210a Sunday, February 16, 2014

such as a protein, across a lipid bilayer. To create a membrane, twosub-microliter, lipid-encased aqueous droplets are contacted - termed a dropletinterface bilayer (DIB). The peptides adsorb to the protein cargo non-covalently and somehow ‘‘carry’’ the protein from one droplet to the otherthrough the membrane. We then assay the translocated cargo through a fluoro-genic assay. The DIB method recapitulates the findings of earlier studiesinvolving Pep-1, including the dependence of protein transport on voltageand membrane charge, while also contributing new insights. Specifically, wefound that the symmetry of the bilayer membrane may play a role in Pep-1-mediated protein translocation. In addition, we used a newly developed peptidetransduction domain mimic (PTDM) as a protein carrier, which exhibiteddistinct differences compared to Pep-1’s mechanism. We’ve also used theDIB system to monitor the translocation of proteins through pores, such asthe anthrax toxin. We anticipate that the DIB method may be useful for a va-riety of transport-based studies; in particular those which must make use oftiny quantities of purified species.

1058-Pos Board B813Cobaltabisdicarbollide Macroanion is able to Diffuse across the LipidMembrane; Study of Kinetics and TransportCarmina Verdia Baguena1, Antonio Alcaraz1, Clara Vinas2,Francesc Teixidor2, Vicente M. Aguilella1.1Universitat Jaume I, Castellon de la Plana, Spain, 22Institut de Ciencia deMaterials de Barcelona (ICMAB-CSIC), Barcelona, Spain.The ability to form membranes is not restricted to polar lipid molecules or evento organic molecules, as the boron-based molecule cobaltabisdicarbollide,[Co(C2B9H11)2]

-, (commonly known as cosan) can also form vesicles andmembranes. Here we show that cosan and its derivatives can interact with phos-pholipid membranes.Cosan is a monoanionic metallabisdicarbollide, which is soluble in both waterand oils. Structurally, cosan has no similarity to the polarized lipid moleculesthat make up biological membranes. It comprises a cobalt atom sandwichedby two carboranyl clusters [1-3]. Although these clusters are hydrophobic,the metal ion imparts a dispersed negative charge spread over the whole mole-cule (fig.1). As a consequence of this, the exposed B-H and C-H bonds of thecarboranyl clusters possess weak dipoles and form intermolecular attractionsthat give the molecule its unusual physico-chemical property of being simulta-neously hydrophobic and hydrophilic. This duality allows cosan to interact withlipid membranes.Using membrane electrophysiology recordings and direct measurements ofCOSAN concentrations by inductively coupled plasma mass spectrometry(ICP-MS) we show that COSAN transits cell-free artificial lipid membranes.We study the kinetics of the transport and try to get insights on the mechanismby which COSAN crosses planar phospholipid membranes.[1] C.E. Housecroft et al., Encyclopedia of Inorganic Chemistry. John Wiley &Sons; 2008.[2] M.F. Hawthorne et al, J. Am. Chem. Soc 1968;90:879-896.[3] R.N. Grimes, Carboranes. Academic Press: Burlington, MA; 2011.

1059-Pos Board B814Experimental Observation of Surface Charge Inversion in a BiologicalNanopore in Presence of Monovalent and Multivalent CationsMarıa L. Lopez-Peris, Marıa Queralt-Martın, Vicente M. Aguilella,Antonio Alcaraz.Physics, University Jaume I, Castellon, Spain.The electric double layer formed at the pore surface produces the exclusion ofcoions and the accumulation of counterions. A particularly captivating situationoccurs when interfacial charges attract counterions in excess of their own nom-inal charge, thus leading to an effective charge inversion of the system. Thisphenomenon has been reported in such diverse systems like lipid vesicles, col-loids, Langmuir monolayers, membranes, flexible polyelectrolytes and othersynthetic nanodevices that are in contact with an aqueous solution containingmultivalent ions. Here, we report experimental evidence of charge inversionin the bacterial channel OmpF of E. coli, not only in presence of multivalentcations but also in presence of monovalent ones. We perform reversal potentialexperiments in different conditions of pH and salt concentration to analyze boththe effect of cation type and size in the selectivity changes.

