A new microfluidic method enabling the generation of multi-layered tissues-on-chips using skin cells as a proof of concept

Microfluidic-based tissues-on-chips (TOCs) have thus far been restricted to modelling simple epithelia as a single cell layer, but likely due to technical difficulties, no TOCs have been reported to include both an epithelial and a stromal component despite the biological importance of the stroma for the structure and function of human tissues. We present, for the first time, a novel approach to generate 3D multilayer tissue models in microfluidic platforms. As a proof of concept, we modelled skin, including a dermal and an epidermal compartment. To accomplish this, we developed a parallel flow method enabling the deposition of bilayer tissue in the upper chamber, which was subsequently maintained under dynamic nutrient flow conditions through the lower chamber, mimicking the function of a blood vessel. We also designed and built an inexpensive, easy-to-implement, versatile, and robust vinyl-based device that overcomes some of the drawbacks present in PDMS-based chips. Preliminary tests indicate that this biochip will allow the development and maintenance of multilayer tissues, which opens the possibility of better modelling of the complex cell–cell and cell–matrix interactions that exist in and between the epithelium and mesenchyme, allowing for better-grounded tissue modelling and drug screening.This work was supported by the "Programa de Actividades de I+D entre Grupos de Investigación de la Comunidad de Madrid" project S2018/BAA-4480, Biopieltec-CM and the Cátedra Fundación Ramón Areces

Aluminum Nanoholes for Optical Biosensing

[EN] Sub-wavelength diameter holes in thin metal layers can exhibit remarkable optical features that make them highly suitable for (bio)sensing applications. Either as efficient light scattering centers for surface plasmon excitation or metal-clad optical waveguides, they are able to form strongly localized optical fields that can effectively interact with biomolecules and/or nanoparticles on the nanoscale. As the metal of choice, aluminum exhibits good optical and electrical properties, is easy to manufacture and process and, unlike gold and silver, its low cost makes it very promising for commercial applications. However, aluminum has been scarcely used for biosensing purposes due to corrosion and pitting issues. In this short review, we show our recent achievements on aluminum nanohole platforms for (bio)sensing. These include a method to circumvent aluminum degradation-which has been successfully applied to the demonstration of aluminum nanohole array (NHA) immunosensors based on both, glass and polycarbonate compact discs supports-the use of aluminum nanoholes operating as optical waveguides for synthesizing submicron-sized molecularly imprinted polymers by local photopolymerization, and a technique for fabricating transferable aluminum NHAs onto flexible pressure-sensitive adhesive tapes, which could facilitate the development of a wearable technology based on aluminum NHAs.The authors gratefully acknowledge financial support from MINECO projects, Spain (TEC2012-31145, CTQ2012-37573-C02 and CTQ 2013-45875-R).Angulo Barrios, C.; Canalejas Tejero, V.; Herranz, S.; Urraca, J.; Moreno-Bondi, MC.; Avella-Oliver, M.; Maquieira Catala, Á.... (2015). Aluminum Nanoholes for Optical Biosensing. Biosensors. 5(3):417-431. https://doi.org/10.3390/bios50304174174315

Desarrollo de superficies nanoestructuradas para biosensores ópticos y sensores biomiméticos

