of topography and FSS to cell populations. The platform shows great promise to enhance cell function studies, speed drug development, and provide a pathway to regenerative medicine therapies.References: [1] Teixeira AI. Biomaterials. 2006:3945-3954. [2] Dalby MJ. Nature Mat. 2007: 997-1002.
3:15 PM - II5.3
Fabrication and Characterization of a Polymeric Microdevice for Cell Loading with Controllable Strain Distribution.
Qian Wang 1 , Yi Zhao 1
1 Biomedical Engineering Department, Ohio State University, Columbus, Ohio, United States
Show AbstractLive cells are constantly subjected to mechanical signals, which are critical for regulating cellular functions under various physiological conditions. To quantitatively understand the effects of these mechanical signals, many engineered methods are developed for applying mechanical loads to cells. Among these methods, applying mechanical strains by deforming thin polymeric membranes is widely used. Nonetheless, this method is primarily used at conventional scale where a large number of cells are strained at the same time. Straining a selected group of cells in culture, which is essential for intercellular mechanotransduction study, however, is not yet implemented.This paper reports development of a microdevice that can deliver controllable bi-axial mechanical strains to selected cells in culture. To address the non-uniform strain field in microscale polymer membranes, a microfabrication strategy is developed to tune the strain gradient. With the ease of mechanical stimulation and the controllable strain gradient, this work promises a potential in quantitative intercellular mechanotransduction study.Similar as the counterparts at the conventional scale, mechanical strains are applied to cells by deforming a polymeric membrane. Thin polydimethylsiloxane (PDMS) membranes are fabricated using soft-lithography and connected with microfluidic channels. Each PDMS membrane is 500μm in diameter and 60μm in thickness. Upon actuation, fluid in the microchannels deforms the PDMS membranes and delivers controllable strain to cells. Each membrane is independently deformable so that strain with desired magnitudes can be delivered independently to each membrane. However, since the PDMS membrane has a constant thickness, a highly non-uniform strain profile is generated upon a differential pressure. Cells on the membrane are thus subjected to different magnitudes of strain. This may complicate the subsequent analysis. Finite element analysis shows that strain gradient can be controlled by designing PDMS membrane with varying thickness. For example, a membrane with a large thickness in the center and a small thickness in the peripheral area can lead to a more uniform strain profile. Such a membrane with a varying thickness is fabricated by using a deformed PDMS membrane as the master template mold and transferring the deformed profile to a second PDMS substrate using soft-lithography. After fabrication, the actual strain profile upon actuation is experimentally determined by monitoring the displacements of the microdots patterned on the membranes. The results agree well with the finite element analysis, where the membrane with a constant thickness has a large strain gradient, and the membrane with a varying thickness exhibits a small strain gradient change in the majority area of the membrane. This work provides a starting point for designing more complicated strain gradient profile for cellular mechanotransduction studies.
3:30 PM - **II5.4
Opto-Mechanical Platforms for Cell Force Study.
Xin Zhang 1
1 Mechanical Engineering, Boston University, Boston, Massachusetts, United States
Show AbstractMicrosystems are providing key advances in studying single-cell mechanical behaviors. The mechanical interaction of cells with their extracellular matrix is fundamentally important for cell migration, division, phagocytes and apoptosis. As the displacement and scales of cellular phenomena is comparable to optical wavelength, optical metrology offers superior resolution and real-time imaging capabilities to measure cell forces and subcellular behavior as compared to its traditional counterparts. In this talk, I will present new advances in cellular force measurement based on opto-mechanical-based methods and discuss its unique capacities in studying cell mechano transductions.
4:30 PM - **II5.5
Microtechnologies for Studying Cell Mechanobiology.
Craig Simmons 1 2
1 Department of Mechanical & Industrial Engineering, University of Toronto, Toronto, Ontario, Canada, 2 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
Show AbstractCells reside in three-dimensional, soft extracellular matrices where they interact with other cells and, in the case of cardiovascular and musculoskeletal tissues, are subjected to dynamic mechanical loading. However, in traditional cell culture platforms (e.g., microtiter well plates), cells are grown on rigid, static two-dimensional surfaces. Thus, current platforms for studying cardiovascular and musculoskeletal cell biology poorly represent the in vivo environment, which limits the novelty and translatability of the biological information they generate. In this talk, I will describe some of the microtechnologies that we are developing to address these limitations. These microfluidic platforms are designed to allow precise control over the cellular microenvironment, including matrix stiffness and proteins, soluble proteins, cell-cell interactions, and biophysical forces. Compared with standard culture platforms, these microsystems better mimic key components of the in vivo cellular mechanobiological microenvironment, offer more precise control over microenvironmental cues, and avoid some of the confounding factors associated with application of mechanical forces at the macroscale. They are also compatible with on-chip imaging and are highly parallelizable, and therefore can be used in high-content and high-throughput screening applications. Current applications include fundamental studies of cell-matrix and cell-cell interactions in mechanically active environments and screening of biomaterial properties for stem cell-based tissue regeneration.
5:00 PM - **II5.6
Wetting Phenomena of Phospholipid Films for Electroporating Membranes.
Evelyn Wang 1 , H. Jeremy Cho 1 , Shalabh Maroo 1
1 Mechanical Engineering, MIT, Cambridge, Massachusetts, United States
Show AbstractWe investigated wetting phenomena of phospholipid films for the development of tunable electroporating membranes. Electroporation is widely used to permeabilize the cell membrane with an electric field for applications such as DNA transfection, gene therapy, and targeted drug delivery. More recently, electroporation has received interest to actively control artificial membranes made of supported lipid bilayer films for cell and chemical separations in bioMEMS applications. To successfully achieve such control, however, a thorough molecular understanding of lipid-fluid interactions is needed. In this work, we experimentally quantified surface energies associated with the tail of DPPC lipid monolayers on mica and glass substrates. We studied the quality of these films, orientation of lipids, and wetting behavior at varying surface pressures. The surface morphology of these films was characterized using atomic force microscopy. Three different probe liquids of varying polarity, water, formamide, and diiodomethane, were used. Advancing contact angles ranging from 70o to 100° were obtained for all fluids. However, the high contact angle hysteresis (~30-50°) and large effect on adhesional stability of the films suggests the strong interaction between polar liquids (water and formamide) and the hydrophilic substrates (mica and glass). Meanwhile, the minimal hysteresis (~10°) and little change in surface morphology indicates the weak interaction between the nonpolar diiodomethane and mica. To facilitate understanding of these experimental results, molecular dynamics simulations were performed on supported DPPC monolayers, which suggest how changes in film morphology occur at different surface pressures. This work offers a first step towards quantifying surface energies of different lipids and providing the molecular understanding necessary to tune pore size in electroporating membranes.
5:30 PM - II5.7
On-Chip Measurements of Cell Compressibility Using Acoustic Standing Wave.
Deny Hartono 1 , Yang Liu 2 , Kian-Meng Lim 2 3 , Lin-Yue Lanry Yung 1
1 Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore Singapore, 2 Singapore-MIT Alliance, National University of Singapore, Singapore Singapore, 3 Department of Mechanical Engineering, National University of Singapore, Singapore Singapore
Show AbstractMeasurements of mechanical properties of biological cells are of great importance for investigating different stages of cell differentiation, for examining health status of cells and for facilitating cell separation. Up to date, however, direct measurements of bulk modulus or compressibility of cells are very limited. Herein, we report on-chip measurements of the compressibility of a series of cell lines and water-borne parasites using acoustic radiation for gaining further insights on the mechanobiological meaning of cell compressibility. To achieve this, we have developed a new class of acoustic-based tool that permits fast and continuous cell compressibility measurements. The tool exploits the formation of acoustic standing wave within a straight microchannel. When polystyrene beads and cells are introduced into the channel, the acoustic radiation force moves them to the acoustic pressure node. By fitting the experimental and theoretical trajectories of the beads and the cells, the acoustic energy density and subsequently the compressibility of the cells can be obtained respectively. We find that the compressibility of various cancer cells (>4 x 10-10 Pa-1) is higher than that of normal fibroblast cells (<4 x 10-10 Pa-1). In addition, we find that the compressibilities of parasites, namely Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, are surprisingly low (<3 x 10-10 Pa-1). This work demonstrates not only on-chip measurements of the compressibility of some important biological cells but also the development of a novel acoustic-based tool for cell compressibility measurements and for complementing existing toolbox for measuring mechanical properties of biological cells.
5:45 PM - II5.8
Using Dielectric Spectroscopy as a Tool for Mitochondrial Membrane Potential Studies.
Divya Padmaraj 1 2 , Rohit Pande 1 2 , Jarek Wosik 1 2 , John Miller 2 3 , Wanda Zagozdzon-Wosik 1
1 Electrical and Computer Engineering, University of Houston, Houston, Texas, United States, 2 Texas Center for Superconductivity, University of Houston, Houston, Texas, United States, 3 Physics Department, University of Houston, Houston, Texas, United States
Show AbstractMitochondria are energy-converting organelles which produce Adenosine Tri Phosphate. They play a significant role in determining cell death, cell signaling, modulation of intracellular Ca2+ flux and aging. Hence they are vital in our physiology, and an understanding of their structure and operation is important in studying life, health and pathology of various diseases. The mitochondrial membrane potential, Ψm, (generated by electrogenic H+ pumps in the inner membrane) is a highly sensitive indicator of the energetic state of mitochondria and health of cells. It can be used to investigate the activity of the proton pump, electron transport system, and the state of mitochondrial permeability. We intend to develop a new technique to monitor Ψm by dielectric spectroscopy and enforce it by studies of ionic activity of mitochondria (using ISFETs) in parallel with dielectric measurements (impedance spectroscopy) to gain a better understanding of the mitochondrial membrane. We propose to develop a noninvasive technique for mitochondrial membrane studies by observing trends in impedance spectroscopy with the addition of ionophores and inhibitors.Impedance spectroscopy (1 KHz to 10 MHz) was done on mice cardiac mitochondria (conc. 1 mg/ml). The mitochondrial membrane was seen to relax at about 100 kHz, and had a capacitive behavior. In order to investigate correlation between mitochondrial membrane potential and measured impedance, we added FCCP ionophore and glucose to the suspension as they are both known to influence Ψm. In both cases the measured impedance of the suspension increased. Glucose addition increases the mitochondria respiration rate and reduces Ψm. FCCP depolarizes the membrane and also reduces Ψm. These results show that the impedance of the suspension is sensitive to mitochondrial membrane properties. We developed an equivalent electrical circuit for the mitochondrial sample, and modeled the mitochondria, buffer solution and electrode/electrolyte interface in detail. As we are concerned more with the inner membrane, the effect of changes in inner membrane capacitance on impedance was simulated. The charge gradient across the inner membrane is reflected in the capacitance across it; hence the inner membrane capacitance (Cim) is a good parameter to study Ψm. We saw that Cim controls the mid-frequency impedance, and by increasing it by 1 nF, we get an impedance drop of 295 Ω (at 8 kHz). This correlates with the experimental results showing an increase in impedance when charge stored was dissipated (reducing membrane capacitance) using FCCP. Uncouplers influence on mitochondrial membrane was confirmed by ionic measurements. From the presented results it can be concluded that dielectric spectroscopy has the potential for being used as a real-time and efficient tool for Ψm studies. Further development of the technique such as simultaneous measurement of Ψm and pH would aid diagnosis and study of mitochondrial diseases.
