Rong Fan Yale University
Jianping Fu University of Michigan
Jianhua Qin Chinese Academy of Sciences
Aleksandra Radenovic EPFL – STI/SV – IBI – LBEN
AA2: Sensing and Molecular Analysis I
Tuesday AM, April 26, 2011
Room 3008 (Moscone West)
2:30 PM - **AA8.1
Ultra-high-throughput Screening with Drop-based Microfluidics.
David Weitz 1 Show Abstract
1 School of Engineering and Applied Sciences, Department of Physics and Kavli Institute for Bionano Science and Technology, Harvard University, Cambridge, Massachusetts, United States
This talk will describe the use of drop-based microfluidics to perform very high throughput bioassays. The microfluidic system will be described and some applications will be discussed.
3:00 PM - AA8.2
Three Stage High Pressure and High Temperature Continuous Microflow Synthesis of InP Quantum Dots.
Jinyoung Baek 1 , Peter Allen 2 , Bawendi Moungi 2 , Klavs Jensen 1 Show Abstract
1 Chemical Engineering, MIT, Cambridge, Massachusetts, United States, 2 Chemistry, MIT, Cambridge, Massachusetts, United States
InP quantum dots (QDs) are of technological interest in optoelectronic devices as a replacement for CdSe QDs. However, current InP nanocrystal synthesis have not been reached the sophisticated level of the synthesis of CdSe or PbSe QDs with narrow size distributions.We have utilized a continuous high temperature and high pressure microfluidic system to enable the use of various solvents which have not been traditionally accessible in QD synthesis, such as supercritical octane. The use of supercritical phase with low molecular weight solvents provides excellent mixing which is essential to obtain high quality QDs. In addition, the microfluidic system provides precise control of synthetic conditions to enable fast screening of growth conditions. Truly continuous multi-stage microfluidic system allows to separate reaction conditions such as mixing and aging steps, and thus enables systematic investigation of the synthesis of InP QDs.This work investigates synthesis of InP QDs with a truly continuous 3-stage high temperature and pressure microreactor system without including any batch manipulation between synthesis steps. By separating the mixing process from the following aging process, we have found that InP QD synthesis are primarily dominated by non-molecular inter-particle processes such as ripening process or coalescence process. We also have probed the effects of concentrations, and indium to fatty acid ratio. We have found that adjusting the indium to fatty ratio has largest effect on particle size due to enhanced inter-particle process. Operating conditions of the microfluidic reactor system above 320 C and above 65 Bar with supercritical octane as a solvent has enabled to obtain high quality InP QDs in as little as 2 minutes. We also have synthesized larger InP QDs through 6 sequential monomer injections, similar to SILAR (Successive Ion Layer Adsorption and Reaction) method, in 1.5 to 4 minutes, and have obtained InP QDs with size distributions as narrow or narrower than the InP QDs synthesized via the ripening process.
3:15 PM - AA8.3
Microcapsules with Tunable Dimensions and Mechanical Properties Made Using Microfluidics.
Philipp Chen 1 , Randall Erb 1 , André Studart 1 Show Abstract
1 Complex Materials, Department of Materials, ETH Zürich, Zürich Switzerland
Filled capsules feature prominently in applications involving delivery and controlled release of materials in medicine, cosmetics and the food industry. They are routinely fabricated by emulsification of immiscible liquids and subsequent interfacial polymerization. Deliberately tuning the size, shell thickness and composition of capsules is crucial to control their strength and permeability, but often difficult to achieve using traditional emulsification techniques.We exploit a recently developed microfluidic technique to form microcapsules with tailored dimensions and properties using double emulsions as monodisperse templates. In this method, a fluid is dripped from an orifice into a second, immiscible fluid in a co-flow configuration within a glass microcapillary device. Both fluids are in turn engulfed by a third fluid, which flow-focuses all phases into a collecting orifice, forming double emulsions. To stabilize the emulsions, polymeric surfactants such as poly(vinyl alcohol) are added to the inner and outer fluids. The size of each double emulsion phase can be coarsely tuned through the orifice diameters and finely adjusted with the respective fluid flow rates. Using suitable acrylate monomers as the middle fluid and in situ photopolymerization, we can produce monodisperse capsules of diameters varying from 60–250 µm and shell thicknesses in the range of 7–50 µm. To predict our droplet and microcapsule sizes, we developed an analytical model that describes the flow-induced dripping based on a balance between shear forces and interfacial tension. We found that despite the complex flow behavior in a microfluidic device, droplet size can be predicted with established break-up dynamics of isolated droplets. The model accurately describes a wide range of experimental data from both our work and literature without relying on fitting parameters.Using different chemical compositions for the middle fluid, we can tune the permeability and mechanical properties of the capsule shell. Monomers whose respective homopolymers possess low glass transition temperatures (Tg), such as 2-phenoxyethyl acrylate, yield elastic and ductile capsules. In contrast, high Tg systems like isobornyl acrylate form stiff and brittle capsules. The spectrum in between these extreme cases can be covered using monomer blends. By adding difunctional monomers, a cross-linked shell network can be formed which decreases its permeability. The shell can be further modified by the addition of reinforcing or functional nanoparticles to the middle phase, thereby creating composite microcapsules.The proposed model to predict droplet sizes combined with the wide range of materials that can be used in this microfluidic approach allow for unprecedented control over both the dimensions and the properties of microcapsules, enabling the design and fabrication of capsules for specific mechanical conditions.
3:30 PM - AA8.4
Surface-tension-driven Synthesis of Uniform Complex Particles Using Confined Polymeric Fluids.
Chang-Hyung Choi 1 , Heon-Ho Jeong 1 , Jinkee Lee 2 , Anubhav Tripathi 2 , Howard Stone 3 , David Weitz 4 , Chang-Soo Lee 1 Show Abstract
1 Chemical Enginnering, Chungnam National University, Deajeon Korea (the Republic of), 2 , Brown University, Providence, Rhode Island, United States, 3 , Princeton University, Princeton, New Jersey, United States, 4 , Harvard University, Cambridge, Massachusetts, United States
This study demonstrates a novel method for synthesizing monodisperse complex particles through surface-tension-driven flow. We suggest two routes for the generation of uniformly sized polymeric particle with different morphologies such as convex and flat-top shapes. A photocurable solution (Polyethylene glycol diacrylate; PEG-DA) and a nonphotocurable wetting solution (n-hexadecane) are sequentially loaded into a micromold. The different processes in a loading sequence of the two solutions of PEG-DA and hexadecane resulted in formation of different contacting interfaces of n-hexadecane/PEG-DA and air/PEG-DA, respectively. Additionally, we provide examples of particles with diverse shapes such as bullets, cylinder, discs, hemispheres, heart, twin cylinder, twin donut, hexagon with open or closed ends. Furthermore, combinations of two different routes show the feasibility for the fabrication of Janus particles having compartments with different properties. The complex particles fabricated by our method can be exploited as anisotropic building blocks for fabrication of complex systems.
AA4: Materials Assembly and Function
Tuesday PM, April 26, 2011
Room 3008 (Moscone West)
4:15 PM - **AA9.1
Molecular Detection, Analysis and Sorting in Nanofluidic Systems.
Harold Craighead 1 Show Abstract
1 Applied Physics, Cornell University, Ithaca, New York, United States
We have used a variety of lithographic and non-lithographic methods to engineer structures of controlled geometry for integration of fluidics, optics and electronics in analytical systems. We have for some time been studying the biophysics of nucleic acids in confined geometries, targeting individual nucleic acid molecules for sequencing and analysis. We recently have begun activity on single-molecule epigenetic analysis by identification of labeled epigenetic marks on individual chromatin fragments. In addition to identification and quantification of the presence of labeled marks we are automatically sorting and recovering individually selected fragments for subsequent sequencing. Similar fluidic devices have been applied to synthesis of nucleic acid aptamers using miniaturized systems for rapid identification of molecular configurations that bind selectively to protein or other target molecules. The talk will address the technologies and approaches we are exploring for this and related studies using micro and nanofluidic systems for nucleic acid analysis, selection and synthesis.
4:45 PM - **AA9.2
Teaching Old Polymers New Microfluidic Tricks: Aqueous Two Phase Systems for Cell and Reagent Micropatterning.
Shuichi Takayama 1 2 Show Abstract
1 Biomedical Engineering, University of Michigan, Ann ARbor, Michigan, United States, 2 WCU Program, UNIST, Ulsan Korea (the Republic of)
Aqueous two phase systems (ATPS) comprised of polymers such as polyethylene glycol (PEG) and dextran (DEX) have been used for decades to perform cell, protein, and other biomolecular separations. Here, we describe the use of such polymer-derived aqueous two phase systems to perform microfluidic cell and reagent patterning. Adherent cells are cultured in PEG containing media and micropatterns of reagent containing DEX phase is printed over the cells. For example, solution-based addressable cell transfection microarrays can be created that localize reagents to only the DEX phase. Formation of small droplets of the reagent-confining phase on cells cultured in the reagent-excluding phase enables selective delivery of genetic material and other reagents to discrete groups of cells grown on any substrate and allows flexible timing of reagent delivery. We show effectiveness of this method with readily analyzed gene expression demonstrations and localized gene knockdown. Another example is microprinting of embryonic stem cells over feeders cells to engineer stem cell niches that guide differentiation. Yet other applications include patterning bacterial microcolonies and high throughput screens for cell migration. The method provides capabilities similar to patterning of cells and reagents using multiple laminar streams but without need for channels, or flow, or worry of diffusion of reagents. The phases stay separated stably and reagents also partition stably in just the one phase with appropriate formulation. ATPS-based micropatterning is highly flexible in its cell and reagent delivery capabilities. It is also a low-cost microfluidic technology that can be utilized in any laboratory with minimal equipments.
