Phillip Kuhn

Implementing Enzyme-Linked Immunosorbent Assays on a Microfluidic Chip To Quantify Intracellular Molecules in Single Cells

Cell-to-cell differences play a key role in the ability of cell populations to adapt and evolve, and they are considered to impact the development of several diseases. Recent advances in microsystem technology provide promising solutions for single-cell studies. However, the quantitative chemical analysis of single-cell lysates remains difficult. Here, we combine a microfluidic device with the analytical strength of enzyme-linked immunosorbent assays (ELISA) for single-cell studies to reliably identify intracellular proteins, secondary messengers, or metabolites. The microfluidic device allows parallel single-cell trapping and isolation in 625-pL microchambers, repeated treatment and washing steps, subsequent lysis and analysis by ELISA. Using a sandwich ELISA, we quantitatively determined the concentration of the enzyme GAPDH in single U937 cells and HEK 293 cells, and found amounts within a range of a few (1-4) attomol per cell. Furthermore, a competitive ELISA is performed to determine the concentration of the secondary messenger cyclic adenosine monophosphate (cAMP) in MLT cells, in response to the hormone lutropin. We found the half maximal effective concentration (EC50) of lutropin to have an average value of 2.51 ± 0.44 ng/mL. Surprisingly, there were large cell-to-cell variations for all supplied lutropin concentrations, ranging from 36 to 536 attomol cAMP for nonstimulated cells and from 80 to 1040 attomol cAMP for a concentration around the EC50 (3 ng/mL). Because of the high sensitivity and specificity of ELISA and the large number of antibodies available, we believe that our device provides a new, powerful means for single-cell proteomics and metabolomics.

Tip-enhanced Raman spectroscopic imaging of patterned thiol monolayers.

Summary Full spectroscopic imaging by means of tip-enhanced Raman spectroscopy (TERS) was used to measure the distribution of two isomeric thiols (2-mercaptopyridine (2-PySH) and 4-mercaptopyridine (4-PySH)) in a self-assembled monolayer (SAM) on a gold surface. From a patterned sample created by microcontact printing, an image with full spectral information in every pixel was acquired. The spectroscopic data is in good agreement with the expected molecular distribution on the sample surface due to the microcontact printing process. Using specific marker bands at 1000 cm−1 for 2-PySH and 1100 cm−1 for 4-PySH, both isomers could be localized on the surface and semi-quantitative information was deduced from the band intensities. Even though nanometer size resolution information was not required, the large signal enhancement of TERS was employed here to detect a monolayer coverage of weakly scattering analytes that were not detectable with normal Raman spectroscopy, emphasizing the usefulness of TERS.

Microfluidic trapping of giant unilamellar vesicles to study transport through a membrane pore

We present a microfluidic platform able to trap single GUVs in parallel. GUVs are used as model membranes across many fields of biophysics including lipid rafts, membrane fusion, and nanotubes. While their creation is relatively facile, handling and addressing single vesicles remains challenging. The PDMS microchip used herein contains 60 chambers, each with posts able to passively capture single GUVs without compromising their integrity. The design allows for circular valves to be lowered from the channel ceiling to isolate the vesicles from rest of the channel network. GUVs containing calcein were trapped and by rapidly opening the valves, the membrane pore protein α-hemolysin (αHL) was introduced to the membrane. Confocal microscopy revealed the kinetics of the small molecule efflux for different protein concentrations. This microfluidic approach greatly improves the number of experiments possible and can be applied to a wide range of biophysical applications.

A microfluidic vesicle screening platform: monitoring the lipid membrane permeability of tetracyclines.