Symposium Organizers
Mehmet R. Dokmeci Harvard Medical School
Brigham and Women's Hospital
Junji Fukuda University of Tsukuba
Ali Khademhosseini Harvard-MIT Division of Health Sciences and Technology
Hirokazu Kaji Tohoku University
II6: Functional Materials for High Throughput Studies
Session Chairs
Wednesday AM, November 30, 2011
Room 206 (Hynes)
9:30 AM - **II6.1
Development of Microfluidic Biochip Applied to Cell-Based Assay for Drug Discovery.
Toshiyuki Kanamori 1 , Shinji Sugiura 1 , Koji Hattori 1 , Kimio Sumaru 1
1 Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Japan
Show AbstractThe R&D cost for drug discovery is recently rising more and more, leading to the depression in newly-launched drugs. To overcome this issue, it is very important to streamline and accelerate the earlier stage of drug discovery, especially lead optimization. For this purpose, so-called cell-based assay is utilized in the screening of lead compounds even at this moment, but the reliability is insufficiency because the results obtained with the existing technologies are not well corresponding to the downstream evaluations such as animal and clinical studies. We have pointed out the following two defects of the existing cell-based assay technologies; 1) standard cells for the assay is not obtainable, leading to the variation due to the individual variability of the cells, 2) the natural function of the cells does not develop.To make the function of the cells closer to that in vivo, we suppose that the micro environment of cell culture should be similar to that in vivo. We are now looking at micro process to cultivate cells, because it is easy to precisely control the cell-culture environment in the micro process. Additionally, high throughput screening, which is necessary for drug discovery, is easily feasible on a microchip, where many micro processes can be integrated.In this talk, I introduce three new technologies to manipulate cells in a microprocess, i) perfusion culture microchamber [1], ii) protein micro-patterning [2], iii) photo-responsive surface [3], and the practical cell-based assay, where these technologies are utilized.Reference1) Sugiura S., et al.: Biotech.Bioeng., 100, 1156 (2008)2) Hattori K., et al.: Biomicrofluidics, in press3) Sumaru K., et al.: Biosens.Bioelectron., 22, 2356 (2007)
10:00 AM - II6.2
Hydrogel-Filled Silicon Stamps for Generating Multiplexed Protein Microarrays.
Erhan Bat 1 , Pascal Jonkheijm 1 , Jurriaan Huskens 1
1 Molecular Nanofabrication, University of Twente, Enschede, Overijssel, Netherlands
Show AbstractProtein microarrays have recently gained great interest as they are suited for performing bioanalytical applications such as in vitro diagnostics, proteomics, antibody characterization and drug screening. Several techniques such as ink-jet printing, dip-pen nanolithography and microcontact printing have been used to generate protein microarrays. Inkjet printing and dip pen nanolithography allow printing of multiple proteins however these are serial techniques, control over the spot geometry/size and reproducibility is challenging. Moreover the most commonly applied technique of inkjet printing does not allow printing of proteins with less than 50 µm resolution. Microcontact printing is a soft lithography technique that allows parallel arraying of a single biomolecule over a large surface area with high resolution. Microarrays with smaller feature sizes are desired as they would allow rapid, highly sensitive, and direct determination of analyte concentration using a small amount of sample. Microcontact printing is a low cost, simple and reproducible method but usually re-inking of the stamp is required in each replication step, which is time consuming and costly and creating a multiplexed array is still a challenge. In this project, we aim at addressing the multiplexicity and inking challenges of microcontact printing. We present hydrogel-filled silicon stamps having individually addressable ink reservoirs. With this design, multiplexicity can be achieved while avoiding the need for re-inking. To prepare the stamps, we fabricated silicon microstructures having separate wells (320×320×380 µm) and each well having a 25 µm thick membrane (144 microchannels measuring 5 µm in diameter) on the printing side. The reservoirs and the microchannels of the silicon microstructures were filled with macroporous poly(2-hydroxyethyl methacrylate-co-ethylene glycol dimethacrylate) hydrogels. Methacryloxypropyl silane functionalized wells allowed covalent binding of hydrogels to the surface of the silicon. Using these hydrogel-filled stamps, we printed arrays of fluorescently labeled immunoglobulins on polydimethylsiloxane substrates reproducibly at least up to twenty times without re-inking. The spot sizes were very close to 5 µm in agreement with the stamp design. The fluorescence intensities of the printed spots were comparable for each printing step indicating that the amount of transferred protein did not vary significantly. With the help of an inkjet spotter, we addressed three immunoglobulins each labelled with a different fluorophore to separate reservoirs of the stamp. In this manner, multiplexed protein microarrays were obtained. We have also evaluated this printing system in an antibody-antigen assay. These hydrogel-filled silicon stamps hold great promise for multiplexed bioanalytical applications as well as for patterning of bioactive molecules for tissue engineering applications.
10:15 AM - **II6.3
2-D and 3-D Cell Microarrays-Based Functional Screening.
Xavier Gidrol 1 , Eric Sulpice 1 , Patricia Obeid 1 , Stephanie Combe 1 , Frederique Kermarrec 1 , Lamya Ghemin 1 , Stephanie Porte 1 , Amandine Pitaval 1 , Manuel Thery 2 , Nathalie Picollet-D'hahan 1
1 Laboratoire Biomics, CEA, Grenoble France, 2 Laboratoire de Physiologie Cellulaire et Vegetale, CEA/CNRS/UJF/INRA, Grenoble Cedex 09 France
Show AbstractTo analyze the phenotypic consequences of perturbing mammalian cells with drugs or RNAi, there is an increasing need for systematic cell-based assays in an HTS format. Cell microarrays provide an attractive solution as they offer more than a simple miniaturization and mechanization of conventional microtiter plates. While standard monolayer two-dimensional culture conditions are poor mimics of the cellular environment in-situ, microfabricated systems enable three-dimensional organotypic cell cultures or constrained microenvironments and have the potential to provide biological insight not achievable before. This presentation compares different cell microarray formats and evaluates their potential use in functional screening.
10:45 AM - II6.4
Microfabricated Polyester Microwell Device for Stem Cell Culture Experiments.
Seila Selimovic 1 2 , Francesco Piraino 1 2 3 , Hojae Bae 1 2 , Marco Rasponi 3 , Alberto Redaelli 3 , Ali Khademhosseini 1 2 4
1 Medicine, Brigham & Women's Hospital, Cambridge, Massachusetts, United States, 2 , Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, United States, 3 Bioengineering, Politecnico di Milano, Milan Italy, 4 , Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, United States
Show AbstractLately much effort has been expended in reducing wet lab experimental systems to a microscale, especially by applying microfluidic technologies. High throughput screening solutions have also seen a strong research focus, in particular in stem cell applications. In this context, microwells are employed as a standard device element for cell culture applications. Common fabrication techniques (photo- and soft lithography and etching) have several drawbacks: costly equipment, time- and effort intensive fabrication, and lack of flexibility in the design process. We demonstrate an adaptable, affordable, and rapid method for generating microwells for stem cell culture experiments by laser engraving of a sticky polyester film. Laser power and speed were adjusted to control microwell diameter, and by stacking several film layers different microwell depths were generated. In this fashion a 1000-microwell chip was fabricated in a few minutes for stem cell culture applications. We seeded murine embryonic stem cells and human hepatoblastoma cells in such a device and observed cell aggregate formation within 3 days of culture. The aggregates were viable after 9 days in culture and could be successfully retrieved. We did not observe any negative effects of the sticky film coating on cell proliferation and viability. Our results indicate that the polyester microwell device is useful for stem cell culture studies, especially as its fabrication requires little technological skill and equipment.
11:30 AM - **II6.5
Towards Next-Generation Proteomic Assays: Functional Materials as Sieving Matrices and Binding Scaffolds.
Amy Herr 1 , Dohyun Kim 1 , Mei He 1 , Samuel Tia 1 , Alex Hughes 1 , Chenlu Hou 1
1 Bioengineering, UC Berkeley, Berkeley, California, United States
Show AbstractWhile the genomics revolution has had sweeping impact on our understanding of life processes, the arguably more important “proteomics revolution” remains unrealized. Proteins are more directly linked to function than genes, but proteins are also dynamic and more biochemically complex. Consequently, protein analysis often demands multi-stage biochemical assays to measure not one, but multiple physicochemical properties (e.g., Western blot, 2D electrophoresis). Unfortunately, benchtop assays consume significant resources, making the biological sciences protein ‘data limited’. To surmount these challenges and realize an era of high throughput proteomics, innovation in instrumentation is needed. Microfluidic technology has advanced separations science, yet progress in multi-stage separations has lagged. Accepted multi-stage design approaches suffer from inherent information loss owing to strategies that ‘discretize’ first-stage separations by mapping readouts to discrete compartments in a second-stage. At UC Berkeley, we are introducing novel non-discretizing integration strategies. This talk will highlight multi-stage assays uniquely enabled by our ‘μMosaic’ fabrication technique: an approach that allows us to spatially and spatiotemporally pattern microchannels and chambers with heterogeneous, discrete nanomaterials. Our design strategy yields low-dispersion, near lossless electrokinetic material transport between disparate assay stages. In one example, I will summarize our recent progress towards fast, hands-free and perhaps even quantitative Western blotting, employed here for analysis of specimens from clinical sample repositories. Our ultimate goal being to advance the understanding of life processes – including development and disease – through quantitative bioinstrumentation.
12:00 PM - II6.6
Biohybrid Thin Films for Cardiac Valve Safety Pharmacology Assays.
Kartik Balachandran 1 , Matthew Hemphill 1 , Leila Deravi 1 , Kevin Parker 1
1 Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractRecent clinical case studies have highlighted the detrimental and often disastrous functional effects of neurological drugs such as Fenfluramine-Phentermine, anti-Parkinson’s medication and 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") on cardiac valves. These patients present with valves that have altered rigidity, curvature, and excessive fibrosis ultimately compromising their function. Considering these effects, there is currently no high-throughput method for screening the functional effects of pharmacological agents on valve tissue function in vitro. We therefore developed a novel polymer thin film-based drug contractility assay comprised of valve interstitial cells innervated with primary neurons. We utilized this assay to probe for alterations in valve contractile tone due to anti-Parkinson’s drugs. A monolayer of aligned valve interstitial cells (VIC) were constructed on a detachable polydimethylsiloxane (PDMS) membrane by micropatterning 20μm lines of fibronectin. Primary neonatal rat neurons were then seeded on the VIC monolayer and axonal projections were allowed to develop for 7 days. Thin films were then attached to PTFE posts in a Tyrode’s solution bath and subject to increasing concentrations of ropinirole hydrochloride (anti-Parkinson’s), fluoxetine hydrochloride (Prozac/SSRI drug), dopamine, serotonin, and HA-1077, while being monitored under a stereo miscroscope. Changes in tissue contractility were calculated based on observed changes in thin film radius of curvature and were compared against control samples, which were not co-cultured with neurons. We observed significant dose-dependent tissue contraction to dopamine and serotonin. The potent rho-kinase inhibitor HA-1077 elicited strong tissue relaxation, which enabled measurement of tissue basal tone. We are currently investigating if the contractile response of these thin films to the aforementioned drugs and neurotransmitters is increased when VICs and neurons are co-cultured.
12:15 PM - II6.7
High Throughput Magnetic Cytometry in Native Biological Samples.