5:15 PM - AA9.3
Droplet Microfluidics for Material Synthesis and Bioanalysis.
Nick Carroll 1 , Peter Crowder 1 , Robert Applegate 1 , Steven Graves 1 , Jeremy Edwards 2 , Plamen Atanassov 1 , David Weitz 3 , Dimiter Petsev 1 Show Abstract
1 Chemical and Nuclear Engineering, University fo New Mexico, Albuquerque, New Mexico, United States, 2 Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, New Mexico, United States, 3 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Microfluidics allows for the generation of small micrometer sized monodisperse droplets. Such droplets can be used as tiny reactors for material synthesis or analytical applications on a large scale. In this study we report data on using droplet microfluidics for fabrication of mesoporous silica particles with porosity that varies between a few nanometers and a few tens of nanometers. Aqueous droplets containing silica precursor and surfactants are formed in an oil continuous phase. The water is then expelled out of the droplets leading to solidification of the silica. The concentrated surfactants form structures, which are templated by the oxide forming a network of pores. Varying the conditions like surfactants type and concentrations and/or presence of electrolytes allows obtaining a wide range of pore morphologies. Certain surfactant compositions lead to sharp drop of the interfacial tension, which leads to formation of microemulsions. Tuning of the microemulsion phase that is used for templating, may result in biporous particles. The monodisperse mesoporous spheres can be ordered in 2D and 3D structures that may have an additional level of porosity corresponding to the void spaces between the particles. Another application of microfluidic generated droplets is large scale analysis of nucleic acid samples. Each droplet represents a tiny test-tube with a sample that is analyzed. The analysis can be performed on chip or off chip and is very convenient to identify the presence of specific exons in a RNA fragment. The proposed method has a great potential for early diagnostics of diseases that are characterized by expressing particular proteins.
5:30 PM - AA9.4
Design and Fabrication of Functionalized Microspheres from Microfluidic Approaches.
Weijia Wen 1 Show Abstract
1 Physics, HKUST, Kowloon Hong Kong
We report the successful fabrication of different types of functionalized microspheres by using the microfluidic flow-focusing (MFF) approach. The magnetic nanoparticle-based core-shell/solid microspheres can be realized by embedding the magnetic nanoparticles within the shell or core, thereby enabling the microspheres to deform under an applied magnetic field. As an example for core-shell microsphere, by encapsulating drug inside the microspheres, we demonstrate drug release under the compression-extension oscillations of the microspheres induced by an AC magnetic field. This active pumping mode of drug release can be controlled by varying the frequency and magnitude of applied magnetic field, as well as magnetic field’s time profile. Other kind of microspheres, like monodispersed hollow titania microspheres can be also achieved from microfluidic droplet-templated approach.
Rong Fan Yale University
Jianping Fu University of Michigan
Jianhua Qin Chinese Academy of Sciences
Aleksandra Radenovic EPFL – STI/SV – IBI – LBEN
AA8: Materials Synthesis and Characterization
Wednesday PM, April 27, 2011
Room 3008 (Moscone West)
9:45 AM - **AA11.1
Federal Funding Opportunities to Support the Development of your Innovative Biomedical Technology.
Mark Lim 1 Show Abstract
1 , Strategic Analysis Inc., West Lawn, Pennsylvania, United States
There are many funding opportunities available for physical scientistsinterested in developing novel technology platforms that can be used to research, prevent, diagnose, and/or treat disease. Discussed is a brief overview of several funding sources from the NIH and other federal agencies that aim to support the pipeline of biomedical technology innovation - from proof-of-concept approaches to the development of an applied prototype - that aim to accelerate clinical care and/or research. The session will conclude with perspectives and tips on how to find such publicly available information.
10:15 AM - AA11.2
Smart Microspheres for Detection and Delivery.
Wynter Duncanson 1 , David Weitz 1 Show Abstract
1 , Harvard University, Cambridge, Massachusetts, United States
In the medical field and the oil industry, there is a growing interest in smarter microspheres designed to seek out specific sites and to be triggered to deliver payloads or enhance contrast. Microspheres with high porosity or core-shell structures are typically formed by blending liquids containing surface active compounds with gases, cosmetics, foods, or drugs under high-shear. The chaotic production conditions produce microspheres with broad distributions in sizes, porosities, and shell thicknesses which require further processing or filtering to obtain the desired structures. This highlights the need for greater control during microsphere fabrication. Microfluidics offers greater control over the size distribution of these emulsions relative to conventional preparation methods. The flexibility of microfluidics allows us to use a variety of materials including self-assembling materials and unique surfactants to make smarter materials for contrast enhancement as well as payload delivery. We fabricate monodisperse porous and core-shell microspheres using sophisticated microfluidic technologies, self-assembling, and triggerable materials. These advanced techniques coupled with the benefits of the smart materials create new opportunities to not only produce simple microspheres, but also highly complex functional microspheres.
10:30 AM - AA11.3
Optofluidic Fabrication of Charge Selective Hydrogel for PCR-free Signal Amplification of DNA in Microfluidic Environment.
Hyoki Kim 1 , Junhoi Kim 1 , Eun-Geun Kim 1 , Austen Heinz 1 , Sunghoon Kwon 1 , Honggu Chun 2 Show Abstract
1 Electrical Engineering and Computer Science, Seoul National University, Seoul Korea (the Republic of), 2 Advanced Institutes of Convergence Technology, Seoul National University, Suwon Korea (the Republic of)
Miniaturized system for the detection of DNA has attracted much attention because of a substantial importance to forensic science, clinical diagnostics. In usual case in microfluidic detection, however, a small amount of DNA and the short path length make sensitive detection challenging. To overcome the limitations, the conventional way for enhancing the sensitivity of DNA detection is to amplify the copy number of DNA by chemical amplification through polymerase chain reaction (PCR). However, enzymes, primers, and precise temperature control unit should be incorporated in PCR. Also, subsequent purification process should be necessary before analysis, which makes difficulties to miniaturization. For the alternative over chemical amplification, it would be a promising candidate if one can increase amount of DNA in the local area of interest by concentrating the DNA without any chemical treatment. Since DNA has a uniform charge to size ratio, it is efficient to make preconcentration by charge selective way.Here, we present that DNA can be effectively concentrated in a small spot in a glass microfluidic environment using a negatively charged nanoporous hydrogel as a charge selective molecular filter. Material that we used for nanoporous hydrogel is poly-AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid), and an optofluidic maskless photochemical curing device was developed for producing a charge selective molecular filter. In optofluidic maskless lithography system, a high power UV source, digital mirror device (DMD) and digital control unit enables fast polymerization process, and any desired shapes of hydrogels can be produced in a microfluidic channel. The key advantage of this optofluidic fabrication over conventional contact mask lithography is to prevent diffraction induce pattern blurring, thus it enables to produce very high resolution features in a typical microfluidic environment. Also, three dimensional analysis of preconcentration dynamics was monitored for the first time (to our knowledge) through a confocal microscope. Finally, ssDNA preconcentration was demonstrated for PCR-free signal enhancement. Cy-3 labeled 15 base pair single stranded oligonucleotide of was used for amplification monitoring. The presenting technique shown here does not suffer from the device size, because the local spot where the DNA are accumulated is located in very near the ion depletion boundary. Also, the technique shown here is easy to be incorporated with larger system for the subsequent analysis such as a DNA length selection.
10:45 AM - AA11.4
Electrical Detection of Biological Molecules Using Streaming Current Measurements in Micro and Nanofluidics.
Diogo Martins 1 2 , Virginia Chu 1 , Duarte Prazeres 2 3 , João Conde 1 3 Show Abstract
1 , INESC Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Lisboa Portugal, 2 IBB-Institute for Biotechnology and Bioengineering, Instituto Superior Técnico, Lisbon Portugal, 3 Department of Chemical and Biological Engineering, Instituto Superior Técnico, Lisbon Portugal
In an aqueous solution the inner walls of a hydrophilic microchannel are usually charged and, consequently, the solution adjacent to those surfaces will form an electrical double layer with an excess of counter-ions and a deficit of co-ions. Under flow conditions, the bulk solution in the microchannel drags the mobile portion of the electrical double layer, thus creating an ionic current. This current can be measured via electrodes positioned at both ends of the microchannel. This current depends only on the charge density and zeta-potential of the surface and is a powerful method to probe the immobilized surface charge in a micro or nanochannel. Functionalization of the surface with reactive molecules and the immobilization of both DNA and proteins were successfully detected in a microfluidic device by streaming current measurements.Hybrid PDMS/glass microchannels with Au/Cr electrodes were fabricated. PDMS microchannels were fabricated using soft-lithography with typical dimensions of 20 micron height and 200 micron width. Solutions were pumped into the microchannels with a syringe pump. The streaming currents in a very low ionic force solution (DI water, pH~5.5) are measured using a picoammeter. DI water was used to increase the sensitivity of the measurement by increasing the thickness of the charged electrical double layer. The streaming current is measured for flow rates of 5 microliters per minute. The streaming current was measured in PDMS-glass microchannels confirming the expected negative surface charge due to the presence of ionized hydroxyl groups on the surface. Next, the channels were functionalized with APTES (covalently) or polylysine (adsorption). In both cases the streaming current showed a change in direction relative to the PDMS-glass measurement indicating a surface charge inversion, as expected, since at pH 5.5 both APTES and polylysine are positively charged. The surface functionalization was confirmed by fluorescence measurements. Next, DNA and immunoglobulin G (IgG) were immobilized on the functionalized surfaces and successfully detected with the streaming current method and confirmed by fluorescence microscopy. Ongoing work is focused on applying this method to the detection of the presence of specific target biomolecules (DNA and antigens) in solution by molecular recognition with probes immobilized on the microchannel surface. The main challenge is the control of non-specific binding. A second goal is to reduce the height of the channels to the nanoscale (50 nm) to increase the sensitivity of the measurement by reducing the contribution of the neutral bulk flow. As shown above, the measurement of streaming currents has the potential to allow label-free electrical detection of biomolecules in microfluidic devices.