For many drugs including antibiotics such as tetracyclines it is crucial that the molecule has the ability to quickly and passively permeate lipid membranes. Hence, the understanding of the permeability in relation to the molecular structure is an important aspect to rationally design novel pharmaceutically active compounds with high bioavailability. Here, we present a versatile method to study the kinetics of tetracycline permeation across liposome membranes on a microchip. Liposomes are immobilized onto the glass surface in a stripe pattern via an avidin-biotin bond and covered by microchannels to allow continuous delivery of tetracycline and buffer. The fluid flow provides a constant concentration profile and thereby resembles the drug transport via blood in the human body. Total internal reflection fluorescence (TIRF) microscopy was used to image the formation of a fluorescent drug-europium complex inside the liposomes. The permeation rates of various tetracyclines were investigated and the results compared to a conventional method (water-octanol partitioning). The findings largely confirm the correlation between membrane permeability and lipophilicity of the permeating molecules (Overtons rule). However, slight deviations reveal that lipophilicity is an important but not the exclusive parameter for the prediction of permeation. The method is fast enough to study the permeation of unstable tetracyclines such as rolitetracycline. Additionally, with the use of different cholesterol concentrations, the influence of membrane composition on the permeation rate can be investigated conveniently. The microfluidic approach can be easily applied to investigate the kinetics of other processes such as ligand-membrane receptor association and dissociation, provided that the process can be visualized by means of fluorescence spectroscopy.

A facile protocol for the immobilisation of vesicles, virus particles, bacteria, and yeast cells

Immobilisation of liposomes and cells is often a prerequisite for long-term observations. The most common immobilisation approaches rely on surface modifications, encapsulation in porous materials or trapping in microfluidic channels by means of hurdle-like structures. While these approaches are useful for larger mammalian cells, the immobilisation of smaller organisms like bacteria and yeast or membrane model systems such as liposomes typically requires modification of their outer membrane to ensure that they are stably arrested at a defined position. Here, we present a protocol to immobilise biological objects, which can interact with hydrophobic cholesterol. A water-soluble molecule (cholesterol-PEG-biotin) is used as a linker, which can bind via avidin to biotinylated BSA (bBSA) previously absorbed on a glass surface. For better visualization, bBSA is arranged in a dot pattern by means of microcontact printing, and a microfluidic channel is used for sample supply. We show that our approach can be used to successfully immobilise artificial liposomes of different sizes, native (cell-derived) vesicles, vaccinia virions, Saccharomyces cerevisiae and Escherichia coli, simply by flushing the objects through the channel. Under these conditions, small liposomes and biological objects are stably arrested at high flow rates, while larger cells and liposomes can be released again by application of high shear stress. This protocol can be applied for long-term studies where fluids must be changed repeatedly, for measuring fast kinetics where rapid fluid exchange is essential, and to study the effects of shear stress.

Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times

Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements.

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

A microfluidic device for the delivery of enzymes into cells by liposome fusion

Liposomes are versatile carriers of drugs or biomolecules and are ideally suited to transport molecules into cells. However, mechanistic studies to understand and improve the fusion of liposomes with cell membranes and endosomes are difficult. Here, we report a method that allows for stable coimmobilization of liposomes and living cells, thereby bringing the membranes into close contact, which is essential for membrane fusion. The small unilamellar liposomes are tethered to the surface by a linker so that no modification of the liposome membrane for cell binding is required. The cells are positioned above the liposomes by posts that are integrated into the microfluidic device, and a pH drop induces the fusion of the cell‐liposome membranes. Both membrane fusion and release of molecules into the cytosol are visualized by fluorescence dequenching assays. Furthermore, we proved the efficient delivery of the enzyme β‐galactosidase into the cells when a fusogenic liposome composition was used. The device could be used for fusion studies but is also a versatile means for cell transfection.

Towards nanowire sensors on a microfluidic platform: In-situ formation, positioning and sizing of nanowire bundles