David Issadore 1 , Jaehoon Chung 1 , Huilin Shao 1 , Ralph Weissleder 1 , Hakho Lee 1
1 Center for Systems Biology, Massachusetts General Hospital, Boston, Massachusetts, United States
Show AbstractWe have developed a miniaturized Hall (µHall) cytometer for the high-throughput detection and profiling of individual cells in native biological samples. The system is a hybrid microfluidic / semiconductor chip, consisting of an epitaxial GaAs Hall sensor-array and a microfluidic channel built on top. The fluidic channel employs hydrodynamic flow-focusing structures to form a focused stream of individual cells that pass over the µHall sensors one-by-one. For the detection by µHall sensors, biological cells are labeled with molecular-specific magnetic nanoparticles (MNPs); individual cells thereby assume magnetic moments proportional to the expression levels of target biomarkers. Subsequent µHall detection not only counts the number of targeted cells but also provides their molecular signature. Furthermore, with their capacity for highly localized magnetic detection, µHall sensors can perform measurements on native biological samples (e.g., whole blood) and without the need to remove excess MNPs. The entire cytometry thus can be carried out in a self-contained, portable chip format. Here we present the first µHall prototype that is capable of processing ~100,000 cells/sec with a resolution of 500 MNPs/cell. The clinical potential of the system is demonstrated by molecularly profiling cells for known tumor markers and by detecting as few as ~20 tumor cells in 500 µL of whole blood.
12:30 PM - II6.8
Plasmonic Interferometers for High-Throughput, Real-Time, Optical Detection of Biochemical Analytes.
Jing Feng 1 , Vince Siu 1 , Alec Roelke 1 , Vihang Mehta 1 , Steve Rhieu 1 , Tayhas Palmore 1 , Domenico Pacifici 1
1 school of engineering, brown university, Providence, Rhode Island, United States
Show AbstractPlasmonics is a rapidly emerging field of nanophotonics enabling the manipulation of light in devices with a footprint much smaller than the incident wavelength. With appropriately designed metal-dielectric structures, light at optical frequencies can be efficiently coupled to surface plasmon polaritons (SPPs), which are electromagnetic waves coupled to free electron oscillations in a metal. Being confined at the metal surface, SPPs are very sensitive to dielectric properties of the materials they propagate through, which gives SPPs the potential to sense the presence of chemical and biological analytes at the surface. Here we demonstrate an optical nano-sensor for real-time monitoring of biochemical analytes, such as NaCl, glucose, and various cytokines. In this work, we took advantage of SPP-based three-beam interference to enhance device sensitivity to small refractive index change. We fabricated thousands of groove-slit-groove nano-scale structures per square centimeter on a 300 nm-thick metal substrate by means of focused ion beam lithography. The groove/slit length was 10 µm. The silt width was 100 nm, while the groove was 200 nm wide. The distance between one of the two grooves and the central slit was fixed at 566 nm. The other groove was etched at a varying distance from the slit, from 0.25 to 10 µm in step of 25nm. A lab-on-a-chip system was set up to perform spectral measurements on the structures, using a polydimethylsilozane (PDMS) micro-fluidic channel to guide the analytes in and out of the structures. We made use of a white light illumination source to measure transmission through each plasmonic interferometer in the 400-800 nm wavelength range. By designing structure parameters properly, we were able to enhance the device sensitivity, compared to other, more conventional methods. For example, setting the changing groove-slit distance to 9.75 µm, we detected a 10-µM concentration of glucose in solution, which is the lower bound of physiological values typically found in human saliva. A sensitivity of 23,000 nm/RIU at 590 nm was recorded, far larger than what has been reported so far. Taking into account the skin depth of SPPs and the submicron-scale length of both arms of our plasmonic interferometer, our approach allows detection of analytes using a sensing volume in the femtoliter range. Moreover, by functionalizing the device surface with different linkers, we were able to improve the sensor specificity. In addition, we also simulated transmission of the structures in air and with refractive index change using an interference model, which confirmed the experimental findings. Simulated and experimental data as well as design strategies to improve the device sensitivity will also be discussed. All of these factors show the potential of our compact plasmonic interferometers to monitor levels of clinically-relevant analytes, such as glucose, in blood serum, or even saliva, in real time, with high sensitivity and high throughput.
12:45 PM - II6.9
Thermoset Polyester Droplet-Based Microfluidic Devices for High Frequency Generation.
Jin-Young Kim 1 , Andrew deMello 2 , Soo-Ik Chang 3 , Jongin Hong 4 , Danny O'Hare 1
1 Bioengineering, Imperial College London, London United Kingdom, 2 Chemistry, Imperial College London, London United Kingdom, 3 Biochemistry, Chung-buk National University, Cheongju Korea (the Republic of), 4 Chemistry, Chung-ang University, Seoul Korea (the Republic of)
Show AbstractIn recent years, numerous studies have assessed microfabricated systems as tools for performing rapid measurements on minute chemical and biological samples due to significant advantages in terms of speed, throughput, yield, selectivity and control. Recently, the manipulation of multiphase (or segmented) flow within microfluidic channels has been considered a promising route for large-scale experimentation in biology and chemistry. Importantly, such an approach allows the compartmentalization of reagent volumes ranging from a few femtolitres to hundreds of nanolitres in a continuous immiscible fluid, the production of monodisperse droplets at kHz frequencies and accurate control of reactions. Whilst most droplet-based microfluidic devices are made from polydimethylsiloxane (PDMS), it is not suitable for high frequency droplet generation that requires high operating pressure due to its low shear modulus. In this study, droplet based microfluidic devices were fabricated in thermoset polyester (TPE) which offers many of the attractive properties of PDMS but has superior resistance to high pressure when fully-cured. A novel process flow has been developed to optimise bonding which nonetheless allows rapid prototyping using conventional soft lithography techniques. Substrate transparency was assessed for the compatibility with optical detection and substrate resistance to pressure. The TPE device bonded with an oxygen plasma treated PET substrate at 76°C is shown to function efficiently at pressures up to18 MPa, which is approximately 18 times higher than typical PDMS devices. This attractive property enabled investigation of high frequency (kHz) droplet generation as a function of a wide range of flow-rates with three different oils as continuous phase.
II7: Materials and Devices for Implantable Systems
Session Chairs
Wednesday PM, November 30, 2011
Room 206 (Hynes)
2:30 PM - **II7.1
Wireless Feedback-Controlled Drug Delivery Pumps for Small Animal Research.
Ellis Meng 1 , Roya Sheybani 1 , Heidi Gensler 1 , Christian Gutierrez 1
1 Biomedical Engineering, University of Southern California, Los Angeles, California, United States
Show AbstractBiomedical microelectromechanical systems (bioMEMS) enables microfabricated devices for next generation drug delivery applications. Specifically, advanced devices suitable for use in small research animals that can provide on-demand delivery of drugs directly to the site of therapy are not currently available. Most laboratory studies involving drug administration in small animals are conducted using needle injection and therefore involve manual animal handling. Existing technologies for drug delivery in small animals are also insufficient. External pumps require tethers that impede animal movement and suffer from risk of entanglement. Implantable pumps include peristaltic and osmotic pumps but feature only limited drug payload and device lifetime, slow infusion, and limited delivery modes. To achieve chronic administration of drug with only a single surgical procedure, wirelessly-operated bioMEMS pumps were developed. These pumps feature on-demand dosing with operation over a wide dynamic range of flow rates, remote and wireless operation, accurate electrolysis-based pumping, a refillable drug reservoir, miniature form factor, and broad drug compatibility. In addition, these pumps have been integrated with sensors for electrochemical dose measurement that enables closed-loop drug delivery. Accurate, real-time tracking of drug dose and on-the-fly flow rate adjustments were demonstrated. The sensing method was also successfully applied to leak and occlusion detection. To date, there are no implantable closed-loop pumps for small animal research. Our advanced bioMEMS pumps achieve chronic temporal and spatial control of delivered compounds to maximize bioavailability and therapeutic efficacy.
3:00 PM - II7.2
Conductive Polymers for Localized Controlled Release of Therapeutic Agents for Treatment of Open Wounds.
Joseph Mbugua 2 , Eve Fabrizio 2 , Mark Chase 2 , Theo Nicholson III 2 , June Jung 2 , Jeffrey Maskrod 2 , Sigiang Zhu 2 , Ramil Mercado 2
2 , Crosslink, Springfield, Missouri, United States
Show AbstractA systemic issue with administering multiple drugs is mitigated through the use of an electronically controlled drug delivery system (DDS) that delivers one or more drugs directly to the wound site through electronic controlled release using biocompatible conductive polymers. The controlled release of model drug compounds such as sodium salicylate, Lincomycin HCl, and Clindamycin HCl along with other relevant drugs has been demonstrated using polymeric electrodes.The DDSs are easily prepared by the deposition of polymeric biocompatible films via chemical-polymerization or electropolymerization of pyrroles or thiophenes and their derivatives. Other monomers are also being investigated. Some derivatives of pyrrole never before reported as polymers have been polymerized and their role as potential drug delivery electrodes investigated. Biocompatibilities of the polymers with drug candidates and/or electrolyte systems were tested on three different mammalian cell lines. Loading of the drug molecules was done by either in situ polymerization of the monomers in the drug/monomer solution or utilizing the redox properties of the polymer in presence of the target drug. Apart from techniques applied in loading and release of drug molecules, the amount of drug loaded varied significantly with molecular size. For more electronic controlled release, the DDS was polymerized onto conductive substrates such as carbon (fiber mats, graphene or printed carbon ink) and gold (plated or printed onto flexible substrates). The release was triggered by oxidation of the polymer for positively charged drugs or by reduction of the polymer for negatively charged drugs. Real time release profile of salicylate using a stepped electronic waveform to control the release into 0.1M phosphate buffer saline (PBS) at pH 7.4 has been demonstrated. Prior to the first electronic bias of the film, very little passive release of the drug was observed. For each electronic bias, release of salicylate was only observed during negative bias. Once the film was turned off by a positive bias, the drug concentration stabilized demonstrating precise controlled release of the drug.Key words: Controlled release, Drugs, Conductive polymers, multiple drugs, Biocompatible, DDSCrosslink, Inc. would like to acknowledge the US Army Medical Research and Materiel Command (USAMRMC: W81XWH-10-2-0051) for funding and support of this project.
3:15 PM - II7.3
A Microfluidic Dialysis Cell for the Characterization of Interactions in Soft Biomaterials.
Jan Scrimgeour 1 2 , Jae Kyu Cho 3 , Victor Breedveld 3 , Jennifer Curtis 1 2
1 School of Physics, Georgia Institute of Technology, Atlanta, Georgia, United States, 2 Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, United States, 3 School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
Show AbstractCharacterization of solvent responsive interactions is increasingly important in understanding the function of native biomaterials and in the design of biomimetic “smart” materials. We present a microfluidic dialysis cell containing a novel low molecular weight cut-off membrane that is designed to facilitate the analysis of macromolecule interactions in responsive materials. The microfluidic device achieves long term retention of samples while allowing timely changes to be made in the sample’s supporting solvent. In addition, the dialysis membranes, fabricated by coating a porous solid-state membrane with a thin layer of hydrogel, effectively suppress the transmission of fluid flow from the flow cell to the neighboring sample chamber. This eliminates background flow in the sample and enables analysis of molecular interactions and material properties to be carried out in situ using a range of micro-characterization techniques such as fluorescence recovery after photobleaching and microrheology. Measurements of pH-sensitive binding between the protein, neurocan, and polysaccharide, hyaluronan, both components of the extracellular matrix in the central nervous system, demonstrate the integration of macromolecule retention, solvent switching and in-situ micro-characterization in the dialysis cell.
3:30 PM - II7.4
Nanoporous Gold: A Biomaterial for Microfabricated Drug-Delivery Platforms.