AA9: Novel Functional Fluidics I
Wednesday PM, April 27, 2011
Room 3008 (Moscone West)
2:30 PM - **AA13.1
Real Time DNA Sequencing From Single Polymerase Molecules.
Stephen Turner 1 , Jonas Korlach 1 Show Abstract
1 , Pacific Biosciences, Inc., Menlo Park, California, United States
SMRT (single molecule real time) DNA sequencing is a high-throughput method for eavesdropping on template-directed synthesis by DNA polymerase in real time. Pacific Biosciences has developed two important technology components which enable the commercial application of this process: The first is the zero-mode waveguide (ZMW) confinement technology, a nanophotonic device for the confinement of optical observation that allows single-molecule detection at concentrations of labeled nucleotides relevant to the enzyme. The second important component is phospholinked nucleotides where, in contrast to other sequencing approaches, the fluorescent label is attached to the terminal phosphate rather than the base. The enzyme cleaves away the fluorophore as part of the incorporation process, leaving behind completely natural double-stranded DNA Through the combination of these innovations, our technology allows the speed, processivity, efficiency and fidelity of the enzyme to be exploited. We apply this technology to shotgun sequencing using a fast and simple sample preparation concept that facilitates whole-genome sequencing directly from genomic DNA.
3:00 PM - AA13.2
Slaven Garaj 1 , Daniel Branton 2 , Jene Golovchenko 1 3 Show Abstract
1 Department of Physics, Harvard University, Cambridge, Massachusetts, United States, 2 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States, 3 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Nanopore-bases devices are used to detect and analyze individual biomolecules, and they offer prospects for inexpensive and rapid DNA sequencing. In such device, a biopolymer is electrophoretically threaded through a nanoscale pore, transiently modulating the ionic current thought the nanopore. To spatially resolve geometrical or chemical features along the translocating molecule, the active part of the membrane has to be sufficiently thin. We use graphene as atomically thin membrane that insulates two ionic solutions, and we demonstrate that it can sustain significant perpendicular electric fields without degradation. After fabricating a single nanometer-scale pore through graphene, we use it to detect and characterize many individual DNA molecules. By measuring ionic flow through the graphene nanopore, we demonstrate that the effective thickness of graphene in water is less then one nanometer. This indicates, in collusion with theoretical modeling, that the graphene nanopore is potentially capable of discerning between sequential nucleotides along the DNA molecule, making it a candidate for next-generation sequencing platform. Excellent electrical conductivity of the graphene opens up a possibility to use the graphene nanopore concomitantly as an electrical sensor and as an electrode for controlling the DNA translocation dynamics.
3:15 PM - AA13.3
The Mechanism of Nanopore Drilling by Focused Electron Beam for DNA Translocation Control.
Hyun-Mi Kim 1 , Min-Hyun Lee 2 , Ki-Bum Kim 2 3 Show Abstract
1 Research Institute of Advanced Materials, Seoul National Univ., Seoul Korea (the Republic of), 2 Department of Materials Science and Engineering, Seoul National Univ., Seoul Korea (the Republic of), 3 WCU Hybrid Materials Program, Seoul National Univ., Seoul Korea (the Republic of)
The fabrication of sub-10 nm solid state nanopore to detect and control the transport of DNA by a focused electron beam in a transmission electron microscope has now become a common practice adopted by many research groups. However, it is still not yet clear how the membrane structure is perforated by the focused electron beam. We believe that understanding this mechanism is quite important in order to make a drastic progress in this area. It is more critical since the nanopore structure will be more complicated in a future with the incorporation of few electrodes in the membrane.In this presentation, we will undertake a theoretical consideration of the energy transfer from the fast electrons to the solid through such mechanisms as elastic and inelastic scattering just in order to explain the sculpturing of nanopore by electrons. From the theoretical consideration, we can calculate the cross section of elastic scattering resulting the direct atomic displacement and that of inelastic scattering which results in the ionization, excitation and the temperature increment. Based on the calculation, we can extract that the incident electron energy is the critical parameter in order to explain the nanopore drilling phenomenon. Then, we preformed nanopore drilling in a Si3N4 membrane using two different electron energies, 200kV and 300kV, to identify the drilling mechanism since the calculation of the scattering cross section clearly reveals that the cross section of direct atomic displacement increases with increasing incident electron energy, while the ionization cross section and temperature increment decrease. The experimental results of the nanopore drilling on the incident electron energy strongly support that the nanopore is perforated by the direct atom displacement. We can also calculate the direct atomic displacement energy of various materials from the movement of contrast curve which is introduced as characteristic plot of normalized drilling volume as a function of electron dose.
3:30 PM - AA13.4
Single Molecule Study of Transcription Using Nanopore Based Force Spectroscopy.
Camille Raillon 1 , Nouria Hernandez 2 , Aleksandra Radenovic 1 Show Abstract
1 LBEN, EPFL, Switzerland, Lausanne Switzerland, 2 CIG, UNIL, Lausanne Switzerland
Transcription is a crucial step in gene expression and regulation, which is defined at the molecular level as the transformation of genes (or operons) into functional proteins and enzymes (1). We use nanopore based force spectroscopy (2) (3) to understand dynamics of transcription: how RNAP translocation is applied in molecular and mechanistic terms and how DNA responds to local application of the force. To do so we combined a microfluidics system, a force measuring optical trap, a low-noise current-voltage amplifier and a Si3N4 membrane containing a nanopore. To investigate RNAP interaction with DNA we varied the nanopore diameter from 4 to 30 nm, nanopore material and modulated the interaction strength by designing appropriate promoters.
3:45 PM - AA13.5
Microfluidics for Permeability Monitoring of Single Aquaporin Proteoliposomes.
Gabriel Ohlsson 1 , Seyed Ruhollah Tabaei Aghda 1 , Magnus Branden 1 , Jonas Tegenfeldt 2 , Fredrik Hook 1 Show Abstract
1 Department of Applied Physics, Chalmers University of Technology, Göteborg Sweden, 2 Department of Physics, University of Gothenburg, Göteborg Sweden
A microfluidic setup has been designed for observing membrane protein mediated transport on the level of single proteoliposomes. Observing transport events at the single liposome level removes the ensemble average and allows for examination of hidden heterogeneities in the system. The device is capable of rapid (sub 10 ms) switching of the solution over surface-attached liposomes. A switch to a hyper-osmotic solution containing a permeable solute leads first to liposome shrinkage due to water efflux and subsequently to liposome swelling due to solute permeation into the liposomes. Calcein-loaded proteoliposomes (POPC and 1% biotin-modified PE, made by extrusion through a 200 nm filter) were immobilized to a NeutrAvidin modified surface of the microfluidic device. Time resolved changes in fluorescence-emission intensity upon volume-change induced self quenching of calcein were measured in an inverted microscope operated in either EPI or TIRF mode using a camera running at 125 frames/sec. The microfluidic channels were made by soft lithography, with a PDMS structure bonded on top of a thin microscope glass slide. A membrane vacuum pump and a fast electronic valve (sub 10 ms) were used to accomplish rapid switching of the solution over the measurement area.The change in fluorescence intensity, monitored using surface-sensitive TIRF mode, upon a switch above the detected area from a solution without to a solution with carboxyfluorescein showed was completed within the time resolution of the camera (8 ms), indicating sub 10 ms microfluidic switching. The applicability of the device to monitor the rapid transport events mediated by certain membrane channels was evaluated by studying the water transfer mediated by the human aquaporin 5 (AQP5). The AQP5-mediated transfer in single surface-attached liposomes was recorded by simultaneously monitoring the changes in the self quenching of dyes encapsulated in multiple proteoliposomes. Single channel conductance of aquaporins were calculated and shown to be in very good agreement with literature data based on ensemble measurements. This offers the potential to probe dynamics and heterogeneities within the liposome population.
Rong Fan Yale University
Jianping Fu University of Michigan
Jianhua Qin Chinese Academy of Sciences
Aleksandra Radenovic EPFL – STI/SV – IBI – LBEN
AA13: Nanopores and Nanoholes
Thursday PM, April 28, 2011
Room 3008 (Moscone West)
1:00 AM -
AA10.3 Transferred to AA5.11
AA15: Poster Session III
Thursday PM, April 28, 2011
Salons 7-9 (Marriott)
1:00 AM -
AA15.5 Transferred to AA17.2
AA11: Sensing and Molecular Analysis II
Thursday AM, April 28, 2011
Room 3008 (Moscone West)
11:30 AM - AA17.1
Study of an In Situ Protein Membrane in a Microfluidics System.