VV would like to acknowledge funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 801809).Funding from the European Research Council under the 7th Framework Programme (ERC Strating Grant no. 203428 nµLIPIDS) is gratefully acknowledgedThis project has been funded by European Union within the Seventh Framework Programme of R&D.Trabajo presentado en Aquaculture Europe 19 (Our future, growing from water), celebrado en Berlin del 7 al 10 de octubre de 2019.Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Santiago de Compostela.--2017-07-21.--Sobresaliente Cum LaudeOur aim is to provide insights into some basic facts of US government debt management by introducing simple financial frictions in a Ramsey model of fiscal policy. We find that the share of short bonds in total U.S. debt is large, persistent, and highly correlated with total debt. A well known literature argues that optimal debt management should behave very differently: long term debt provides fiscal insurance, hence short bonds should not be issued and the position on short debt is volatile and negatively correlated with total debt. We show that this result hinges on the assumption that governments buy back the entire stock of previously issued long bonds each year, which is very far from observed debt management. We document how the U.S. Treasury rarely has repurchased bonds before 10 years after issuance. When we impose in the model that the government does not buy back old bonds the puzzle disappears and the optimal bond portfolio matches the facts mentioned above. The reason is that issuing only long term debt under no buyback would lead to a lumpiness in debt service payments, short bonds help offset this by smoothing out interest payments and tax rates. The same reasoning helps explain why governments issue coupon-paying bonds. Solving dynamic stochastic models of optimal policy with a portfolio choice is computationally challenging. A separate contribution of this paper is to propose computational tools that enable this broad class of models to be solved. In particular we propose two significant extensions to the PEA class of computational methods which overcome problems due to the size of the model. These methods should be useful to many applications with portfolio problems and large state spaces.Este documento contiene la edicion critica, estudio e indices de la traduccion latina del Coran preparada para el cardenal italiano Egidio de Viterbo (c. 1465 – 1532). El texto fue traducido por primera vez en 1518 aunque su copia completa y corregida data del ano 1621. El estudio preliminar, que constituye la primera parte de la introduccion, incluye una sinopsis de las traducciones conocidas del Coran al latin, una edicion del prologo a uno de los manuscritos, presentacion de las personas historicas involucradas intelectualmente en la elaboracion y estudio de esta traduccion y tambien un estudio de algunas glosas. La segunda parte del estudio de la introduccion esta dedicada a los manuscritos y a las normas de edicion, y profundiza en el problema de la transmision del texto. La edicion critica esta fijada a partir de los dos manuscritos conservados, Cambridge ms. Mm. v. 26 (C) y Milan ms. D 100 inf. (M). El material editado ha requerido la lectura de 936 folios en total, que han sido editados en 708 paginas. El aparato critico recoge los variantes entre C y M, correcciones y aclaraciones anadidas entre lineas en ambos manuscritos, correcciones y anotaciones anadidas por la segunda mano de C y algunas glosas de caracter filologico o cultural relacionadas con la traduccion. Los indices elaborados en base a la edicion latina recogen nombres propios y palabras redactadas tambien en idiomas que no sean latin. Estos indices estan divididos en cuatro listas: los nombres propios (Index nominum personarum, locorum et rerum), las palabras arabes (Index verborum arabicorum), las palabras griegas (Index verborum graecorum) y las palabras hebreas (Index verborum hebraeorum).The structural phase behavior of high-quality single crystals of methylammonium lead iodide (CH₃NH₃PbI₃ or MAPbI₃) was revisited by combining Raman scattering and photoluminescence (PL) measurements under high hydrostatic pressure up to ca. 10 GPa. The single crystals were specially grown with the final thickness needed for pressure experiments, retaining their high quality due to a less invasive preparation procedure, which avoids sample thinning. Both PL and Raman spectra show simultaneous changes in their profiles that indicate the occurrence of three phase transitions subsequently at around 0.4, 2.7, and 3.3 GPa. At the second phase transition, the Raman spectra exhibit a pronounced reduction in the line width of the phonon modes of the inorganic cage, similar to the changes observed at the tetragonal-to-orthorhombic phase transition occurring at around 160 K but ambient pressure. This behavior is interpreted