Erkin Seker 1 2 , Yevgeny Berdichevsky 3 , Kevin Staley 3 , Martin Yarmush 1 2
1 Center for Engineering in Medicine, Harvard Medical School & Massachusetts General Hospital, Boston, Massachusetts, United States, 2 , Shriners Hospitals for Children, Boston, Massachusetts, United States, 3 Neurology, Harvard Medical School & Massachusetts General Hospital, Boston, Massachusetts, United States
Show AbstractThere is constant demand for expanding the functionality of BioMEMS devices to create more effective tools for monitoring and modulating biological processes. Even though the integration of nanostructured materials in BioMEMS tools has already been shown to improve device capabilities, the need for novel materials is ever present. Nanoporous gold (np-Au), with its open-pore structure with a characteristic length of tens of nanometers, is a promising candidate nanomaterial with many desirable properties, including large surface area-to-volume ratio, corrosion resistance, high conductivity, and well-studied thiol-based surface chemistry. While np-Au has been used in a variety of applications, from fuel cells to electrochemical sensors, its interface with biology, where many of its exciting applications lie, is surprisingly non-existent. This is for the most part due to the lack of knowledge of its compatibility with living cells and tissues. Towards filling this knowledge gap, this paper demonstrates, for the first time, the biocompatibility of np-Au coatings with several key cell types and illustrates drug delivery from its porous network to modulate cell behavior in situ. We report the viable culture of various cell types (astrocytes, cortical neurons, microglia, dermal fibroblasts, and endothelial cells) and organotypic hippocampus slices on microfabricated np-Au films. We discuss the effect of nanoporous gold surface topography, cell culture conditions, and surface treatment on cell adhesion and viability. The results include scanning electron microscope images exemplifying the interaction of cells with the nanostructured surface, as well as immunostained samples revealing the cytoskeletal structure in response to nanotopographical features. We present molecular release profiles from np-Au coatings for various parameters including film thickness and molecular size. As an in vitro demonstration of the performance of np-Au coatings in modulating cell behavior, we illustrate the concentration-dependent inhibition of astrocyte proliferation on np-Au films that were loaded with an anti-mitotic pharmaceutical at different concentrations. We expect that establishing np-Au as a biomaterial with drug delivery capabilities will create new opportunities for engineering advanced BioMEMS devices that can monitor and modulate biological processes in both in vivo and in vitro settings.
3:45 PM - II7.5
Assembly of Bio-Photonic Materials Using Proteins Isolated from Cuttlefish Sepia Officinalis.
Leila Deravi 1 , George Bell 2 , Andrew Magyar 3 , Holly McIlwee 1 , Lydia Mathger 2 , Alan Kuzirian 2 , Evelyn Hu 3 , Roger Hanlon 2 , Kevin Parker 1
1 Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States, 2 , Marine Biological Labs, Woods Hole, Massachusetts, United States, 3 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractCurrent high performance photonic devices are composed of inorganic, dielectric semiconductors or metal oxides that are frequently manufactured under harsh reaction conditions. While these devices have been optimized to rapidly modulate light, they are limited in flexibility. Nature offers evolutionarily derived solutions to the current limitations of these devices. Cephalopods, such as the cuttlefish Sepia officinalis, are capable of manipulating their total body coloration and patterning by varying the state and local distribution of their dermal pigments and proteins to reflect, absorb and transmit light; however, the molecular components responsible for this coloration are still not well understood. To identify these components, we first isolated S. officinalis dermal tissue and identified the major proteins within the tissue using tandem mass spectrometry. Identified proteins of interest had homology to the reflectin (<50%) and crystallin (~10%) family of proteins. Thus it appears that S. officinalis camouflage is due in part to the interplay between reflective and light-modulating proteins and pigments. We hypothesized that optical textiles assembled from this skin homogenate would offer photonic properties similar to the native cuttlefish tissue. To test this hypothesis, protein nanofibers (440±210 nm) were manufactured from cuttlefish skin using Rotary Jet Spinning (RJS), a process based on solution extrusion through a perforated reservoir using centrifugal forces. The optical properties of these fabricated materials were analyzed using micro-photoluminescence (PL) spectroscopy. Micro-PL revealed a broad emission centered at ~650 nm from the cuttlefish pigments and proteins, a property which has not been reported for intact skin of S. officinalis. Materials manufactured using these proteins and pigments offer a unique platform to design novel photonic materials potentially useful for adaptive coloration.
4:30 PM - **II7.6
Applications of Sensing and Actuation Materials in Medical Micro-Instruments.
Yogesh Gianchandani 1
1 EECS Department, University of Michigan, Ann Arbor, Michigan, United States
Show AbstractOver the years, medical instruments have provided significant motivation for microsystems research. Implantable devices, in particular, benefit from the small form factor and low power consumption that are signatures of microsystems technology. Silicon microstructures have been investigated for various kinds of neural probes, cochlear prostheses, etc. Progress in microfabrication methods for other materials has opened up a number of new possibilities for micro-instruments. This talk will outline some of the emerging technologies and applications that are being explored in the Wireless Integrated Microsystems (WIMS) Institute at the University of Michigan. For example, antenna-stents fabricated from stainless steel may be instrumented with silicon-micromachined capacitive pressure sensors for applications in which the disease is correlated to intraluminal pressure. Biliary stents can benefit from magneto-elastic sensors for sludge accumulation. Piezoelectric transducers offer the capability for ultrasonic sensing of tissue density at the tip of a biopsy needle. Piezoelectric actuation can also be used for ultrasonic heat generation – for the purpose of tissue ablation and cauterization. A more conventional use of piezoelectric actuation is in valves that are integrated into a drug delivery pump. The use of electronically controlled micro-valve manifolds, with embedded sensors for pressure and flow, promises to transform drug delivery devices.
5:00 PM - II7.7
Nanofluidic/Plasmonic Biosensors for Point of Care Virus Diagnostics and Detection of Biomarkers with the Naked Eye.
Ahmet Yanik 1 2 5 , Min Huang 1 2 , Arif Cetin 1 2 , Alp Artar 1 2 , Hossein Mousavi 4 , Alexander Khanikaev 4 , Gennady Shvets 4 , John Connor 3 2 , Hatice Altug 1 2
1 Electrical and Computer Engineering, Boston Univ, Boston, Massachusetts, United States, 2 Photonics Center, Boston University, Boston, Massachusetts, United States, 5 Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States, 4 Physics, University of Texas, Austin, Boston, Massachusetts, United States, 3 Microbiology, Boston University Medical School, Boston, Massachusetts, United States
Show AbstractWe introduce an ultrasensitive label free biodetection technique based on asymmetric Fano resonances in optofluidic/plasmonic nanoholes with far reaching implications for point-of-care diagnostics [1]. Our sensors bring a number of advantages over commonly available label free sensing techniques: (i) ultrasensitive detection limits surpassing gold standard Biacore sensors, (ii) detection of biomarker molecules with “the naked eye” without using any labeling agents, spectrometer or a camera, (iii) massive multiplexing capabilities, (iv) targeted delivery of analytes to overcome mass transport limitations, and (v) extra degree of freedom in microfluidic design enabling 3-D integration. Normal excitation of surface plasmon polaritons by grating-coupling mechanisms in nanopatterned plasmonic surfaces holds the much promise for massive multiplexing. However, grating coupled plasmonic sensors have not been widely adapted as a result of broader resonance line-widths and lower spectral resolutions due to larger radiative losses. By exploiting extraordinary light transmission phenomena through high quality factor sub-radiant dark modes, we experimentally demonstrate for the first time record sensitivities surpassing the gold standard Biacore devices. Steep dispersion of the plasmonic Fano resonance profiles in high quality plasmonic sensors exhibit dramatic light intensity changes to the slightest perturbations within their local environment. As a spectacular demonstration of the extraordinary sensitivity and the quality of the fabricated biosensors, we show direct detection of a single monolayer of biomolecules with naked eye using these Fano resonances. The demonstrated label free sensing platform offers unique opportunities for point-of-care diagnostics in resource poor settings by eliminating the need for fluorescent labeling and optical detection instrumentation (camera, spectrometer, etc.) as well as mechanical and light isolation.Performances of biosensors are often limited by the depletion zones created around the sensing area which impede the effective analyte transport. One of the main conceptual constraints in previous approaches is that microfluidics and biosensing are always considered as different parts of a sensing platform completing each other but not a fully merged single entity. Our hybrid nanoplasmonic/nanofluidic sensors enable dramatic improvements in mass transport efficiency. Unlike previous approaches where the analytes simply stream pass over the surface, our platform enables targetted delivery of analytes to biosensor surface. The nanoholes in our devices act as ultrasensitive label free sensors as well as nanofluidic channels enabling multilayered lab-on-chip systems and 3D control of fluidic control. We also demonstrate direct detection live viruses from biological media at clinically relevant concentrations with little to no sample preparation. [1] Yanik et al. Proc. Natl. Acad. Sci. in press (2011)
5:15 PM - II7.8
Antimicrobial Peptide Functionalized Graphene Nanosensors for Highly Sensitive Pathogen Detection.
Manu Sebastian Mannoor 1 , Jefferson Clayton 1 , Michael McAlpine 1
1 Mechanical & Aerospace Engineering, Princeton University, Princeton , New Jersey, United States
Show AbstractThe development of a robust and portable sensor for the detection of pathogenic bacteria could impact areas ranging from real-time water quality testing to hospital sanitation monitoring. Of particular interest are biosensors which combine the natural specificity of biological recognition with sensitive, label-free sensors providing electronic read-out. Evolution has tailored antimicrobial peptides to exhibit broad-spectrum activity against pathogenic bacteria, while retaining a high degree of compactness and robustness. We have demonstrated selective and sensitive detection of a variety of infectious agents via electronic detection based on antimicrobial peptide-functionalized nanosensor arrays. The hybrid device was able to demonstrate both Gram-selective detection as well as pathogenic vs. non-pathogenic strain differentiation. We have simulated a “water-sampling” chip, consisting of a microfluidic flow cell integrated onto the nanosensor, which demonstrates real-time on-chip monitoring of the interaction of pathogens with the antimicrobial peptides. Further, via bifunctional self-assembly of peptides onto graphene, we show the synergistic coupling of antimicrobial peptides to graphene nanosensors for detection of bacteria down to single-cell levels. Finally, the incorporation of a parallel resonant circuit with the graphene allows for the formation of a telemetry system which eliminates the need for onboard power or external connections
5:30 PM - II7.9
Supersonic Cluster Beam Implantation: A Novel Approach for Producing Compliant and Biocompatible Micro-Circuits on Elastomers.
Gabriele Corbelli 1 2 , Cristian Ghisleri 1 2 , Paolo Milani 1 2 , Luca Ravagnan 2
1 CIMAINA & Physics Department, Università degli Studi di Milano, Milano Italy, 2 , WISE s.r.l., Milano Italy
Show AbstractThe interest for micro- and nano-manufacturing of polymeric materials is continuously increasing driven by different fields such as polymer-based BioMEMS, stretchable electronics, bioelectronics, conformable sensors and actuators. The need for polymer-based micro-devices requires the integration of micrometric electrodes, circuits and interconnections on soft and compliant polymeric substrates. Unfortunately, the standard approaches used for producing such structures have many drawbacks in terms of layer adhesion, electrical functionality under stretching, attainable lateral resolution, sample heating and biocompatibility of the obtained materials. Recently we developed a new method for polymer metallization: the Supersonic Cluster Beam Implantation (SCBI) of neutral metal clusters in a polymer substrate. The clusters are produced in the form of a supersonic beam by a Pulsed Microplasma Cluster Source (PMCS) [1] and are implanted at RT in the polymer substrate forming a metal-polymer nanocomposite layer [2, 3]. This process avoids both sample heating and sample charging, enabling the metallization of ultracompliant soft polymeric materials. Furthermore, thanks to the high collimation of the cluster beam, patterning of circuits with micrometric resolution can be achieved already with stencil masks, allowing also to form circuits on non planar polymeric substrates (as for example molded parts of MEMS).Here we present the application of SCBI for the fabrication of a biocompatible elastomer-based nanocomposite material made by gold clusters implanted in a polydimethylsiloxane (PDMS) matrix. At odd with electrodes obtained by standard approaches, the ones produced by SCBI are able to withstand thousands of uni-axial stretching cycles (over 50000 at 40% strain), showing an improvement (i.e. the decrease) of the electrical resistance at maximum strain as the number of stretching cycles increases, and only a very small increase of the rest resistance (i.e. at 0% strain). Furthermore, the piezoresistivity of the nanocomposite can be tailored with the amount of implanted clusters, enabling to obtain both high gauge factor films for strain sensing applications and low gauge factor films for stretchable electronics.Biocompatibility tests indicate that neuronal cells adhesion, vitality and differentiation improve on the nanocomposite (even without the use of NGF), proving the high biocompatibility and functionality of this novel material, as required for the production of BioMEMS. These results indicate that SCBI can be considered a promising tool for the fabrication of complex microelectronic circuits and interconnects for polymer-based BioMEMS and for next generation polymer-based implantable biomedical devices.[1] K. Wegner, et al., J. Phys. D: Appl. Phys. 39, R439 (2006). [2] L. Ravagnan, et al., J. Phys. D: Appl. Phys. 42, 082002 (2009).[3] M. Marelli, et al., J. Micromech. Microeng. 21 045013 (2011).