Hong Chang 1 , Zimei Rong 1 , Steve Dunn 1 , Pankaj Vadgama 1 Show Abstract
1 School of Engineering and Materials Science, Queen Mary university of london, London United Kingdom
Microfluidic membranes are used in myriad applications, including use in microbioreactor. They serve as bio-catalyst surfaces or allow cell adhesion. However, creating such membranes requires complex manufacturing processes including multi- step self assembles. Recently, a nylon membrane was produced in situ in a flow channel . This process is completed rapidly (within a few minutes), but such membranes are essentially only gas permeable. Control of the thickness and inclusion of porosity is important for effective membrane permeability for general solute transfer and could be sensitive for a given size range of molecules. In the present work, a simplified in situ fabrication technique using two liquid phases has been used to produce a robust and novel protein micro-membrane . The membrane was produced at the liquid interface using cross linking agents and protein solution via fast interfacial polymerization. The thickness of the protein membrane was dependent on the reaction time. The proteins studied were albumin and fibrinogen with an acyl chloride to achieve protein crosslinking. Three acyl chloride crosslinkers were tested in these experiments: terephthaloyl chloride, sebacoyl chloride and isophthaloyl dichloride. It was shown by SEM that surface facing the protein solution has a rough structure whereas the crosslinker (acyl chloride) side had a smooth surface. Each crosslinker also generated unique surface morphologies and cross section morphological structures. Permeability of these membranes was tested by diffusion studies using dye molecules as well as the membrance electrochemical activities. A link between the diffusion rate and the structure of the protein membrane was established. A simplified approach of using ethanol to further modify the porosity of the membrane was established. This indicated that the diffusivity of the membrane increases with ethanol due to swelling leading to increased porosity. Tensile tests on the membranes showed that there was variation in membrane strength that was related to the crosslink of molecule, porosity and was also related to permeability. Hisamoto H, Shimizu Y, Uchiyama K, Tokeshi M, Kikutani Y, Hibara A and Kitamori T 2003 Chemicofunctional membrane for integrated chemical processes on a microchip Anal. Chem. 75 350-4 Hong Chang, Rachel Khan, Zimei Rong, Andrei Sapelkin and Pankaj Vadgama 2010 Study of albumin and fibrinogen membranes formed by interfacial crosslinking using microfluidic flow Biofabrication 2 035002
11:45 AM - AA17.2
Artificial Cells for Mass Production of Metal Nanoparticles Using the Combination of Cell Extracts and Droplets in Microfluidic Device.
Kyoung G. Lee 1 4 , Jong In Hong 2 , Kyeon Won Wang 1 , Tae Jung Park 3 , Do Hyun Kim 1 , Sang Yup Lee 1 3 5 , Seok Jae Lee 4 Show Abstract
1 Department of Chemical & Biomolecular Engineering, KAIST, Daejeon Korea (the Republic of), 4 NTD&I Team, National Nanofab Center, Daejeon Korea (the Republic of), 2 Department of Materials Science & Engineering, KAIST, Daejeon Korea (the Republic of), 3 BioProcess Engineering Research Center, KAIST, Daejeon Korea (the Republic of), 5 Department of Bio & Brain Engineering, Department of Biological Sciences, and Bioinformatics Research Center, KAIST, Daejeon Korea (the Republic of)
The major advantages of using poly(N-isopropylacrylamide) as an artificial cell membrane would be relieved the potential contamination of media, which is one of major component for the growing of cells or microorganism. In this reason, it is much convenient to use as an artificial cell membrane to fabricate polymer beads for the synthesis of nanoparticles. The mimic of cellular structure is difficult to fabricate due to the complexity of biostructures and mechanisms of cells. Several important factors should be considered to fabricate virtual cells inducing mass transfer between membrane and surroundings. In order to control the mass transfer of chemical components, the artificial membrane should have been small pores to transfer chemicals. In this study, we developed a new technique for continuous mass production of monodisperse artificial cells using the combination of a hydrogel polymer and microfluidic device. The uniform size polymer particles are produced by sequentially encapsulating cellular extracts, hydrogel, and oil into the device. We demonstrated an efficient way to encapsulate cellular extracts, which produce metal nanoparticles.
12:00 PM - AA17.3
Optimized Multiplexed Cell Capture using Parallel Bioactivated Microfluidic Channels
Mehdi Javanmard 1 , Farbod Babrzadeh 1 , Ronald Davis 1 Show Abstract
1 Stanford Genome Technology Center, Stanford University, Stanford, California, United States
Optimization of targeted cell capture with microfluidic devices continues to be a challenge. On the one hand, microfluidics allow working with microliter volumes of liquids, whereas various applications in the real world require detection of target analyte in large volumes, such as capture of rare cell types in several ml of blood. This contrast of volumes (microliter vs. ml) has prevented the emergence of microfluidic cell capture sensors in the clinical setting. Here we present a generalized methodology for maximizing test solution volumes while taking advantage of the benefits microfluidics has to offer, using parallel bioactivated microfluidic channels. The device consists of channels in parallel with each other tied to a single channel. Each channel is functionalized with receptor proteins. The parallel architecture allows for high capture rates while using high flow rates. Parallel channels allow low Reynolds number flow and minimize formation of bubbles, something that’s difficult to achieve with several millimeter wide devices. In this presentation, we discuss a generalized method for optimizing cell capture in microfluidic devices. Afterwards, we present the model we developed based on monte carlo simulations. Afterwards we discuss fabrication, and testing of our devices, and show the ability for multiplexed detection of target cells.
12:15 PM - AA17.4
Continuous Droplet Motion via Constant Voltage Electrowetting.
C. Lynch 1 , C. Nelson 1 , M. Khodayari 1 , A. Volinsky 1 , Nathan Crane 1 Show Abstract
1 Mechanical Engineering, University of South Florida, Tampa, Florida, United States
Droplet-based microfluidics devices have typically required an array of electrodes to actuate the droplets. This paper will present a method for continuous actuation of a droplet through application of a constant DC voltage. This novel characteristic is achieved through implementation of on-chip diode elements to create an electrowetting response that depends on voltage polarity. Electrochemical- diodes are implemented for easy fabrication with a single-mask photolithograph process. Droplet velocity in excess of 10 mm/s has been measured using this actuation method.
12:30 PM - AA17.5
Fabrication of a Novel Hollow Micro Needles for Biological Applications.
Zeinab Sanaee 1 , Sahar Mehrvarz 1 , Mahdieh Mehran 1 , Shams Mohajerzadeh 1 Show Abstract
1 Electrical engineering, University of Tehran, Tehran Iran (the Islamic Republic of)
Microneedles have applications in drug delivery and biotechnology. In addition, needle-like structures on Si substrates were used to realize miniaturized ionizers on silicon substrates which could be incorporated in portable mass spectrometers. We report a novel needle-like hollow cylindrical structure as a base for the growth of carbon nanotubes (CNT) to form a cage-like structure. By placing a cover on the CNTs, one can arrive at structures with great applications in biological experiments. Such features can be used to entrap biological species. The formation of hollow structures is feasible on Si-membranes. After proper patterning of the masking layer (Ni), the hollow-cylindrical structures are formed on the front-side of Si using DRIE, leading to well-defined vertical features with desired shapes and depths. By a small angle deposition method for the masking layer and proper treatments, the cylindrical structures can convert into hollow needles with ultra small features. Using this technique we have arrived at features of the order of 3um with wall thicknesses less than 70nm. The aspect ratio of the vertical structures has been more than 100 which is a record value. By continuing the etching process, we made fully hollow structures on Si. The needle-like structures can be used to grow CNTs on their rim top-surfaces. The growth of CNTs is possible using DC-PECVD method with H2 and C2H2 gases at 650oC. For this experiment, we have adjusted the growth conditions to achieve CNTs with 50-70 nm width and height of 3-4µm. Using this approach, we have realized a cage-like structure on top of hollow-cylindrical features. The very top surface of the whole structure can be blocked using a thick nickel which is deposited on the CNT-holding needles. To be able to do this, we have applied photoresist coating followed by proper spinning and treatments. Through depositing a nickel layer (300-400 nm thick), and followed by removing the underlying resist, we have achieved cage-like structures where the top side is covered by Ni. The walls of such a structure has opening of the order of 100-200 nm, controlled by the spacing of the original CNTs grown on the rim-side. The hollow structures were used to study the vapor transport through their tiny holes where a considerable differentiation between different vapors was observed. Further elaboration on this phenomenon is being pursued. In addition, we have used the hollow structures to realize low frequency capacitive sensors where a liquid has been incorporated inside the tiny tubes. The deleterious effect of capillary force has been investigated. Since the proposed structure contains a porous wall while the top side is fully clogged, it can be used as a filter for fluids containing small particles. The size of the holes can be tailored to entrap desired biological species. The study on entrapping cancerous cells on the microstructures is underway. Preliminary results on this study will be presented.
AA14: Sensing and Molecular Analysis III
Thursday PM, April 28, 2011
Room 3008 (Moscone West)
4:30 PM - AA4.1
Plasmonic Photothermal Heating on Microfluidic Chips.