1060-Pos Board B815Influences on Cellular Adhesion of Nanoparticles under Blood Flow-LikeConditionsEllen Broda, Ulrich Lachelt, Frauke Mickler, Ernst Wagner,Christoph Brauchle.LMU Munchen, Munchen, Germany.Nanocarrier-mediated drug and gene delivery are novel strategies to treat dis-eases, for example cancer, neurological disorders, infectious and cardiovascu-

lar diseases. Sophisticated drug delivery and gene therapy systems are typicallyequipped with targeting ligands to improve the specific binding to diseasedcells within the human body. These targeting moieties are usually small mole-cules, peptides or proteins which are able to specifically bind receptors on thecell surface. In many diseased cells certain receptors are overexpressed andtherefore represent suitable targets.In our experiments we focus on the specific adhesion of nanocarriers to targetcells. Therefore, we use fluorescent labeled polystyrene beads as model parti-cles, shield them with PEG (polyethylene glycol) and mount a ligand - thetransferrin receptor (TfR) binding peptide B6 - to mimic the surface of thera-peutical nanocarriers. The binding of these targeted beads is directly comparedto the adhesion of non-targeted beads on TfR overexpressing HuH7 cells. Asnon-targeted beads we use three different types, one with hydroxyl groups onthe surface, another with scrambled B6 peptide (same amino acids, but differentorder) and a third type with modified B6 peptide (all positively charged aminoacids are replaced by neutral ones). To include dynamics and determine theimpact of shear stress, the binding study is performed under laminar flow con-ditions, i.e. the beads are flushed over a cell monolayer within a microfluidicchannel. After fixation of the cells, highly-sensitive fluorescence widefield mi-croscopy is performed to analyze the adhesion of beads on a single cell level.With this approach we are able to directly analyze the effect of the targetingligand. In addition, the influences of electrostatics and shear stress on cellularparticle binding are investigated.

1061-Pos Board B816Surface Interactions in Suspensions of Swimming CellsVasily Kantsler1, Jorn Dunkel2, Raymond E. Goldstein3.1Department of Physics, University of Warwick, Coventry, United Kingdom,2Department of Mathematics, Massachusetts Institute of Technology,Cambridge, MA, USA, 3DAMTP, University of Cambridge, Cambridge,United Kingdom.Interactions between swimming cells and surfaces are essential to many micro-biological processes, from bacterial biofilm formation to human fertilization.However, despite their fundamental importance, relatively little is known aboutthe physical mechanisms that govern the scattering of flagellated or ciliatedcells from solid surfaces. A more detailed understanding of these interactionspromises not only new biological insights into structure and dynamics offlagella and cilia but may also lead to new microfluidic techniques for control-ling cell motility and microbial locomotion, with potential applications rangingfrom diagnostic tools to therapeutic protein synthesis and photosynthetic bio-fuel production. Due to fundamental differences in physiology and swimmingstrategies, it is an open question of whether microfluidic transport and rectifi-cation schemes that have recently been demonstrated for pusher-type micro-swimmers such as bacteria and sperm cells, can be transferred to puller-typealgae and other motile eukaryotes, because it is not known whether long-range hydrodynamic or short-range mechanical forces dominate the surfaceinteractions of these microorganisms. Here, using high-speed microscopic im-aging, we present direct experimental evidence that the surface scattering ofboth mammalian sperm cells and unicellular green algae is primarily governedby direct ciliary contact interactions. Building on this insight, we predict andexperimentally verify the existence of optimal microfluidic ratchets that maxi-mize rectification of initially uniform Chlamydomonas reinhardtii suspensions.Because mechano-elastic properties of cilia are conserved across eukaryoticspecies, we expect that our results apply to a wide range of swimmingmicroorganisms.

1062-Pos Board B817Selective Growth of Neural Networks on Micro-Patterned GrapheneSandeep Keshavan1, Matteo Lorenzoni1, Fernando Brandi1, Andrea Giugni2,Francesca Cella Zanacchi1, Bruno Torre1, Silvia Dante1.1Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy, 2Nanostructures,Istituto Italiano di Tecnologia, Genova, Italy.Single crystal graphene is the ideal candidate for next generation of electronicdevices and of biosensors. Interfacing graphene with neural cells could be high-ly advantageous in exploring their electrical behaviour and promising forseveral biomedical applications, including neural regeneration and artificialretina. Here, we present a straightforward fabrication technique based on laserablation to obtain patterned substrates promoting ordered neuron growth.Chemical vapor deposition (CVD) single layer graphene (SLG) was machinedby means of single pulse UV laser ablation technique at the lowest effectivelaser fluence in order to minimize laser damage effects (1). The obtainedpatterned substrates, with alternating micro-sized stripes of ablated and not-ablated SLG, were uniformly coated with poly-D-lysine; primary embryonichippocampal neurons were cultured on the functionalized substrates. As moni-tored by time-lapse imaging, neurons adhered on both regions of the pattern,