5:45 PM - II7.10
AL2O3 Gate Dielectric with Ion Impermeability for In Vivo Biocircuitry.
Anisha Ramesh 1 , Fang Ren 1 , Patricia Casal 2 , Andy Theiss 2 , Samit Gupta 2 , Stephen Lee 2 , Paul Berger 1 3
1 Electrical and Computer Engineering, The Ohio State University, Columbus, Ohio, United States, 2 Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, United States, 3 Department of Physics, The Ohio State University, Columbus, Ohio, United States
Show AbstractThe key feature needed for in vivo biocircuitry is that it allows stable transistor operation in high salt buffers with an impermeability to mobile alkaline ions that will cause the metal-oxide-semiconductor (MOS) threshold voltage to drift. Permeation of such mobile buffer ions into traditional silicon-based device results is a classic contaminant that would interfere with analyte biosensing, for example. We present here a low cost Si MOS capacitor, stable to buffer-mediated drift, by incorporating atomic layer deposition (ALD) of high-k barrier layers on the device surface, to mitigate ion permeation. Different thicknesses of Al2O3 MOS capacitor have been fabricated. The MOS capacitors were dipped in sterile PBS solution for increasing intervals of time starting from 30 minutes up to 24 hours. The triangular voltage sweep (TVS) method was used to characterize the Na+ ion penetration. No sodium ion (Na+) penetration was observed for the Al2O3 capacitors. By contrast, the dose of Na+ ion penetration into silicon dioxide MOS capacitors increased with increasing soak times in the PBS solution. Further, no evidence of Na+ ion response was observed for varying Al2O3 thickness of 10nm, 25nm, 50nm, 100nm.
Symposium Organizers
Mehmet R. Dokmeci Harvard Medical School
Brigham and Women's Hospital
Junji Fukuda University of Tsukuba
Ali Khademhosseini Harvard-MIT Division of Health Sciences and Technology
Hirokazu Kaji Tohoku University
II8: Microfluidics for Cellular Studies
Session Chairs
Thursday AM, December 01, 2011
Room 206 (Hynes)
9:30 AM - **II8.1
Microfluidic Production of Cell Spheroid and Functional Fibers for Bottom-up Engineering of Tissue.
SangHoon Lee 1
1 Biomedical Engineering, Korea University, Seoul Korea (the Republic of)
Show Abstract3D engineering of tissue from dispersed cell has been a great interest of biomedical researchers for the creation of bioartificial organ and regenerative medicine. For this end, the macro-scale scaffold was fabricated as extracellular matrix, and by seeding and culturing cells on the scaffold, the engineered tissue was created. Despite extensive studies, it is still challenging to produce artificial tissue in vitro environment, and the major region may be from the difficulty in controlling spatiotemporal cell-cell interaction and microenvironment. The top-down engineering of tissue has limit to overcome these problems. Here, we propose the bottom-up engineering of 3D tissue using cell spheroids based on two following key technologies: (1) Concave microwell based spheroid formation of diverse cell including rodent embryonic stem cell, hepatocyte and hepatic stellate cell, islet and cancer cells. All these cells aggregated well forming spheroid in concave microstructure; (2) Microfluidic production of multi-functional microfiber encoded with different chemicals, morphology and cells, and these fibers were used for the creation of microvascular structures in the 3D tissues. For the spheroid generation, micro concave well arrays were fabricated using deflection of thin PDMS membrane. Besides, spheroid generation microfluidic chip was developed for the semi-automatic production of cell spheroid without manual intervention. The 3 types of cell spheroid were produced and their functions were evaluated: (1) EBs from mES cells were differentiated to neuro-progenitor cells and cardiac cells, (2) The metabolic function of heterospheres (mixture of hepatocyte and hepatic stellate cell) were evaluated by measuring albumin and urea secretion and P450, and the co-cultured spheroid showed enhanced liver function hepatocyte mono-cultured spheroid, (3) Islet spheroid for the glucose control, and their function (glucose level) was evaluated through in vivo test. Using the spheroid and multi-functional microfiber, we generated artificial live tissue whose size is approximately 1 cm3 having microvascular structure to demonstrate the feasibility of bottom-up engineering of 3D tissue, and their metabolic functions will be tested.
10:00 AM - II8.2
Conformal Deposition of Poly(N-isopropylacrylamide) on Elastomeric Microstructures.
Halil Tekin 1 2 3 , Tonia Tsinman 2 4 , Gozde Ozaydin-Ince 5 , Karen Gleason 5 , Melik Demirel 6 , Robert Langer* 3 5 7 , Ali Khademhosseini* 2 7 8
1 Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States, 3 David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 4 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 5 Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 6 Materials Research Institute and Department of Engineering Science, Pennsylvania State University, University Park, Pennsylvania, United States, 7 Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 8 Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts, United States
Show AbstractElastomeric microstructures were shown to be useful for different applications such as three dimensional (3D) cell culture, tissue engineering, and microfluidic devices due to their biocompatibility, gas permeability, and elasticity. Coating elastomeric templates with functional materials can be useful for cell and protein capturing, tissue formation, and controlled retrieval of tissue constructs. Poly(N-isopropylacrylamide) (PNIPAAm) is one of these functional materials which was utilized to form tissues and release them in a temperature dependent manner. Previously offered coating methods use liquid phase polymerization, causing non-conformal coatings on microstructures. Herein, we utilized initiated chemical vapor deposition (iCVD) to generate conformal PNIPAAm coatings on poly(dimethylsiloxane) (PDMS) based microgrooves. PNIPAAm coatings on PDMS templates caused increased roughness and showed approximately 3 times higher volumetric swelling at 24 °C compared to at 37 °C. Furthermore, PNIPAAm coated templates exhibited higher surface energy at 24 °C (contact angle θ = 30° ± 2) compared to at 37 °C (θ = 50° ± 1). Cell adhesion on PNIPAAm coated substrates at 24 °C was lower than at 37 °C. Protein adhesion on PNIPAAm coated and non-coated substrates was similar at both 24 °C and 37 °C. Additionally, cells were docked within PNIPAAm coated microgrooves, elongated through the direction of the microgrooves, and formed tissue constructs after three days of culturing. The resulting modular tissues were retrieved by switching the temperature from 37 °C to 24°C, allowing the PNIPAAm to become hydrophilic and swell, and subsequently releasing out the tissue constructs. Therefore, the rigidity, uniform polymer thickness, and thermo-responsive properties could make the PNIPAAm coated elastomeric microstructures beneficial for various applications such as tissue engineering, 3D cell culture, microfluidics, and drug discovery as ways to form tissue-constructs effectively and efficiently.
10:15 AM - II8.3
Surface Engineering for Rapid Cell Detachment by Using Electrochemical Desorption of a Zwitterionic Oligopeptide Layer.
Takahiro Kakegawa 1 , Hiroaki Suzuki 1 , Junji Fukuda 1
1 , University of Tsukuba, Tsukuba, Ibaraki, Japan
Show AbstractThis study describes a culture surface to which cells are preferably attached and then readily detached by means of an electrical stimulus. Two oligopeptides, CGGGKEKEKEK and CGGGKEKEKEGRGDSP, were designed to form self-assembled monolayers on a gold surface via a gold-thiolate bond and the electrostatic force between the alternating charged glutamic acid (E) and lysine (K) sequence. Owing to ionic solvation in the alternating sequence, the modified surface resisted nonspecific adsorption of proteins, while cells adhered to this surface via the RGD sequence. Indeed, quartz crystal microbalance measurement revealed that the non-specific adsorption of proteins on the peptide-modified surface was significantly reduced as compared to a surface without modification. The application of a negative potential to the gold surface resulted in desorption of the monolayers and following detachment of the attached cells. In the quantitative measurements, it was shown that ~70% of the cells were detached in the first 1 min of potential application and the remaining cells were completely detached in the following 1 min of potential application. To our best knowledge, this is the most rapid cell detachment approach among those based on other principles. This approach could potentially be a new tool for manipulating cells and engineering tissues.[References][1] R. Inaba, A. Khademhosseini, H. Suzuki, J. Fukuda, “Electrochemical desorption of self-assembled monolayers for engineering cellular tissues,” Biomaterials, 30(21), pp. 3573-9, 2009.[2] Y. Seto, R. Inaba, T. Okuyama, F. Sassa, H. Suzuki, J. Fukuda, “Engineering of capillary-like structures in tissue constructs by electrochemical detachment of cells,” Biomaterials, 31(8), pp. 2209-15, 2010.[3] J. Fukuda, Y. Kameoka, H. Suzuki, Spatio-temporal detachment of single cells using microarrayed transparent electrodes, Biomaterials, in press
10:30 AM - **II8.4
Cell and Tissue Culture in Structured Microenvironment.
Teruo Fujii 1 2
1 Institute of Industrial Science, University of Tokyo, Tokyo Japan, 2 , JST CREST, Tokyo Japan
Show AbstractMicrofluidic devices can be used as advanced platforms for cell and tissue culture experiments. In addition to in vivo mimicking microstructures formed in the device, one can apply more sophisticated control on cells and tissues in terms of soluble factors, extra cellular matrices, etc. In other words, cells and tissues can be placed in structured microenvironment, which is completely different from uniform and homogeneous conditions in conventional culture formats such as dishes and bottles. The structured microenvironment is beneficial not only to advanced characterization of cellular functions, but also to the development of automated and high throughput culture systems. In this talk, some examples of microfluidic culture are presented to show the useful features of the “structured microenvironments”. The examples include the cultures of rat primary hepatocytes in in vivo mimicking structure of hepatic cords, directed differentiation processes of mouse iPS and ES cells, and body-on-a-chip type platforms for drug testings.
11:30 AM - II8.5
All-Elastomeric, Multiplexed Microfluidic Phage Display Biopanning.
Kellye Cung 1 , Michael McAlpine 1 , Russell Slater 1 , Yue Cui 1 , Habib Ahmad 2
1 , Princeton University, Princeton, New Jersey, United States, 2 , California Institute of Technology, Pasadena, California, United States
Show AbstractThe development of a method for automated proteomic screening could impact areas ranging from fundamental molecular interactions to the discovery of novel disease markers and therapeutic targets. Surface display techniques allow for efficient handling of large molecular libraries in small volumes. In particular, phage display has emerged as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Although screening against single targets is inherently high thoughput, the process becomes cumbersome when multiple targets are involved. Here, we demonstrate for the first time a fully elastomeric microfluidic chip that can perform phage display biopanning assays on multiple targets in parallel. The chip is shown to be able to identify well-established control consensus sequences while simultaneously identifying undiscovered sequences for clinically important targets. Indeed, the confined parameters of the device allow for highly controlled assay conditions. We anticipate that this easily-fabricated, disposable device has the potential to impact areas ranging from fundamental studies of protein, peptide, and molecular interactions, to applications such as fully automated proteomic screening.