Caihong Fang 1 , Lei Shao 1 , Jianfang Wang 1 Show Abstract
1 , The Chinese University of Hong Kong, Hong Kong China
Microfluidic chips, in which fluids can be controlled and analyzed within microchannels, have received intensive interests from researchers in both industrial and academic communities. The benefits of miniaturization, integration, and automation will facilitate their potential use in various areas, ranging from chemical synthesis, bioanalysis, and drug discovery to medical diagnostics. Thermal control plays a role of vital importance in most of these applications, for which fast, localized and precise temperature management is strongly required. A common method to realize fluid thermal treatment is resistive heating achieved by inserting patterned resistors in microfluidic chips. This simple approach is restricted by the complex fabrication techniques and the inherently limited density of integration. Alternatively, optical means can be applied for localized thermal control, but strongly light-absorbing materials are often needed, which in turn introduces additional fabrication steps and interfering factors to analytes. Therefore, a facile and economic heating method is highly desired to overcome these obstacles.Au nanocrystals (NCs) exhibit excellent photothermal conversion properties due to their plasmonic features. Here, we propose a plasmonic photothermal method for optical heating on microfluidic chips, using poly(dimethylsiloxane) (PDMS) embedded with Au NCs as the light-absorbing microfluidic material. We used thiol-terminated methoxypoly(ethylene glycol) to replace the capping surfactants to protect the Au NCs before dispersing them into the PDMS prepolymers and incorporating them into microfluidic chips. The Au NCs were found to be distributed uniformly in the PDMS matrix. The modified PDMS chips remain transparent and can effectively convert the light into heat under laser illumination. In addition, the spectral window of light absorption can be tailored easily by varying the size of the embedded Au NCs. To examine the optical heating effects of different Au NC-incorporated PDMS chips, rhodamine B solutions were injected into microchannels and their temperature-dependent luminescence spectra were collected. We found that when the plasmon wavelength of the Au NCs is close to the laser wavelength, the temperature can rise up significantly in a short time from room temperature to ~65 degree. Our results clearly indicate that Au NCs can be incorporated in microfluidic chips to realize effective thermal control through the plasmonic photothermal conversion. Since the laser beam can be focused to a very small region, we can envisage that the Au NC-incorporated PDMS will be successfully used to achieve the fast zonal heating on microfluidic chips. We believe that our method will promote the development of the microfluidic techniques involving thermal management.
4:45 PM - AA4.2
SiCHAS: A Platform for Analyzing Multiplexed Single Cell Secretants.
David Martin 1 , Andrew Kummel 2 , Davorka Messmer 3 Show Abstract
1 Electrical & Computer Engineering, University of California San Diego, San Diego, California, United States, 2 Chemistry & Biochemistry, University of California San Diego, San Diego, California, United States, 3 Moores Cancer Center, University of California San Diego, San Diego, California, United States
Investigating cancer from a systems biology perspective has been hindered by the lack of tools for a dynamic multiplexed analysis of surface and secreted proteins from small numbers of tumor cells. While flow cytometry is most often used to measure surface molecules and to detect intracellular levels of secretants, intracellular levels often don’t correlate with amounts secreted from cells and the multiplex capability is limited. Furthermore, the cells need to be fixed and are lost for future studies needing live cells; no time-course measurements can be performed on the same cells. Other flow based methods involve some form of capture of secretants on the cell surface, with subsequent secondary staining. These approaches are limited by the constraints of flow cytometry and are difficult to generalize or multiplex because of either highly specific modalities, or the need for specialized reagents. “Single Cell Hyper Analyzer for Secretants” (SiCHAS) has been designed to enable parallel and time dependent analysis of cell-cell communication. Such a platform could be used to query the cross-talk between cancer cells and cells of their microenvironment and provide measurements of several surface molecules and secretants all at the single cell level. Microfabrication techniques have been used to create planar arrays of “microwells” consisting of 10x10mm patterns in diced Si wafers with well openings of 50x50um, 85um depth, and separated by 10um wide walls. Smooth sidewalls have been reliably produced with greater than 95% well yield. To enable fluorescence microscopy detection of secretants, microwells with transparent bottoms are being fabricated from Si/Pyrex bonded wafers. 200mm thick Si wafers are bonded to a 500mm thick Pyrex wafer, with target microwell patterns being subsequently etched completely through the Si layer to the Pyrex layer with deep silicon cryo-etching. Cell-cell communication can be studied by introducing passages or openings in the sidewalls between wells, allowing secretants to diffuse between microwells. After microwell fabrication, antibody capture arrays will be coated on the transparent Pyrex bottoms of the microwells to enable fluorescence based quantification of secretant levels. Capture antibody surface coatings are being optimized independently on glass slides for IFN-g cytokine detection. Heterogeneous capture antibody arrays will then be investigated for multiplexed analysis of secretants wherein different secretants will be labeled with differently colored fluorescent dyes. The SiCHAS technology will be evaluated on cell lines by measuring cell surface expression and secretants from individual cells over time.
5:00 PM - AA4.3
The Invariance of Electrowetting Contact Angle Saturation To Polymer, Fluid, and Interfacial Materials Properties.
Stephanie Chevalliot 1 , Manjeet Dhindsa 1 , Stein Kuiper 2 , Jason Heikenfeld 1 Show Abstract
1 , Cincinnaty University, Cincinnati, Ohio, United States, 2 , Philips Research, Eindhoven Netherlands
Electrowetting consists of reducing the contact angle formed between a liquid and a hydrophobic dielectric/electrode substrate by electromechanical force through the application of voltage. It has recently received significant interest mainly due to its ability to manipulate small amount of fluids and it is especially promising for “lab on a chip” applications. Obtaining large contact angle modulation at low voltages is desirable for nearly all applications. Basic electrowetting theory predicts that continued increase in applied voltage will allow contact angle modulation to zero degrees. In practice, the effect of contact angle saturation has always been observed to limit the angle modulation, often only down to a contact angle of 60 to 70°. The physical origins of contact angle saturation have not yet been explained successfully and unequivocally. At best, scientists have produced multiple disconnected hypotheses (droplet ejection, charge injection, a thermodynamic limit, etc.) that do not satisfactorily hold for the large body of electrowetting experimental results.We present that with DC voltage, electrowetting contact angle saturation is invariant with electric field (up to 3x increase), contact line profile, interfacial tension (from 7 to 43 mN/m), choice of non-polar insulating fluid (silicone oils, tetradecane, decahydronaphtalene), and type of polar conductive fluid (deionised water, propylene carbonate) or ionic content (sodium chloride, hydrochloric acid, sodium hydroxide and tetrabutylammonium acetate at different concentrations). A contact angle of ~60° was systematically reached at saturation. Our experiments were performed and designed using accepted electrowetting materials, without bias toward supporting a particular theory for saturation. Because the saturation is so invariant to multiple parameters, our experimental results may suggest a new theory for electrowetting saturation: micro-droplet ejection through “Taylor saturation”. This new theory draws upon the physics and materials research performed for Taylor cones and electrospinning/electrospraying of materials. Although our work does not unequivocally prove what does cause contact angle saturation, it reveals what factors play a very limited or no role, and how dominant factors causing saturation may change with time of voltage application. This presentation will therefore provide additional direction to the continued pursuit of a universal theory for electrowetting saturation.
5:15 PM - AA4.4
Assembling of Nanowire Nano-motors Using Electric Tweezers for Manipulation of Flows in Micro/Nanofluidic Devices.
Donglei Fan 1 , Frank Zhu 2 , Soo Hyung Lee 3 , R. Cammarata 3 , C. Chien 3 Show Abstract
1 , University of Texas at Austin, Austin, Texas, United States, 2 , Hitachi GST, San Jose, California, United States, 3 , Johns Hopkins University, baltimore, Maryland, United States
We report a bottom-up approach to efficiently assemble nanowires into arrays of nano-motors on magnetic nano-bearings for manipulation of flows in micro/nanofluidic devices. We assembled nanowire nano-motors using “electric tweezers”, our own invention, which can transport and rotate nano-entities in liquids. The nano-motors consist of nanowires working as rotors, patterned nano-magnets working as bearings, and simple quadrupole micro-electrodes working as stators. By applying appropriate electric fields, arrays of such nano-motors have been assembled and rotated with precisely controlled angles, speed, and chirality. This work will find numerous applications in micro/nanofluidics such as mixing, pumping, and sensing of flows.
5:30 PM - AA4.5
Self-assembly of Uniform Polyhedral Silver Nanocrystals into Densely Packed Supercrystals Using Microfluidic Chambers.
Joel Henzie 1 , Peidong Yang 1 Show Abstract
1 Chemistry, University of California-Berkeley, Berkeley, California, United States
Understanding how polyhedra pack into extended arrangements is integral to the design and discovery of crystalline materials at all length scales. Much progress has recently been made in enumerating and characterizing the densest crystal packings of polyhedral shapes using theoretical methods. However, there are few experimental demonstrations of these ordered arrangements, especially at the nanoscale, where such order can generate novel electronic and optical properties. While molecules and nanoparticles can in principle be induced to organize themselves into densely packed structures, dictating their spatial relationships requires precise control of particle shape, polydispersity, interactions and driving forces. Here we show with experiment and computer simulation that a range of highly uniform, nanoscale Ag polyhedra can self-assemble into their densest known packings under simple gravitational driving forces. Microfluidic chambers enable precise control of the supercrystal dimensions and monitoring by microscopy.Adsorbing polymer prevents irreversible binding between polyhedra, and can also induce depletion attractions that stabilize less dense, ordered packings. In the case of octahedra, controlling polymer concentration allows us to tune between the well-known Minkowski lattice, and a novel packing with complex helical motifs. Because these supercrystals are composed of Ag building blocks, they could be immediately useful as plasmonic metamaterials, with a wide range of applications including label-free chemical and biological sensing, transformation optics, and plasmon-enhanced photocatalysis.