Sunday, February 16, 2014 211a

but they spontaneously migrate to the SLG areas and eventually form an or-dered interconnected neuron pattern perfectly superimposed to the patterndesign. Surface functionalization was then more effective on the SLG, and re-sulted in notably aligned neural network. The described technique could beconsidered a valuable candidate to realize a new generation of highly special-ized biosensors. To gain further insight into the preferential positioning of neu-rons onto SLG, the distribution of focal adhesion proteins on the patterned SLGwas investigated; Stochastic Optical Reconstruction Microscopy (STORM)was employed in order to localize the cellular components for focal adhesion.Super resolution imaging qualitatively confirms that the distribution of vinculinmolecules tagged with Alexa 647 has more affinity towards the SLG regionscompared to the ablated ones.1 Lorenzoni et al, Scientific Reports 3:1954, DOI:10.1038/srep01954.

1063-Pos Board B818Cell-Permissive Protein-Resistant Substrates for Interrogating NeuronalGuidance CuesJoshua A. Maurer, Natalie A. LaFranzo, John T. Walker, Matthew J. Hynes.Department of Chemistry, Washington University, St. Louis, MO, USA.Current in vitro tools for evaluating neuronal guidance cues suffer from severaldrawbacks, including difficult substrate preparation and hard-to-interpret re-sults. Microcontact printing of an alkanethiol self-assembled monolayer(SAM) provides a robust method for producing high-resolution protein patternsby protein adsorption from solution. However, cell and protein resistant glycolterminated monolayers are typically employed in the background, which pre-vents the evaluation of potential neuronal guidance cues. We have developedzwitterionic background monolayers that are protein-resistant, but remaincell-permissive. Using surface plasmon resonance imaging (SPRi) and cell-culture studies, we have demonstrated that these zwitterionic monolayersprovide well-defined, non-receptor mediate cellular attachment through inter-actions with cell-surface glycosylation. Exploiting these properties, we havecreated a monolayer based stripe assay, where the interactions between neurons(cell bodies and neurites) and extracellular matrix (ECM) proteins or guidancecues may be observed and quantified. This system goes beyond current technol-ogies, such as direct protein patterning and microfluidics, and is even capable ofevaluating neuronal response to ECM protein, such as laminin.

1064-Pos Board B819Conductive Milieu on Cellular ElectromechanicsSoonGweon Hong, Albert Kim, Philip Lee, Luke P. Lee.UC Berkeley, Berkeley, CA, USA.Cellular polarity and alignment are significant biophysical factors in tissue ar-chitectures and tissue-to-organ level functions. Multiple biophysical contribu-tors are addressed as cellular alignment factors in mechanical, biochemical andelectrical aspects; however, passive electric conductance has not been dis-cussed. Herein, we report the spontaneous alignment of cardiomyocytes to pre-defined electric conductivity environments as example. Two to three days afterpassaging iPSCs-derived cardiomyocytes on thin gold strips (i.e., 10-nm thick-ness, 10s-mmwidth and 10s-mm period) patterned on a nonconductive substrate(i.e., glass), the most single-cell cardiomyocytes adhere on the nonconductivearea and align themselves parallel to the conductive pattern, without anyexternal stimuli. From control experiments, we can exclude any mechanicalcues as the reasons of the spontaneous alignment, such as surface groove andmechanical stiffness. Currently, we hypothesize diamagnetic effect or Frohlichelectromagnetic effect as the physiological response of cardiomyocyte, whichmay be induced from the electric field coupling between cardiomyocytes andthe gold pattern. Along with the further understanding, this observation willhighlight passive electric elements as important biophysical aspects sincemany cellular components possess a level of electric properties.

1065-Pos Board B820Contractility of Neonatal Cardiomyocytes is Altered with DifferentDensities of Laminin Covalently Attached to MicropostsAlexandre J.S. Ribeiro1,2, Kathia Zaleta-Rivera2,3, Euan A. Ashley2,3,Beth L. Pruitt1,2.1Mechanical Engineering, Stanford University, Stanford, CA, USA,2Cardiovascular Institute, Stanford University, Stanford, CA, USA,3Department of Cardiovascular Medicine, Stanford University School ofMedicine, Stanford, CA, USA.Heart disease is the number one killer in the USA. The collective contractilityof the muscle cells of the myocardium - cardiomyocytes - generates the neces-sary force for the function of a healthy beating heart. Laminin interacts in vivowith cardiomyocytes. Changes in the extracellular concentration and organiza-tion of laminin relate to different types of heart disease. Arrays of polydime-thylsiloxane (PDMS) microposts measure forces generated by adhesivemammalian cells and were here used to characterize the contractility of single