11:45 AM - II8.6
Voltage Controlled Electrostatic Trap for Confining Nanometric Objects in a Fluid.
Ashwin Panday 1 2 , Brandon Lucas 3 , L. Jay Guo 1 2 3
1 Macromolecular Science and Engineering , University of Michigan, Ann Arbor, Michigan, United States, 2 Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, Michigan, United States, 3 Applied Physics, University of Michigan, Ann Arbor, Michigan, United States
Show AbstractThe capability to trap or confine nanometric objects and single molecules in fixed positions can allow researchers to acquire unprecedented detail about chemical interactions, binding events in biological systems, creating nanostructures with high precision etc. Technologies like optical tweezers and electrokinetic traps (ABEL) are widely used to trap particles but trapping small macromolecules for extended periods of time remains challenging due to the photo physical constraints and the randomizing effect of brownian motion. The aim of this work is to explore the active use of electrostatic fields, applied to the solutions containing nanometric objects, in order confine them to desired positions and also to desorb them at will. This system is designed to mimic the biological phenomena of protein enzyme attracted to and diffuse along a DNA molecule. Since all materials carry some amount of surface charge in solution, in principle it should be possible to attract or repel them from a surface having opposite or same surface charges. Our experiment is based on creating a tunable, spatially modulated electrostatic potential profile by biasing a metal nano line in an aqueous solution. The potential profile created near the nano line can be tuned and optimized by changing its voltage. Au nano-lines were made on a substrate by using various techniques like Focussed Ion Beam and conventional lithographic techniques. Charged polystyrene nano particles and polyamidoamine (PAMAM) dendrimers(G-5) are being studied for their interactions. It is noteworthy that 5nm dendrimers are the same size as globular proteins. Solutions with low salt concentrations are used to suspend the particles inside a micro channel. Fluorescent polystyrene particles and fluorescence-labelled dendrimers were imaged in a standard wide-field configuration using 473nm excitation and a 520 LP filter. In addition a total internal reflection (TIRF) microscope was used to capture the surface interactions. The positive charge created on the nano line, using an external power supply, attracts the negatively charged polystyrene particles electrostatically. It has been seen that the particles from the free solution drift towards the voltage biased metal line and line up. The magnitude of the voltage controls the force of attraction. By reversing the polarity of the charge on the nano line, it was seen that the attracted particles desorb from the surface as the force changes from attractive to repulsive. Some irreversible binding events do occur and could be due to some irregularities in the surface morphology. The electrostatic potential profile for a line charge can be easily modeled and helps in optimizing the dimensions of the nano-line. This setup allows for a single active trap which can be used for confining different types of molecules as it is material independent and relies on the charge on the molecule.
12:00 PM - II8.7
Smart Surfaces: Use of Electrokinetics for Selective Modulation of Biomolecular Affinities.
Sam Emaminejad 1 2 , Mehdi Javanmard 2 , Robert Dutton 1 , Ronald Davis 2
1 Electrical Engineering, Stanford University, Stanford, California, United States, 2 Biochemistry, Stanford Genome Technology Center, Stanford, California, United States
Show AbstractA platform has been demonstrated, both with simulation and prototype experiments that offers the potential for performing a bead-based multiplexed immunoassay where in a single channel various regions are immobilized with a different antibody, each targeting a different antigen. In our assay we would like to elute the immuno-bound beads from each individual region for further downstream quantification and analysis. For multiplex analysis, we need to selectively elute beads from each region one by one. We use negative dielectrophoresis (nDEP) in conjunction with shear force, which provides the switch-like behavior necessary to achieve this goal. At an optimal flow rate, nDEP turned on results in bead detachment, whereas when nDEP is off, the beads remain attached. As a proof of concept we demonstrate the ability of nDEP to provide this switching behavior in a singleplex assay. First order vertical nDEP forces along with the drag force applied to the beads for given flow rates were calculated using COMSOL. For proof of concept, we demonstrated the elution of a Protein G–IgG interaction which is on the same order of magnitude in strength as typical antibody-antigen interactions. Polystyrene protein G coated beads (6.7 µm-diameter) were first conjugated to biotinylated anti-IgG and then streptavidin. Biotinylated BSA was physically adsorbed on the channel surface. The beads were incubated in the channel such that they bound to the surface. For each experiment, flow rate was increased gradually until all beads detached. At each flow rate, we quantified the percentage of detached beads relative to total number of initially captured beads (at rest). For the case of 20 Vpp all beads detached as flow rate reached 0.35 µLmin-1, whereas when DEP was off more than 90% still remained attached. With nDEP off, the flow rate had to be increased to 0.95 µLmin-1 in order to remove the majority (70%) of the beads.
12:15 PM - II8.8
Raman Mapping of Surface Distribution and Trace Detection of Common Biomolecules and Stress-Related Biomarkers.
Zachary Combs 1 , Sehoon Chang 1 , Kyle Anderson 1 , Richard Davis 1 , Vladimir Tsukruk 1
1 Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
Show AbstractWe have demonstrated a nanoengineered substrate composed of micropatterned silver or gold nanoparticles to be used for the label-free mapping and trace detection of adsorbed biomolecules. We utilized surface-enhanced Raman scattering (SERS) phenomenon to monitor the known bioanalytes, protein A and human immunoglobulin G (IgG), as well as stress-related catecholamine biomarkers such as dopamine and serotonin. This label-free SERS approach provides accurate, selective, and fast detection of protein A and IgG solutions with a nanomolar concentration, down to below 1 nM for IgG in solution. Also, the trace detection of small biomolecules such as dopamine and serotonin provides a new method for monitoring these important stress-related biomarkers. This label-free method could also be utilized for the facile detection of proteins in field conditions as well as in clinical, forensic, industrial, and environmental laboratories through further development into a multiplexing device.
12:30 PM - II8.9
Combinatorial Identification and Zygosity Discrimination of Apolipoprotein-E Single Nucleotide Polymorphisms in a Microfluidic Electrochemical Device.
Allen Yang 1 , Kuangwen Hsieh 1 , Tom Soh 1
1 , University of California at Santa Barbara, Santa Barbara, California, United States
Show AbstractThere has been a recent growing interest in the detection of single nucleotide polymorphisms (SNP) for the early identification of genetic diseases. As an example, large population genomics studies suggest that people who carry a copy of one of the minor alleles of the Apolipoprotein E (ApoE) gene are at increased risk for late-onset Alzheimer’s disease. There is a greater variability in the ApoE gene which has SNPs at two locations in the gene sequence that code for the three common variants of the protein (one normal and two minor alleles) resulting in six potential homozygous and heterozygous combinations. A diagnosis platform for screening patients for genetic markers for Alzheimer’s would need to address the following requirements: (1) combinatorial identification of target genetic markers, (2) differentiation of allele zygosity, and (3) the ability to detect unpurified PCR-amplified target. While there is considerable research into a variety of techniques for rapid SNP screening such as ligation reaction or molecular beacons, these methods generally only fulfill one of these important requirements and there is no singular technique that can demonstrate all these capabilities. We report here a microfluidic multi-target SNP detection platform that uses electrochemical melt curve analysis for rapid combinatorial identification of ApoE SNP alleles from PCR amplified ssDNA. This detects the electrochemical current changes as hybridized target melts from self-assembled oligonucleotide probes due to an increase in surface electrode temperature. Our specific approach uses methylene blue modified ssDNA probes that allows efficient hybridization with PCR amplicons. The probes generate a redox current that responds to the hybridization and melting of target amplicons. Continuous monitoring of the probe current as electrode temperature increases allows us to construct an electrochemical melt curve. Analysis of melting temperature differences enables a single probe to identify a perfectly matched, mismatched, or heterozygous mix of matched and mismatched target. We show that by using a second probe and electrode in the same microfluidic volume that we can identify in real-time the presence of SNPs at either location in the ApoE sequence and from the melt curves identify the exact ApoE allele pairing of any sample from the six possible combinations. We demonstrate that we have solved critical challenges in SNP detection by demonstrating the combinatorial identification and zygosity discrimination of PCR-amplified Alzheimer’s disease genetic markers.
12:45 PM - II8.10
Stable and Solvent-Less Lipid Bilayers Based on Nano- and Micro-Fabrication.
Ayumi Hirano-Iwata 1 2 , Azusa Oshima 1 , Tomohito Nasu 1 , Yasuo Kimura 1 , Michio Niwano 1
1 , Tohoku University, Sendai Japan, 2 , PRESTO, Japan Science and Technology Agency (JST), Saitama Japan
Show AbstractReconstitution of ion channels in free-standing bilayer lipid membranes (BLMs) provides an excellent system for drug screening and designing an exquisite biosensor. However, mechanical instability of BLMs hinders widespread application of BLM systems. Conventional free-standing BLMs are formed across small apertures in plastic septa. The size of the aperture is commonly in a micrometer range, which seems too large to stably suspend nanometer-thick BLMs. In this presentation, we will show you our recent approaches for preparation of stable BLMs through the combination of nano- and micro-fabrication and BLM formation. Solvent-less BLMs were formed across nanometer-scaled apertures, such as anodic nanoporous alumina films [1] and microfabricated Si chips [2], and the membrane properties were characterized in terms of electric properties, functionality and membrane stability. Nanoporous alumina films were formed by anodization of an aluminum sheet (99.999%) in oxalic acid or phosphoric acid. The remaining aluminum layer was removed in a mixture solution of CuSO4 and HCl. Microapertures (diameter: 20-30 micrometer) were fabricated in a 240 nm thick Si3N4 layer deposited on a FZ Si (100) wafer (>9000 ohm cm, 200 micrometer in thickness). The apertures were formed by isotropic etching in hot phosphoric acid. BLMs were prepared by the monolayer-folding method and current recordings were performed with an Axopatch 200B patch-clamp amplifier (Molecular Devices).BLMs spanned over the porous alumina films showed improved membrane stability and background noise currents small enough for recording gramicidin single-channel currents. Much higher stability was observed with BLMs formed in microapertures fabricated in nanometer-thick Si3N4 septa. Since the edge of the aperture was smoothly tapered, the stress on lipid bilayers at the contact with the septum was minimized owing to the tapered structure of the aperture edge. The BLMs were resistant to applied voltage of ±1 V and the lifetime of the membranes was 15-43 h with and without incorporated channels. The BLM containing gramicidin channel exhibited tolerance to repetitive solution exchanges, though the electric properties of the BLMs are necessary to be improved. The realization of BLMs having both mechanical stability and proper electric properties will open up variety of applications including highly sensitive biosensors and high throughput drug screenings for ion channels. References[1]A. Hirano-Iwata, T. Taira, A. Oshima, Y. Kimura, and M. Niwano, Appl. Phys. Lett., 96, 213706 (2010).[2]A. Hirano-Iwata, K. Aoto, A. Oshima, T. Taira, R. Yamaguchi, Y. Kimura, M. Niwano, Langmuir, 26, 1949–1952 (2010).
II9: BioSensors and Detection Technologies
Session Chairs
Thursday PM, December 01, 2011
Room 206 (Hynes)
2:30 PM - **II9.1
Laser Treated Paper: A Versatile Microsystem Substrate.