5:45 PM - AA4.6
Guided Assembly of Nanowires and Their Integration in Microfluidic Devices.
Josep Puigmarti-Luis 1 , Phillip Kuhn 1 , Petra Dittrich 1 Show Abstract
1 Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich Switzerland
Much effort focuses nowadays on the development of new methodologies for the formation and integration of functional nanostructures by bottom-up technologies. In our group the research focuses on synthesizing and integrating new nano- and micrometer-sized functional wires on a microfluidic platform. Recently, we have presented a new route for a straightforward production of hybrid and coordination polymer nanowires by using microfluidic technologies. We have proven that microfluidic devices operating under laminar flow conditions can guide and tune the formation pathway of the assembled structures just by varying the flow rates of the incoming streams. In this contribution, we will present recent developments to form and integrate hybrid nanowires based on tetrathiafulvalene (TTF) and metallic gold on a microfluidic chip. The formation can be achieved either at the interface of laminar streams of TTF and HAuCl4 precursor solutions or in a microchannel, where both reactants are supplied from either side to mix and react inside at a central location. In order to immobilize these nano- and microwires directly on the microchip at a predefined position, microclamps with pneumatic actuation have been designed and fabricated by means of multilayer soft lithography. Au-TTF-nano- and microwires can be conveniently trapped upon actuation of the clamps, and again released. Different clamp designs are discussed to separate the nanowires from surrounding solution, or to allow functionalization of the nanowires once they are trapped. Furthermore, integration of electrode patterns below the pneumatic clamps facilitates the electrical characterization of the nanowires. Ultimately, the work aims at the construction of nanowire-based sensors on microfluidic platforms with electrical or optical readout. With respect to the latter, we could recently detect a significantly enhanced Raman signal of TTF in Au-TTF nanowires, which promises the use of Au-TTF nanowires as SERS probes.References: J. Puigmartí-Luis, D. Schaffhauser, B. R. Burg, P. S. Dittrich, Adv. Mat. 2010, 22, 2255.  J. Puigmartí-Luis, M. Rubio-Martínez, U. Hartfelder, I. Imaz, D. Maspoch, P. S. Dittrich, submitted.  P. Kuhn, J. Puigmarti-Luis, I. Imaz, D. Maspoch, P. S. Dittrich, Lab Chip, accepted.  J. Puigmartí-Luis, J. Stadler, D. Schaffhauser, Á. Pérez del Pino, B. R. Burg, Petra S. Dittrich, submitted.
AA13: Nanopores and Nanoholes
Thursday PM, April 28, 2011
Room 3008 (Moscone West)
9:00 PM - AA10.1
Water Soluble, Biocompatible Metal Oxide Nanoparticles as Contrasting Agents in MRI.
Kerstin Koll 1 , Thomas Schladt 1 , Filipe Natalio 1 , Wolfgang Tremel 1 Show Abstract
1 Inorganic Chemistry, Johannes-Gutenberg-University Mainz, Mainz Germany
Nanotechnology is a concept that plays a significant role in modern medicine. Quite a numerous number of nanoparticula drugs are already in use, or have entered clinical trials[1,2]. This is due to their large surface to mass ratio and, depending on their chemical nature, their ability to adsorb or carry other compounds such as drugs, proteins or antibodies. We designed magnetic nanoparticles, based on MnO and Fe2O3, respectively. To allow for water solubility, they were functionalized with dopa-poly(ethylene glycol) (PEG)- amine. Dopamine thereby acts as an anchor to bind the nanoparticles to PEG, which allows for solubility in numerous solvents. The amino group further allows binding of a bioactive group such as CpG or Poly(I:C), or binding of a dye to allow visual tracking of the particles in vitro. Furthermore, binding of protoporphyrin IX allowed simultaneous tracking of these particles as well as their use in photodynamic therapy in vitro. The magnetic core however, enables this system to be used as contrasting agents in MRI, which has already been shown in the past[6,7,8].A new approach is to bind the antibody against the tumor suppressor protein 53 (p53), which is over expressed in many cancer cells . Using the DNA-sequence for the light chain of the antibody against p53, we altered this sequence to add additional properties that allow us to effectively separate unbound proteins from nanoparticles and that allows us to establish a method to quantify the amount of bound protein. : R. A. Petros, J.M. DeSimone, Nature Reviews, 2010, 9, 615-627: R. Duncan, Nature reviews, 2006, 6, 688-701: W. H. DeJong, P. JA. Borm, International Journal of Nanomedicine, 2008, 3, 133-149: M.I.Shukoor, et al. Angew. Chem. Int. Ed., 2008, 47, 4748-4752, M. I. Shukoor et al., small, 2007, 3, 1374-1378, J. Rother et al., in preparation: T.D. Schladt et al., Angew. Chem. Int. Ed., 2010, 49, 3976-3980: H. B. Na, T. Hyeon. J. Mater. Chem. 2009, 19, 6267–6273: H. B. Na, et al., Angew. Chem. Int. Ed. 2007, 46, 5397–5401: T.D. Schladt et al., J. Mater. Chem., 2010, 20, doi: 10.1039/c0jm01465f, : Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991), Science, 1991, 253, 49–53
9:00 PM - AA10.2
Engineering Micropatterned Surfaces for Cytokine Detection.
Jeong Hyun Seo 1 , Alexander Revzin 1 Show Abstract
1 Biomedical Engineering, UC Davis, Davis, California, United States
Cytokines are proteins secreted by leukocytes responding to pathogens or infections , therefore, cytokine production correlates with the ability of leukocytes to mount an effective immune response. Our laboratory is interested in developing micropatterned cytometry surfaces and has recently demonstrated a photolithography-like approach for fabricating hydrogel microwells on acrylated glass substrates.(Zhu Analc Chem 2009). Physical adsorption of anti-leukocyte (CD4) and anti-cytokine (IFN-gamma) Abs into the microwells allowed to capture single T-cells and detect cell-secreted cytokines. In the present study we sought to enhance sensitivity of cytokine detection by designing strategies for oriented immobilization of Ab molecules inside hydrogel microwells. To achieve this, glass substrates were functionalized by methoxysilanes carrying acrylate and thiol end groups. The presence of this mixed layer on glass was verified by time of flight (TOF)-SIMS and ellipsometry. This bi-functional silane layer allowed to anchor poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures during photocrosslinking. In addition, thiol groups presented inside the microwells could be activated with a hetero-bifunctional crosslinker to covalently immobilize avidin inside the microwells. Subsequently, biotinylated anti-cytokine antibody molecules were immobilized inside the microwells. Micropatterned surfaces prepared in this manner were sensitive down to 1 ng/ml (60 pM) IFN-gamma and could be used for simultaneous detection of two different cytokine molecules (IFN-gamma and TNF-alpha). In the future, these cytokine sensing micropatterned surfaces will be used for sensitive and multiplexed detection of cytokine release from single leukocytes.
9:00 PM - AA10.4
Transport Properties of Proteins and Quantum Dots in Nanochannels in Multi-gated Field Effect Transistor Configuration.
Louis Tribby 1 , Cornelius Ivory 2 , Sang Han 1 Show Abstract
1 Chemical & Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico, United States, 2 Chemical Engineering & Bioengineerig, Washington State University, Pullman, Washington, United States
The use of nanofluidic architectures as a means of concentrating and separating biomolecules, nanoparticles, and other small species of similar size scale may prove useful in developing new bioseparation and detection technologies. Recognizing this potential, a variety of nanofluidic devices have emerged that utilize enhanced electrokinetic control of fluid and molecular/particle motions at these scales. In our study, we have fabricated an array of slit-like nanochannels (100 nm w x 400 nm d x 15 mm l) in a multi-gated field effect transistor configuration, using interferometric lithography and conventional top-down fabrication techniques. Our main objective in developing such dynamically controllable separation platform is to further increase our ability to rapidly concentrate and separate proteins (or nanoparticles) that have low abundance or require long separation time by conventional methods. In order to produce effective separation strategies, we have first experimentally characterized electrokinetic transport properties of proteins and nanoparticles within our device. Based on this characterization and understanding, we will report a technique to form highly concentrated protein bands in our nanochannels. We will also report observable differences in electrokinetic mobility for semiconductor nanocrystals in aqueous solutions whose surface is functionalized with organic ligands to assume different charges. These results and their implications towards nanofluidic separation techniques will be further discussed.
9:00 PM - AA10.5
Design, Fabrication of High-throughput Microarray Microfluidic Device for Membrane Protein Polyhedral.