as evidence for the locking of the organic cations in the cage voids above 2.7 GPa due to the reduced volume and symmetry of the unit cell. At the third phase transition, reported here for the first time, the PL is greatly affected, whereas the Raman spectrum experiences only subtle changes related to a splitting of some of the peaks. This behavior may indicate a change mostly in the electronic structure with little effect on the crystal structure. Strikingly, the sharp Raman features observed at high pressures do not support amorphization of MAPbI₃ with onset at 3 GPa, as claimed by most of the high-pressure (X-ray) literature. We interpret this apparent discrepancy in terms of the degree of disorder introduced at different length scales in the perovskite lattice by the pressure-induced freeze-out of the methylammonium cation motion.espanolEl presente articulo estudia el tratamiento que la historiografia espanola de epoca Moderna dio a Al-Andalus, y como la integro en una narracion de la historia nacional. En el, trato de ir mas alla de la narrativa, apologetica o critica, de la “Reconquista”, y muestro como Al-Andalus se asocia a una idea del Oriente biblico relacionada con la escritura de la historia sagrada, que acaba introduciendose en el pensamiento critico del s. XVII. EnglishThis paper studies the way in which Early Modern Spanish historiography dealt with Al- Andalus, and how it tried to integrate it into the framework of Spanish national history. I criticize the narratives linked to the “Reconquista” (both critical and apologetic), and argue how Al-Andalus was connected to a more general idea of the Biblical Orient, that was used to write the sacred history of Spain. Finally, I try to show how this idea became part of the 17th-century Spanish critical thought.Trabajo presentado en el XL Congreso de la Sociedad Espanola de Genetica, celebrado en Cordoba del 16 al 18 de septiembre de 2015.XXIII Europhysics Conference on Atomic and Molecular Physics of Ionized Gases, ESCAMPIG XXIII. -- Bratislava, Slovakia, July 12-16, 2016. -- https://www.escampig2016.org/The influence of the humanities on the study of a technological subject like robotics needs to rapidly grow, for the simple reason that robotics is becoming a part of humanity: assisting, interacting, and enabling people in an increasing number of ways in daily life. The robotics research community is well aware of the need for such a crossover with the humanities and many joint ventures are being undertaken, such as forums on “Robotics meets the Humanities” at main robotics conferences, the launching of research projects, and the publication of special issues in scientific journals. This cross-cutting has even led to a new discipline: Roboethics, a subfield of applied ethics studying both the positive and negative implications of robotics for individuals and society, with a view to inspire the moral design, development and use of so-called intelligent/autonomous robots, and help prevent their misuse against humankind. The discipline involves two main areas: legal regulation and ethical education. Regarding the former, institutions such as the European Parliament, the South Korean Robot Ethics Charter, the IEEE Standards Association, and the British Standards Institution are developing regulations for robot designers, programmers, and users. There are many options to integrate ethics education (or Humanities) in technological university degrees, ranging from including a professional ethics course in the syllabus, to allowing students to take certain credits or a minor in a Humanities Department, to even offering a combined degree, like the Computer Science and Philosophy degree at the University of Oxford. Prestigious associations such as IEEE and ACM include 18 knowledge areas in their Computer Science curricula, one of which is “Social Issues and Professional Practice”, so that “students develop an understanding of the relevant social, ethical, legal and professional issues”. To this end, some courses in this area recur to science fiction to exemplify conflictive situations, since narrative is a good way to engage students in safe discussion and reasoning about difficult and emotionally charged issues without making it personal. Some experiences along this line will be described.Los materiales aqui presentados incluyen: la presentacion de la actividad, el guion para el profesorado, un power-point para poner en la sesion, las 30 fichas de cada personaje, asi como un par de objetes representativos.M. E. F. Brollo acknowledges the Brazilian agency CNPq for the grant [232947/2014-7] within the Science without Borders program and the COST action program for the grant [TD1402 – 38989]. The work was supported by the Spanish Government under projects MAT2017-88148-R (AEI/FEDER, UE), MAT2016-76507-R, CTQ2016-78454-C2-2-R, by the Comunidad de Madrid (S2018/NMT-4321 NANOMAGCOST-CM), the