Girish Chitnis 1 3 , Babak Ziaie 2 3 4
1 School of Mechanical Engineering, Purdue University, West Lafayette, Indiana, United States, 3 Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana, United States, 2 School of Electrical and Computer Engineering, Purdue University, West Lafayette, Indiana, United States, 4 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United States
Show AbstractUse of silicon and other electronic substrates to create micro systems such as lab-on-a-chip devices is very common. Although excellent semi-conductor properties of such materials are not particularly useful for such applications, we still need to pay the high price for the substrate, unnecessarily. The purpose of research presented here is to develop inexpensive processes and applications for ultra-low cost substrates like paper. The basic process in this research utilizes change in the surface properties of a hydrophobic substrate due to laser ablation. Any paper with a hydrophobic surface coating (e.g., culinary parchment paper having a silicone-based coating) can be used for this purpose. Although it has been known that laser modification can alter the wettability of the engineering materials by causing structural and chemical changes to the surface, to date no such treatment has been applied to paper. Laser engraving system generates patterns by raster scanning laser across the surface. Since the system directly writes computer generated pattern onto the paper, it is very easy to inscribe complex shapes. Patterns, up to 60 µm, are created reliably, using cheap hydrophobic paper (wax paper, parchment paper etc.) and a CO2 laser (10.6μm wavelength, 120 W maximum power). SEM images show that the laser treatment creates micro/nano scale porous structures on the surface due to physical modification (melting and re-solidification) of hydrophobic coating. XPS spectroscopy reveals that the laser treatment also causes oxidation of the surface chemical resulting in polar hydroxyl groups on the surface which also contributes towards hydrophilic nature of the surface.The patterned substrate is used to controllably deposit various substances such as chemicals and colloids to create functional papers. Since surface roughness of the laser treated area helps in trapping colloidal particles, we are able to pattern hydrocarbon based colloids as well as the aqueous ones. Here we demonstrate patterning of bio-reagent (luminol) and magnetic nanoparticles (aqueous and hydrocarbon based), to create a paper-diagnostic-device and a paper-actuator respectively. Magnetic paper is treated further by reheating up to melting temperature of wax which results in a coating of wax that protects the sample from possible particle detachment. It is important to note that this process can be extended to pattern many more materials to create variety of functionalities.In conclusion, a maskless, simple and inexpensive technique to create pattern functional material on paper is developed. Heat treatment can be used to create a protective layer of wax that makes the pattern more robust and water-resistant. Overall, laser treated hydrophobic paper has a great potential to develop into a versatile substrate for microsystems.
3:00 PM - II9.2
Anisotropic Cellular Alignment on Nano-Wrinkled Polymeric Surface.
Toshinori Fujie 1 2 , Francesco Greco 1 , Silvia Taccola 1 3 , Leonardo Ricotti 3 , Arianna Menciassi 1 3 , Virgilio Mattoli 1
1 Center for MicroBioRobotics IIT@SSSA, Istituto Italiano di Tecnologia, Pontedera, Pisa, Italy, 2 European Biomedical Science Institute (EBSI), Organization for European Studies, Waseda University, Tokyo Japan, 3 Biorobotics Institute, Scuola Superiore Sant’Anna, Polo Sant’Anna Valdera, Pontedera, Pisa, Italy
Show AbstractNano/micro scale topography of materials surface promotes unique cell culture environment, which is a useful tool for understanding of cell biology as well as development of tissue engineering scaffold towards regenerative medicine [1]. Up to date, such intelligent surface has been fabricated by photo-lithography, micro-contact printing, nano/micro imprinting. However, considering the process integration, these approaches were not always adequate in order to produce large dimensional patterns conveniently and rapidly. Recently, Khine et al introduced an ultra-fast and inexpensive approach to prepare nano-wrinkled metal surface without conventional lithographic approach by utilizing shape memory polymer film [2]. The nano-wrinkled surface was induced by leveraging mismatch in stiffness between a prestressed polymer sheet and an overlaying thin metal film. In this study, we evaluated the effect of the nano-wrinkled polymeric interface on the adhesion, proliferation and differentiation properties of murine skeletal muscle cells (C2C12). A tens-of-nm thick layer of poly(3,4-ethylenedioxythiophene) with poly(styrenesulfonate) (PEDOT:PSS) was spincoated on the thermo-retractable polymer film (PolyShrink®). Samples with different periodicity of the nano-wrinkles were produced by thermal treatment of the polymer sheet covered with different thickness of PEDOT:PSS layer. Then, adhesion and proliferation of C2C12 were compared among different samples. The cells preferentially adhered and aligned on low and narrow ridges (1.5 μm height) rather than high and wide ridges (2.5 μm height). Furthermore, we found these trends were also observed in the differentiation stage of C2C12 into the myotube formation. Combination of living cells with tunable nano-wrinkles bearing conductive polymeric surface will provide a unique tool for cell biology, tissue engineering and micro-biodevices.References[1]C. J. Bettinger, R. Langer, J. T. Borenstein. Angew. Chem. Int. Ed. 48, 5406 (2009).[2]C. Fu, A. Grimes, M. Long, C. G. L. Ferri, B. D. Rich, S. Ghosh, S. Ghosh, L. P. Lee, A. Gopinathan, M. Khine. Adv. Mater. 21, 4472 (2009).
3:15 PM - II9.3
A Rapid and Sensitive Detection of Cardiac Markers in Human Serum Using Surface Acoustic Wave Immunosensor.
Joonhyung Lee 1 , Yoon Suk Choi 1 , Yeol Ho Lee 1 , Jung Nam Lee 1 , Hun Joo Lee 1 , Sang Kyu Kim 1 , Kyung Yeon Han 1 , Soo Suk Lee 1 , Jae Chan Park 1
1 BioLab/Samsung Advanced Institute of Technology, Samsung Electronics, Yongin-si, Gyeonggi-do, Korea (the Republic of)
Show Abstract We present a rapid and sensitive surface acoustic wave (SAW) immunosensor that utilizes gold staining as a signal enhancement method. A sandwich immunoassay was performed on the sensing area of SAW sensor and could specifically capture and detect cardiac markers (Cardiac Troponin I (cTnI), Creatine kinase (CK)-MB, and myoglobin). The analytes in human serum were captured on gold nanoparticles (AuNPs) that were conjugated in advance with detection antibody. Addition of these complexes onto capture antibody-immobilized sensor surface resulted in a classic AuNP-based sandwich immunoassay format that has been used for signal amplification. In order to achieve further signal enhancement, gold staining method in this detection strategy was carried out, which proved possible to obtain gold staining-mediated signal augmentation on a mass-sensitive device. In this regard, we investigated the dependence of sensor response due to gold staining as a function of applied cardiac marker concentration in single sensor chip that was Love wave type SAW biosensor designed to operate at the center frequency of 200 MHz. As the concentration of cTnI, CK-MB, and myoglobin increased from 20 pg/ml to 500 ng/ml, from 1.1 ng/ml to 100.5 ng/ml and from 16.0 ng/ml to 1008 ng/ml, respectively so did sensor response due to gold staining. The minimum detectable concentrations of cTnI, CK-MB, and myoglobin were 20 pg/ml, 1.1 ng/ml, and 16.0 ng/ml, respectively, which are lower than clinically relevant concentration (cTnI : 400 pg/ml; CK-MB : 4.3 ng/ml ; Myoglobin : 107 ng/ml). Hence, we demonstrate the potential of the SAW biosensor to perform the sensitive detection of cardiac markers.We validated the effect of a higher operating frequency of SAW sensor on the sensor responses. It has been proven that an operating frequency in acoustic wave sensor depends on mass sensitivity. Furthermore, SAW sensor with a higher operating frequency offers significant advantages in terms of cost as well as ease of construction due to lower thickness of the guiding layer. In order to verify effectiveness of the sensor that was designed to operate at a center frequency of 400 MHz, we investigated the effect of applied cTnI concentration on the sensor response due to gold staining with the same assay protocol as was seen in the 200 MHz sensor. The frequency shift in the 400 MHz sensor was compared with that in the 200 MHz sensor chip. The 400 MHz sensor produced approximately 1.5 to 2 times higher frequency response for cTnI in the range of 0.02-50 ng/ml with a steeper slope of signal versus concentration. This demonstrates the potential that the detection limit can be further improved by SAW sensor with higher operating frequency. Due to its size and high sensitivity, we expect this method to be useful in development of devices for point of care diagnostics.
3:30 PM - II9.4
Efficient Capture and Detection of HIV Subtypes by Oriented Antibody Immobilization in a Microchip for Enhanced HIV Viral Load Detection Applications.
Shuqi Wang 1 , Matin Esfahani 1 , Umut Gurkan 1 , Giguelc Francoise 3 , Daniel Kuritzkes 2 , Utkan Demirci 1
1 Harvard-MIT Health Sciences and Technology, Harvard-MIT Health Sciences and Technology, Cambridge, Massachusetts, United States, 3 Infectious Diseases Unit, Massachusetts General Hospital, Boston, Massachusetts, United States, 2 Harvard Medical School, Harvard University, Cambridge, Massachusetts, United States
Show AbstractDetection of viruses from bodily fluids poses significant challenges for the point-of-care (POC) applications. Viral load tests are not performed at the POC, since sample preprocessing free, inexpensive, quantitative POC viral load technologies are not available. Main challenges with developing such technologies are the difficulty to selectively capture virus particles at high efficiency reliably from unprocessed samples. Antibody immobilization is a promising approach given that repeatable and reliable antibody orientation can be achieved on the surfaces. We demonstrated that high-density antibody coating with favorable orientation for antigen interaction using a Protein G-based surface chemistry in microchannels can effectively capture various subtypes of HIV. We have presented that the Protein G-based surface chemistry performs better over the three other immobilization methods of passive adsorption, GMBS-based covalent attachment, and NeutrAvidin-based antibody immobilization. The fluorescence intensity analysis demonstrated that Protein-G elevated the antibody density by more than an order of magnitude on the channel surface compared to the passive adsorption and GMBS-based covalent attachment methods. This was further verified using nanoscale surface characterization by atomic force microscopy. Further, intact HIV particles from subtypes A, B and C at concentrations of 1000, 10,000, and 100,000 copies/mL were captured at a repeatably high efficiency (72.5-87.8%) measured by RT-PCR using protein-G based gp120 antibody immobilization. The immuno-sensing device with uniformed antibody orientation can potentially enable the development of POC devices to monitor antiretroviral treatment (ART) in resource-limited settings.
3:45 PM - II9.5
Enhanced Biosensing Using Delay Path Modifications in Shear Horizontal Surface Acoustic Wave Sensors.