Hsin-Jui Wu 1 , Yiwei Yan 1 , Yung-Chen "YC" Lee 1 , Michael Stowell 1 2 Show Abstract
1 Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado, United States, 2 Molecular Cellular, and Development Biology, University of Colorado at Boulder, Boulder, Colorado, United States
We would like to design and fabricate the High-throughput Microfluidic device for membrane protein polyhedral. The 2D Membrane protein will be our approach target and it will be our better choice because there are several benefits of using 2D electron crystallography versus 3D crystallography for membrane proteins. First, membrane proteins require conditions that make their membrane spanning regions soluble. In 3D crystallography, the protein sample must be pure enough to form crystals of only protein. In 2D crystallography, the membrane protein is solublized by lipids and will form sheets of crystallized protein within lipid bilayers. Second, membrane proteins can form crystals fairly rapidly using 2D crystallography. In 3D crystallography, pure proteins are placed in specific crystallization conditions and left to sit until crystal formed, this could take weeks to months if crystals do form at all. In 2D crystallography, crystals are formed rapidly because the limiting step is detergent removal. Upon detergent removal, lipids can form bilayers where proteins can gain order and form crystals. Third, 2D crystallography followed by high-resolution electron microscopy can produce atomic level 3D images of membrane proteins. Comparison of the current method for 2D and our ideal approach, the amount of the conditions in the current method which can generate 96 conditions by hand pipette. Our approach is that we want to reach thousand different conditions without hand pipette. Secondly for the liquid volume, we would like to reduce from milliliter to micro/nano scale. The advantage is that we can reduce the time consumption because no hand pipette and quick diffusion for small liquid volume. Finally, the device size could be reduced to in one small chip which is around 8cm X 3cm X1cm. Thus, we can use soft-lithograph of the MEMS (Micro Electro Mechanical System) technique to achieve this final goal in membrane protein polyhedral research.
9:00 PM - AA10.6
Design, Fabrication and Characterization of PS-PMMA Nanoporous Membrane for Membrane Protein.
Yiwei Yan 1 , Hsin-Jui Wu 1 , Nathan Feaver 2 , Mark Stoykovich 2 , Yung-Cheng Lee 1 , Michael Stowell 3 1 Show Abstract
1 Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado, United States, 2 Chemical and Biological Engineering, University of Colorado at Boulder, Boulder, Colorado, United States, 3 Molecular,Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado, United States
In biology and pharmacology area, membrane proteins play an important role in representing the primary targets for prescribed and new drugs because of their unique role at the apex of cellular signaling processes. Advances in drug discovery and drug design would be greatly improved with structural information about membrane protein targets. In this project, we are developing a high-throughput microfludic devices and methods for the formation and analysis of novel membrane protein nanocrystals which will allow for rapid structural determination of membrane proteins. An important part of the work is to design and develop a functionalized biocompatible ultra-thin nanoporous membrane with uniform size pores, which can down to 2nm. The PS-PMMA block copolymer membrane, which integrates with PDMS microfluidic channel device, was developed for the membrane protein formation. Atomic layer deposition (ALD) was used to fine-tuning the pore size of the membrane, which can be down to magnitude of angstroms. The end goal of this project is to identify and develop a functionalized nanoporous membrane with uniform pore size down to 2nm for membrane protein nanocrystal formation.
AA15: Poster Session III
Thursday PM, April 28, 2011
Salons 7-9 (Marriott)
9:00 PM - AA15.1
Microfluidic Synthesis of Microfibers by Regeneration of Cellulose from Ionic Liquid.
Sung Tae Kim 1 , Sung-rheb Cho 1 , Suk Tai Chang 1 Show Abstract
1 Chemical Engineering and Materials Science, Chung-Ang University, Seoul Korea (the Republic of)
We have developed an approach for the fabrication of cellulose microfibers in a hydrodynamic flow-focusing microfluidic system. The method is simple, efficient, and environmentally friendly over current processing of cellulose into fibers. The synthesis of cellulose microfibers is based on the regeneration of cellulose from ionic liquids by simply contacting with a glycerol-containing water phase. Unlike other microfluidic synthesis of microfibers, rectangular-shaped microfibers were produced in our device. The shape and size of cellulose fibers were controlled by changing the flow rates of the cellulosic solution and the surrounding sheath fluid and the fluid viscosities. Folded rectangular cellulose microfibers were fabricated in certain range of flow rates by oscillation to form bends in the diverging channel. Colloidal particles or biological cells can be also easily immobilized in this fiber structure. The rectangular cellulose microfibers could find applications in biosensing fibers, scaffolds for tissue engineering, and cellulose-based carbon fibers.This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 20100011214).
9:00 PM - AA15.10
Fabrication of Three-dimensional Micro-vascular ``Single-walled Carbon Nanotubes/5-Ethylidene-2-norbornene” Nanocomposite Beams by Microfluidic Infiltration.
Brahim Aissa 1 2 , Daniel Therriault 2 , Rouhollah Dermanaki Farahani 2 , Emile Haddad 1 , Wes Jamroz 1 , Philippe Merle 3 Show Abstract
1 Smart Materials and Sensors for Space Applications, MPB Technologies INC., Montreal, Quebec, Canada, 2 Center for Applied Research on Polymers (CREPEC) Mechanical Engineering Department, Ecole Polytechnique de Montréal, Montreal, Quebec, Canada, 3 Quality Engineering Test Establishment , Department of National Defence Ottawa, Ottawa, Ontario, Canada
Self healing materials consisting of three-dimensional (3D) micro structured beams reinforced with a single-walled carbon nanotube (C-SWNT)/5-Ethylidene-2-norbornene (5E2N) nanocomposites were fabricated by 3D micro-vascular network infiltration. The 3D micro vascular network was fabricated by the direct-write assembly method. First, the robotized deposition of fugitive ink filaments on an epoxy substrate was performed to form a 3D scaffold. Epoxy resin/Grubbs catalyst matrix material was then prepared using a three-roll mixing mill to encapsulate the 3D ink structure. After the encapsulation and solidification of the structure, the fugitive ink was liquefied and removed, resulting in a 3D micro vascular network of interconnected micro channels. This micro vascular network was then infiltrated by a 5E2N loaded with C-SWNTs. The final samples consist of rectangular beams having a complex 3D structure of C-SWNT/5E2N with potential healing capability. Prior to their incorporation, the kinetic of the 5E2N- Ring Opening Metathesis Polymerization (ROMP) was studied as a function of the temperature and Grubbs catalyst to the 5E2N monomer ratio. Our results demonstrated that the ROMP reaction still effective in a relatively large temperature domain (-15 to 40° C ) with an excellent % monomer conversion (> 90 %) in very short time (less than 1 min at 40° C). Dynamic mechanical and micro/nano-indentation analyses performed on the (C-SWNT)/5E2N healing nanocomposites materials after its ROMP polymerization showed an increase in the stiffness - with respect to the C-SWNT loads. This approach demonstrated here opens new prospects for using carbon nanotube/healing agent nanocomposite materials for self-repair functionality.
9:00 PM - AA15.2
Computational and Experimental Study on Solute Transport in Biomimetic Solar and Fuel Cells with Soft Matter Microfludic Networks.
Hyung-Jun Koo 1 , Orlin Velev 1 Show Abstract
1 Chemical & Biomolecular Engineering, NC State University, Raleigh, North Carolina, United States
Soft matter where reagents are distributed by “microfluidic” networks with vascular capillaries mimics the essential functionality of a number of live tissues such as plant leaves and animal lungs. Such functionality can be replicated in biomimetic structures that can deliver reagents in a wide range of energy-generating or harvesting devices, such as fuel cells or innovative solar energy cells. We explore how a soluble reagent could be supplied rapidly and efficiently through microfluidic channel networks of various designs embedded in semipermeable soft matter. Numerical modeling of the reagent transport and distribution in devices with microfluidic channel networks embedded in hydrogel was performed. The computational model takes into account both the fluid transport in porous media and the solute convection and diffusion to simulate the reagent distribution and outflux with time. Quantitative evaluation of the efficiency of various channel designs was performed on the basis of the required time and the amounts of the supplied and lost reagent for specific coverage of the reagent in the gel media. The rate and efficiency of reagent distribution in three different channel geometries - linear, T-shaped and fractal, were compared. The effect of the length of the channels and the branches was characterized and an optimized channel configuration has been established. We tested the results experimentally by a new class of “soft” microfluidic devices made of agarose, a common polysaccharide gel. Channels were formed in the gel by a replica molding method. The penetration of a water soluble dye was followed by optical imaging. The data for its temporal distribution is in excellent correlation with the simulations. Experiments in progress would prove how the hydrogel microfluidic device can be used for new energy harvesting systems.
9:00 PM - AA15.3
Functionalized Surfaces for Attachment and Fluorescence Imaging of Single DNA Molecules.
Geuntak Lee 1 , Matthew Walsh 1 , Xiaohua Huang 1 Show Abstract
1 Department of Bioengineering, University of California, San Diego, La Jolla, California, United States
Many optical and mechanical studies of single biomolecules require solid surfaces with very low nonspecific binding, well-defined surface properties, and control over the density of biomolecules. We report the development of surfaces derivatized with mixed polymer brushes for the covalent attachment and single molecule fluorescence imaging of DNA molecules. Defined ratios of an unfunctionalized polyethylene glycol (PEG) to a longer and functionalized PEG are used to produce polymer brushes on glass surfaces. Our method entails only three simple steps that can be performed in situ in a flowcell containing a glass coverslip. These include the silanization and functionlization of the glass surface, the grafting of PEG to the surfaces, and the covalent attachment of DNA biomolecules. We demonstrate that it is possible to control the density of the functional groups and thus the DNA molecules by varying the ratio of functionalized to the unfunctionalized polymers. We also show that the glass surfaces with the functionalized polymer brushes remain homogeneous and exhibit very low nonspecific binding to fluorescent dyes, fluorescently-labeled nucleotides and proteins, making them suitable for single molecule fluorescence imaging. Various factors, including polymer density and ratios, that affect the behaviors of the surfaces will be discussed.