European Research Council through ERC-AG grant 340177. IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686). The characterization was performed using the ICMM/CSIC general services (X-ray, ICP). ACS analyses were performed at RISE Acreo in Gothenburg, Sweden and cryo-TEM images were taken by Dr. Francisco Javier Chichon at the Cryo-EM facility (CiB-CNB-CSIC) placed on the National Center for Biotechnology in Madrid, Spain.Oral presentation given at the XII Reunion Cientifica de la Sociedad Espanola de Astronomia, held in Bilbao (Spain), on July 18-22th, 2016.This dossier is the result of a project funded by European Research Council under the European Union Seventh Framework Programme (FP7/2007-2013)/ ERC Grant Agreement number 323316, project CORPI “Conversion, Overlapping Religiosities, Polemics, Interaction. Early Modern Iberia and Beyond,” IP: Mercedes Garcia-ArenaD. G. and Y. K. C would like to acknowledge sup-port from the project ND-PHOT jointly funded by CSIC and CNRS. D. G. and P.C. acknowledge financial support from Agencia Estatal de Investigacion (AEI,Spain) and Fondo Europeo de Desarrollo Regional un-der Project SuMaEco, grant number: RTI2018-095441-B-C22 (AEI/FEDER,UE) and Agencia Estatal de Investigacion through Maria de Maeztu Program for Units o fExcellence in R&D (MDM-2017-0711). Y. K. C. also ac-knowledges funding from the European Research Council through the projects NextPhase & Versyt, from the Centre National d’Etudes Spatiales(CNES) through the project SHYRO, and from the University of Maryland.Trabajo presentado en el 57th European High Pressure Research Group Meeting on High Pressure Science and Technology ( EHPRG 2019), celebrado en Praga (Republica Checa), del 1 al 6 de septiembre de 2019Este trabajo de tesis ha sido posible gracias a una beca predoctoral del Subprograma de Formacion del Personal Investigador (FPI) del Ministerio de Economia y Competitividad asociada al proyecto SAF2011-26273. La investigacion ha sido financiada por los siguientes proyectos de investigacion: “Caracterizacion de los mecanismos moleculares involucrados en la generacion de neuronas serotonergicas”. Ministerio de Economia y Competitividad. Plan Nacional I+D (SAF2011-26273). “Estudio de los mecanismos transcripcionales que regulan la diferenciacion de las neuronas monoaminergicas y su conservacion evolutiva”. Ministerio de Economia y Competitividad. Plan Nacional I+D (SAF2014-56877-R). “Dissecting the gene regulatory mechanisms that generate serotonergic neurons and their link to mental disorders”. European Research Council. Starting Grant.Funding from the European Research Council under the 7th Framework Programme (ERC Starting Grant, project no. 203428, “nμ-LIPIDS”) and the ETH for the fellowship to J.P.-L. is gratefully acknowledged.V.V. would like to acknowledge funding from the European Research Council (ERC) under European Union’s Horizon 2020 research and innovation programme (grant agreement No 801809)In this technical report we present a framework for the empirical validation of a physical cloth model. First, we introduce the model to be validated and its parameters, next we explain how to obtain real world data of the motion of a textile and finally we show how to fit the model to the experimental data. Institut de Robòtica i Informàtica Industrial (IRI) Consejo Superior de Investigaciones Cient́ıficas (CSIC) Universitat Politècnica de Catalunya (UPC) Llorens i Artigas 4-6, 08028, Barcelona, Spain Tel (fax): +34 93 401 5750 (5751) http://www.iri.upc.edu Corresponding author: Franco Coltraro tel: +34 93 401 5750 fcoltraro@iri.upc.edu http://www.iri.upc.edu/staff/fcoltraroLa linea de investigacion principal de este trabajo se desarrolla en el marco del «Los Caminos del Neolitico» (HAR2009‑09027), concedido por la Subdireccion General de Proyectos de Investigacion / Direccion General de investigacion y gestion del Plan Nacional de I+D+I / Secretaria de Estado de Investigacion, del Ministerio de Ciencia e Innovacion. Del mismo modo, la parte analitica se enmarca en el proyecto denominado «Origins and spread of agriculture in the western Mediterranean region» (ERC‑AdG 230561), financiado por el ERCFRAGMENT has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 773051). Carlos Perez Garcia- Pando also acknowledges support by the AXA Research Fund, and the Spanish Ministry of Science, Innovation and Universities (RYC-2015-18690 and CGL2017-88911- R)Trabajo presentado en Aquaculture Europe 19 (Our future, growing from water), celebrado en Berlin del 7 al 10 de octubre de 2019.We thank the National Museum of Nairobi for permits to study the Leakey collection from SHK. Funding by the NSF (BCS-0852292) and the European Research Council-Starting Grants (283366) is gratefully acknowledgedEuropean Research Council under the European Unions Seventh Framework Programme (FP7/2007-2013) ERC grant agreement no 340863 / JAE-PRE program