Subramanian Sankaranarayanan 1 , Stefan Cular 2 , Reetu Singh 2 , Venkat Bhethanabotla 2
1 Center for Nanoscale Materials, Argonne National Lab, Argonne, Illinois, United States, 2 Chemical and Biomedical engineering, USF, Tampa, Florida, United States
Show AbstractSurface acoustic wave devices have been used as sensors for many years; however, the devices used are typically non-optimized designs or have been optimized for a different uses such as band-pass filters . Although these devices work, there are many modifications that can be done to improve the device for sensor applications. We report on the development of novel micro-cavity based acoustic sensors with dramatically enhanced acoustic entrapment that can potentially detect sub-picogram level mass loadings. In this work, we present a finite element analysis study of square, circle, triangle and pentagon shaped micro-cavities filled with polystyrene to vary the sensitivity and operational frequency of shear horizontal surface acoustic wave sensors. The micro-cavities are based on λ/2 side length/diameter and λ/8 depth etched into a 36° YX LiTaO3 substrate. By altering the shape of the micro-cavities the angle at which the surface wave hits the micro-cavities edges is altered for the primary wave shear horizontal wave as well as leaky waves (bulk waves) and subsequent reflective waves. The polystyrene filling of the micro-cavities, similar to a Love-wave device, acts to entrap energy; these inhomogeneous waveguide structures for surface acoustic wave sensors have not been previously reported in literature.Four designs polystyrene filled micro-cavities were measured for characteristics common to surface acoustic wave devices and sensor. The transmitted energy plots for the square, circle, triangle and pentagon designs suggest that the general band-pass characteristics of the devices are similar with the pentagon device several decibels greater. A 5 dB increase in energy transmission is clearly observed for the pentagon over the circle and square designs. Based on our simulation data, we find that Love waves incident on a shallow groove or cavity on the surface of a piezoelectric substrate filled with another elastically isotropic material such as polystyrene is weakly scattered with a part of the energy contained in the reflected and the transmitted Love waves and the other part converted into the bulk shear waves that propagate into the substrate. Our analysis of displacement vector plots for the each of the four designs indicate that these improvements (pentagon>triangle>square>circle) are brought about by a larger coherent reflection of the incident Love wave and subsequent reduced conversions into bulk shear modes which radiate into the substrate. Our simulation results also suggest that although more energy is lost by the square design than the pentagon design, the square design is more sensitive to mass additions to the surface of the delay path. The present study indicates that micro-cavity based sensor designs are a significant improvement over the typical sensor designs based on conventional surface acoustic wave propagation.
4:30 PM - **II9.6
Microfabricated Electrochemical Devices for the Determination of Compounds Related to Cell Metabolism.
Hiroaki Suzuki 1
1 Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
Show Abstract Over the past 40 years, a marked progress has been made in microfabrication of electrochemical sensors. In a recent trend to downsize the entire system and incorporate multiple functions, our group has developed devices particularly aiming for the monitoring of compounds related to cell metabolism, considering the application to cell engineering, environmental technology, and food science. The ability to handle a small volume of samples and/or necessary reagents has enabled measurements which were previously difficult to conduct. In a device developed for the monitoring of ammonia metabolism of hepatocytes, a small volume of a medium of the nL order was extracted using an integrated microfluidic system with electrowetting-based valves. Unlike the conventional analysis that uses a culture dish, the decrease in the concentration of ammonia could be measured in detail using an integrated ammonia electrode. Continuous monitoring is another option. Ammonia metabolism of active sludge was monitored continuously using an integrated NH4+ ion-selective electrode. Here, a critical point is the durability of the thin-film Ag/AgCl reference electrode used as a potential standard. To realize a long lifespan, we used a protecting layer with pinholes for a silver layer and applied a small constant current to the electrode to grow AgCl gradually to stabilize the potential. Clear unique tendencies were observed for two days, which clearly depended on signal molecules added to the medium. Depending on the cases, mixing of reagents is required for the analysis. Freshness of fish was measured by detecting ATP in a plug-based microfluidic system that facilitated processing of solutions including mixing. The decrease in the concentration of ATP in tuna or jack mackerel was clearly detected electrochemically using two enzymes, which agreed well with data obtained by HPLC.
5:00 PM - II9.7
Template-Stripped Plasmonic Nanohole Arrays for Surface Plasmon Resonance Biosensing.
Hyungsoon Im 1 , Si-Hoon Lee 2 , Nathan Wittenberg 1 , Timothy Johnson 1 , Nathan Lindquist 1 , Prashant Nagpal 3 , David Norris 4 , Sang-Hyun Oh 1 2
1 Electrical and Computer Engineering, University of Minnesota, Minneapolis, Minnesota, United States, 2 Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, United States, 3 , Los Alamos National Laboratory, Los Alamos, New Mexico, United States, 4 , ETH Zürich, Zürich Switzerland
Show AbstractReproducible high-throughput fabrication of nanometric apertures in metallic films can benefit many applications in plasmonics, spectroscopy, lithography, imaging, and sensing. Here we use template stripping to replicate periodic nanohole arrays in optically thick metal films for surface plasmon resonance (SPR) biosensing. The films are stripped from reusable silicon master templates prepared using nanoimprint lithography. The template-stripped smooth metallic surface is then encapsulated with a thin silica overlayer, which can be readily modified with biomolecules for affinity biosensing. The silica-coated silver nanohole array chip can also be bonded with polydimethylsiloxane (PDMS) microfluidic channels for fluorescence imaging, formation of supported lipid bilayers, and surface plasmon resonance sensing. The silica-coated nanohole arrays are used to characterize binding kinetics between streptavidin and biotinylated lipid membranes in a real-time, label-free manner. Because nearly centimeter-sized chips can be produced inexpensively without using any additional lithography, etching or lift-off, this method can facilitate widespread applications of metallic nanohole arrays for plasmonics and biosensing.
5:15 PM - II9.8
Miniature Magnetic Resonance System for Point-of-Care Diagnostics.
Changwook Min 1 , David Issadore 1 , Jaehoon Chung 1 , Huilin Shao 1 , Monty Liong 1 , Ralph Weissleder 1 2 , Hakho Lee 1
1 Center for Systems Biology, Massachusetts General Hospital, Boston, Massachusetts, United States, 2 Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States
Show AbstractRobust, sensitive and easy-to-use biosensors for cellular analyses will have significant applications in both basic research and clinical practice. We have recently developed a microNMR (µNMR) technology as a new diagnostic platform to perform rapid, multiplexed, and accurate measurements of cells, proteins and small molecules. The system utilizes targeted magnetic nanoparticles (MNPs) to amplify the analytical signals in NMR detection. When molecularly-specific MNPs identify their targets, the particles induce large, amplified changes in the transverse relaxation of water protons by producing local magnetic fields. As most biological entities have negligible magnetic susceptibilities, DMR measurements can be performed with minimal or no sample purification steps, allowing for fast assays. Here we present our latest µNMR system, specifically designed for clinical applications in point-of-care settings. A major challenge in achieving reliable DMR detection is the fluctuation of NMR frequency (fo) with temperature, which originates from the temperature-dependent drift of the magnetic field from the permanent magnet in the system. As an alternative to using costly and bulky heaters to control magnet temperature, we have implemented a new, automated feedback system that keeps track of fo and reconfigures measurement settings. The mechanism enables robust µNMR measurements in realistic clinical environments (4–50 °C). Furthermore, the new system interfaces with mobile phones for its operation, a feature that maximizes the system portability as well as facilitates data management. The clinical utility of the new µNMR system is demonstrated by detecting and molecularly profiling cancer cells from patient samples. We also present highly sensitive and rapid (<30 min) detection of pathogens (Staphylococcus aureus).
5:30 PM - II9.9
Design, Fabrication, and Characterization of Nanogap Capacitive Sensors for Ultrasensitive Biosensing Applications.
Oguz Hanoglu 1 , Handan Acar 2 , Selim Sulek 2 , Firat Yilmaz 1 , Mustafa Yuksel 4 , Sedat Agan 3 , Necmi Biyikli 2 , Mustafa Guler 2 , Ali Okyay 1 2
1 Electrical and Electronics Engineering, Bilkent University, Ankara Turkey, 2 UNAM – Institute of Materials Science and Nanotechnology, Bilkent University, Ankara Turkey, 4 Ankara Vocational School of Health Sciences, Fatih University, Ankara Turkey, 3 Physics, Kirikkale University, Kirikkale Turkey
Show AbstractHigh-performance biosensors can pave the way for early stage diagnosis of critical diseases. Satisfying the required characteristics of commercial-grade biosensors like sensitivity, dynamic range and reproducibility is the main challenge in biosensor materials and device research. Capacitive sensors, being label-free, low-cost, portable and fast, stand out as promising candidates to solve the current problems in this field. However, devices in the literature often have limited capability especially in sensing low concentrations of target molecules.In this study, we show that a metal-insulator-metal (MIM) capacitor with a vertical nanogap is sensitive to the specific binding of streptavidin to biotin. The binding of streptavidin to biotin molecules that are attached to the insulator between the electrodes modifies the effective dielectric medium in the nanogap between the metal electrodes. This result in a significant capacitance change measured between the electrodes.Vertical nanogaps are formed by consecutive deposition of metal and dielectric layer stack of Cr(10nm)/Au(100nm)/Cr(10nm)/SiO2(200nm)/Al2O3(20nm)/Cr(10nm)/Au(120nm) followed by a partial sacrificial etching step. The top 120-nm-thick Au layer is patterned using standard lift-off process. Using this Au layer as an etch mask, SiO2 + Al2O3 layers are partially etched in diluted HF(hydrofluoric acid) solution such that vertical nanogaps are formed at the edges of the electrodes. For active biosensing, the oxide layer is functionalized by a standard surface modification procedure. The bindings of the self assembled monolayer and biotin are detected by XPS(X-ray photoelectron spectroscopy) measurements. Varying molar concentrations of 50µL streptavidin/PBS(phosphate buffered saline) solutions are introduced to samples via a micropipette. All measurements are conducted in DI(de-ionized) water (εDI-Water=80, εstreptavidin-biotin=2.1) after DI-water rinse and nitrogen dry for repeatability. Frequency-dependent capacitance values are measured with a Keithley Semiconductor Characterization System (4200-SCS) with integrated C-V analyzer. When compared to othercapacitive biosensors which can only take measurements at very high frequencies(>100MHz) with relatively expensive instruments to eliminate the parasitic contribution of the ions in the solution, our biosensor utilizing the nanogap configuration can be operated at low-frequencies (1KHz-1MHz). Our measurements showed that device sensitivity has a peak around 10 KHz. At this frequency, we observed more than 11% change in measured capacitance when 0.01mg/mL streptavidin was applied.
5:45 PM - II9.10
Electrical Detection of Surface Enzymatic Reaction in Nanochannels.
Chuanhua Duan 1 , Yu-Feng Chen 2 , Dong-Kwon Kim 3 , Christopher Brown 4 , Charles Craik 4 , Arun Majumdar 1 5
1 Mechanical Engineering, University of California, Berkeley, Berkeley, California, United States, 2 Green Energy and Environment Research Laboratories, Industrial Technology Research Institute, Hsinchu Taiwan, 3 Mechanical Engineering, Ajou University, Suwon Korea (the Republic of), 4 Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, United States, 5 ARPA-E, US Department of Energy, Washington, District of Columbia, United States
Show AbstractWe report label-free electrical detection of surface enzymatic reaction in 1-D confined nanochannel devices. The effects of surface binding reaction on nanochannel conductance have been reported by Karnik et al. in 2005. Although nanochannels show high sensitivity for such reaction, detection of ultra-low concentration analyte based on this concept is still not practical since analytes are continuously consumed on the surface, leading to an ultra long diffusion-limited reaction time. Detection of surface enzymatic reactions could overcome this limit since enzymes are not consumed during the reaction. Moreover, nanochannel can further accelerate enzymatic reactions as enzymes are confined closely to their surface substrates. In this work, trypsin proteolysis reaction was used to demonstrate this strategy. Trypsin (enzyme) cleaves Poly-L-Lysine (PLL) coated on the surface of silica nanochannels, resulting in a change of surface charge density. This change is detected by monitoring the electrical conductance along the nanochannels. We studied the sensitivity and selectivity of this nanofluidic enzyme sensor and also used it to explore enzyme kinetics in confined space. Trypsin concentration down to 5 ng/ml has been detected in 50-nm-deep nanochannels within one hour. The sensitivity of trypsin proteolysis suggests that it may be possible to study enzyme activity with single-molecule resolution. Optimization of this nanochannel sensor could lead to a quick-response, highly-sensitive and label-free enzyme assay.