9:00 PM - AA15.4
Novel High Aspect Ratio Ag Nanowire Solvothermal Synthesis, Fluidic Assembly and Characterization.
Jin Hwan Lee 1 , Junyeob Yeo 1 , Hyun Wook Kang 1 , Sukjoon Hong 1 , Seung Hwan Ko 1 Show Abstract
1 Applied Nano Tech & Science Lab, KAIST, Daejeon Korea (the Republic of)
Nanostructures including quantum dot, nanoparticles, carbon-nanotube(CNT), graphene, and nanowires have been extensively studied and explored for applications such as optical devices, electrical device, and biosensors due to their promising characteristics. Especially, one dimensional novel metal nanowires are vigorously applied through their unique properties which cannot be found in bulk state. For instance, Silver nanowires have been extensively studied since they play important roles in practical devices. Among the various strategies for Ag nanowire synthesis, polyol process is regarded as an ideal method due to its advantages such as rapidity, high yield ratio and repeatability. In spite of these advantages, controlling the length is difficult and the aspect ratio of a silver nanowire is usually limited to 100 ~ 400.In this study, we developed a novel approach to synthesize longer and high aspect ratio Ag nanowires with higher yield. First, We performed extensive parametric studies to find the optimum condition for ultra long Ag nanowire synthesis by controlling the external conditions such different types and combinations of stir bar, RPM, sonication effect and injection speed. Second, novel synthesis approach called a multiple growth is introduced to increase the aspect ratio of silver nanowire even further. It has been confirmed that the silver nanowire, after its initial growth, can continue to grow as long as the Ag-ion rich condition is provided repeatedly. Through the scheme of a multiple growth, we could successfully obtain extremely high aspect ratio (1000 to 3000) silver nanowire of 300 μm in length with 150 nm diameter. This value is almost an order of magnitude enhancement from the previous works and such nanowires are expected to be particularly valuable for flexible and transparent electrodes. Moreover, mean length of silver nanowires is also increased. The synthesized high aspect ratio long Ag nanowires were functionalized with self assembled monolayer (SAM) to be assembled to form a high density aligned Ag nanowire film in a fluid using Langmuir-Blodgett method or microfluidic assembly in a microchannel. The optical, mechanical and electrical properties of the assembled Ag nanowire film were measured. The fluidic assembled Ag nanowire film can be further applied for electronics or optical devices.
9:00 PM - AA15.6
Electrochemical Microfluidic Aptasensors For Detecting Cell-secreted Cytokines.
Ying Liu 1 , Alexander Revzin 1 Show Abstract
1 , UC Davis, Davis, California, United States
Small proteins called cytokines are secreted by the leukocytes in response to pathogens/infections. Therefore, cytokine production provides an important indicator of the body’s ability to mount a vigorous immune response. Our laboratory is interested in developing miniature biosensors that can detect cytokine production at the site of a small cell population or single cells. This presentation details the development of an aptamer-based electrochemical biosensor for detection of IFN-gamma. A thiolated DNA aptamer was conjugated with Methylene Blue (MB) redox tag and was immobilized on a gold electrode by self-assembly. Upon binding of IFN-gamma the aptamer switches conformation from a hairpin to an unfolded structure, resulting in decreased electron-transfer efficiency between MB and the electrode. The change in redox current was quantified using Square Wave Voltammetry (SWV) and was found to be highly sensitive to IFN-gamma concentration. The limit of detection for optimized biosensor was 0.06 nM (1 ng/mL) with linear response extending to 10 nM (160 ng/mL). This aptasensor didn’t respond to non-specific proteins, pointing to specificity for IFN-gamma. Importantly, given chemical stability of DNA, aptasensor could be regenerated by disrupting aptamer-protein complex in urea buffer and then used again with minimal loss of sensitivity. Unlike standard sandwich immunoassays that employ multiple washing steps and reagents, the aptasensor described here enabled direct, real-time detection of IFN-gamma. Furthermore, we designed a miniature IFN-gamma aptasensor that was integrated into a microfluidic device and could be used to monitor in real-time cytokine secretion from human leukocytes captured in the device. The proposed device is envisioned to have applications in immunology, cancer research and infectious disease monitoring.
9:00 PM - AA15.7
Development of a Sensitive Bead-based Assay for Enhanced Monoclonal Antibody Detection.
Manuel Ruidiaz 1 , Natalie Mendez 2 , Ana Sanchez 3 , Bradley Messmer 3 , Andrew Kummel 4 Show Abstract
1 Bioengineering, University of California, San Diego, La Jolla, California, United States, 2 Biological Sciences, University of California, San Diego, La Jolla, California, United States, 3 Moores Cancer Center, University of California, San Diego, La Jolla, California, United States, 4 Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, United States
Monoclonal antibodies are increasingly used in the treatment of cancer due to their enhanced targeting and immune system stimulation properties. Dosage guidelines typically do not take personal cancer load or metabolism into account, which may affect treatment outcome and cause unwanted side effects. The requirement for an assay that can quickly and precisely measure the concentration of the monoclonal antibody in a serum sample of a patient during therapy is needed. The key to detection is compensation for variation in non-specific binding of serum to the assay surface. (Methods) A bead-based assay with peptide antigen mimetics has been developed to rapidly determine the concentration of antibody drug present in serum specimens with high sensitivity. Alemtuzumab (anti-CD52) and rituximab (anti-CD20) antigen peptides, as discovered by phage display, were synthesized on 10 um TentaGel resin beads using conventional solid phase peptide synthesis techniques. The beads were modified to allow for multiplexing and microfluidic handling via fluorescent labeling and magnetic functionalization. The antigen-displaying fluoromagnetic particles were incubated with spiked serum samples which allowed free antibody to be captured. Primary antibody detection was performed on alemtuzumab while rituximab detection was used to compensate for non-specific serum binding to the beads. After washing, the beads were incubated with a fluorescently tagged secondary antibody for detection by flow or image cytometry. (Results) Serum from thirty (30) individual donors with various spiked serum concentrations of antibody drug were assessed using this assay. Analysis of bead fluorescence data allows for a limit of detection down to 0.1ug/ml of serum antibody drug concentration. (Conclusion) Using detection of a antibody known to be absent in serum, an accurate compensation technique for non-specific binding has been developed on multifunctional antibody assay beads in realistic samples. The developed assay is robust against donor serum variation.
9:00 PM - AA15.9
Make Carbon Nanotube-PMMA Composite Thin: Application to Water Quality.
Christina Villeneuve 1 2 , Sebastien Pacchini 1 2 , Monique Dilhan 1 2 , René-David Colin 1 2 , Alexandre Brouzes 3 , Pascal Boulanger 3 , Robert Plana 1 2 Show Abstract
1 , CNRS-LAAS, Toulouse, Haute Garonne, France, 2 , University of Toulouse, Toulouse France, 3 , CEA-IRaMiS, Saclay France
Nanoporous membranes formed by carbon nanotube embedded in polymer matrix are promising for water purification issue. In our case, aligned carbon nanotubes (CNT) inner core is used as the transport channel. For water quality applications, the membrane needs to be thin (around 50µm) and the nanotubes open. In this study, chemical mechanical polishing (CMP) and grinding methods are investigated and compared to thin such an “hard-smooth” hybrid materials based on Multi Wall Carbon Nanotube (MWNTs are “hard” to cut) impregnated by a Poly-methyl methacryate (PMMA is smooth and easy to thin) matrix. Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) are used to characterize the surface and the structure matrix.MWNT has been synthesized by aerosol-assisted Catalytical Chemical Vapour Deposition (CCVD) at 800°C [1-3]. After the growing step, an annealing phase removes all catalysts residues contained in nanotubes . The following step consists in carpet embedding in a PMMA matrix. In each step a great attention is paid to keep good verticality of the nanotube arrays (misalignment ±5°). In this paper, technology process of thinning down is investigated to obtain thin composite film with opened NTC. In primary, CMP and grinding methods are compared in term of removal rate and surface aspect. PM5 CMP tool from Logitech is used for polishing liquid (slurry), composed by water and Al2O3 particles of different sizes (1µm, 9µm and 20µm). Concerning grinding, water is used as liquid. Results demonstrate a better removal rate and surface aspect with CMP process compare to with grinding process. SEM picture demonstrates greater damages by using grinding process: composite matrix is broken, surface is rough and nanotubes are pulled out principally because of the great removing rate (40 µm/min).In conclusion, CMP exhibits better results in terms of surface roughness, the lowest thickness obtained is ~70µm and no breaking of matrix is observed. Removal rate can be increased with the Al2O3 particles sizes. Taking into account these results, a two step process is proposed to obtain thin film composite matrix:-Start with CMP process in lapping configuration, using traditional plate and bigger particles (20µm of Al2O3) in order to remove PMMA main part above NTC-And finish by CMP process in polishing process, using abrasive plate and lower particles (1µm of Al2O3) to remove PMMA and open NTC. These two steps of process are applied on each side to up to open the carbon nanotube. AFM and SEM measurements show opening CNTs and permits to determine that the external diameter is 10-140nm and the internal diameter is around 7nm, that confirm SEM analysis on the carpet itself.