neonatal cardiomyocytes. We used two types of organosilanes to bind lamininto the surface of PDMS microposts: 3-glycidoxypropyltrimethoxysilane and3-aminopropyltriethoxysilane. We acquired videos of contracting cardiomyo-cytes at two different days after cells started to beat and functionally character-ized the contractility of single cells. More specifically, we calculated generatedforces, beating rate, time of contractions and speeds of contraction and relaxa-tion. These parameters varied in time as a function of organosilane surface sta-bility and cardiomyocyte biological changes when cultured in vitro. Higherforces are generated by cardiomyocytes cultured on laminin covalentlyattached to PDMS microposts relative to laminin physisorbed to oxidizedPDMS. We obtained higher laminin density with 3-glycidoxypropyltrimethox-ysilane, which correlated to higher generated forces. We also observed higherbeating rate at the day 1 and a considerable decrease at day 2. Compared to3-glycidoxypropyltrimethoxysilane, higher stability of laminin covalent attach-ment was observed with 3-aminopropyltriethoxysilane. The beating rate andspeeds of contraction and relaxation increased and time of contractionsdecreased at day 2 for neonatal cardiomyocytes cultured on these PDMSmicro-post surfaces. Our results shed light on the potential of in vitro biomechanicalsystems to model extracellular disease conditions of heart pathologies. Futurework will test the contractility of cardiomyocytes with mutations known tooriginate cardiomyopathies.

Micro- and Nanotechnology I

1066-Pos Board B821Flow Injection of DNA in Nanopores : Direct Optical Visualization of aPressure ThresholdThomas Auger1, Jerome Mathe2, Virgile Viasnoff3, Jean-Marc di Meglio1,Loıc Auvray1, Fabien Montel1.1Universite Paris Diderot, Paris, France, 2Universite d’Evry, Evry, France,3MechanoBiology Institute, Singapore, France.In the regime of pores smaller than the radius of gyration, the flow injection ofpolymers and biopolymers exhibit a flow threshold independent of the poreand of the polymer itself. We have developed a new combination of near fieldoptics (zero mode wave guide) and image analysis in order to revisit this pheno-menon. Working at constant pressure we are able to control and observe directlythe transport of individual biomoleculeswith a time resolution of 5ms. In the caseof DNA, we show that the forced transport through the pore can be described asan energetic barrier only dependent on the injected flow. Further application tobiological systems of this barrier measurement will also be discussed.

1067-Pos Board B822Designing Hydrophobic Gates into Biomimetic NanoporesJemma L. Trick1, E. Jayne Wallace2, Hagan Bayley1, Mark S.P. Sansom1.1University of Oxford, Oxford, United Kingdom, 2Oxford NanoporeTechnologies, Oxford, United Kingdom.The use of nanopores is fast being a major scientific tool in molecular analysisand detection due to their ability to detect polynucleotides, proteins and smallmolecules. Biomimetic modelling of pores allows for a specific function to beincorporated into the molecular structure of the nanopore, based on amino acidmotifs found in existing protein structures.An initial beta barrel model was built computationally, based on the transmem-brane domain of 14 stranded beta-barrel pore, alpha-hemolysin. Hydrophobicand hydrophilic residues were built in a specific arrangement within the struc-ture to replicate an hourglass shape cavity with a central constriction. From this,pore conductions were observed via Molecular Dynamics (MD) and selectedmodels were transformed into hybrid pores in which the location of hydropho-bic residues differed to give constricting regions surrounded by hydrophilic res-idues. From All Atom MD simulations, a hydrophobic gating mechanism hasbeen established within these toy models with intermittent water currentsthrough the pore giving an insight into possible biomimetic motifs which couldbe biochemically integrated into the wild type protein.

1068-Pos Board B823Developing a Broadband Amplifier for Analysis of DNA Structural andMolecular CharacteristicsJared S. Becker1, Michael Goryll2, Bertan Bakkaloglu2, Josh Sloan2,Josh Lambert2.1Arizona State University, Tempe, AZ, USA, 2Electrical Engineering,Arizona State University, Tempe, AZ, USA.The recent emergence of DNA-based diagnostics increases the need for rapidDNA sequencing technologies. One method to achieve this is to pass DNAthrough a nanopore, recording the trans-membrane current with a low-noisecurrent amplifier. The challenge presented in this method is that the bandwidthof commercially available current amplifiers is limited to 100kHz, which is not