La presente Tesis Doctoral es de carácter tecnológico y está enfocada al desarrollo de nuevos biosensores y sensores biomiméticos ópticos. El trabajo ha consistido en la fabricación y caracterización de películas micro/nano−estructuradas en distintos materiales para (bio)detección óptica, empleándose como herramienta principal de nanoestructuración la litografía por haz de electrones (EBL). El objetivo general es lograr mejoras y novedades en el campo de los biosensores ópticos, tanto en lo referente a las prestaciones de los dispositivos como al coste de fabricación de los mismos. Este trabajo se puede dividir en tres partes. En la primera parte se expone el trabajo realizado para lograr sensores biomiméticos basados en polímeros de impronta molecular (MIP), micro y nanoestructurados directamente mediante EBL, algo no realizado con anterioridad a esta Tesis, y con fotolitografía UV. Se ha conseguido demostrar que el copolímero P(MAA-co-MAAEMA) se comporta simultáneamente como MIP y resina de EBL, permitiendo la nanoestructuración directa de películas de este material MIP mediante EBL. En el proceso previo a la consecución de esta demostración, se han obtenido otros resultados de relevancia relacionados con las propiedades ópticas (fotoluminiscencia) y tecnológicas (resinas de alta sensibilidad y doble comportamiento) de resinas comerciales de PMMA y PMMA/MA y del copolímero P(HEMA-co-MAAEMA). El segundo bloque contiene el trabajo desarrollado para obtener (bio)sensores plasmónicos sin marcado basados en redes de nanoagujeros realizadas en aluminio, susceptibles de ser interrogados ópticamente y capaces de detectar variaciones muy pequeñas de índice de refracción en el medio circundante. La novedad ha consistido en proteger eficazmente la superficie de aluminio frente a medios acuosos tamponados, frecuentes en bioensayos, obteniendo dispositivos competitivos frente a los fabricados habitualmente con oro, un metal miles de veces más costoso que el aluminio. Las prestaciones y la estabilidad de los dispositivos fabricados se estudiaron con diferentes pruebas de resistencia, experimentos de determinación de la sensibilidad volumétrica al índice de refracción empleando disolventes, y con ensayos de biodetección realizados como prueba de concepto. Las redes de nanoagujeros han sido fabricadas sobre dos tipos de sustratos: vidrio y policarbonato procedente de CDs y DVDs, esto último con el fin de desarrollar una tecnología que permita la integración de estos dispositivos en discos ópticos comerciales. Además, se ha investigado la transferencia de películas metálicas nanoestructuradas fabricadas sobre policarbonato a una cinta adhesiva flexible de uso común, estudiándose la sensibilidad refractométrica de la estructura resultante. En la tercera y última parte se muestra el trabajo realizado destinado a obtener (bio)sensores sin marcado basados en redes de nanopilares de resina comercial SU−8 fabricados con EBL sobre una película de aluminio depositada en sustratos de silicio, tratando de mejorar las prestaciones de estructuras similares previamente publicadas. Para ello, se ha propuesto e investigado una configuración consistente en redes de nanopilares con forma de neiloide truncado obtenidos tras la aplicación de cierto grado de desenfoque durante la litografía. Los nanopilares resultantes presentan un ensanchamiento en la base, creando una capa delgada de resina entre ellos, originando un efecto de resonancia de modo guiado. Este efecto produce una respuesta óptica espectral que cuenta con una resonancia de alto factor de calidad y muy sensible a los cambios de índice de refracción del medio circundante. Considerando tanto la sensibilidad como la calidad de la resonancia, se ha demostrado que el factor de mérito de la estructura propuesta mejora hasta en dos órdenes de magnitud el de dispositivos similares anteriores con mayor superficie sensora, sin añadir mayor coste o complejidad al proceso de fabricación. ABSTRACT The present Doctoral Thesis is a technology−based work focused on the development of novel optical biosensors and biomimetic sensors. The work has consisted of the fabrication and characterization of micro/nano−structured thin films made with different materials for optical (bio)detection, using electron beam lithography (EBL) as the main fabrication tool. The general goal is to achieve improvements and innovations in the optical biosensors field, considering both performance and production cost. This work can be divided in three parts. The first part of the Thesis describes the work performed to achieve biomimetic sensors based on molecularly imprinted polymers (MIP), directly micro and nanostructured using EBL, which has not been done before this Thesis, and also UV photolithography. The copolymer P(MAA-co-MAAEMA) has been demonstrated as a MIP capable of direct nanostructuration with EBL. Other relevant results related with optical (photoluminiscence) and technological (high sensitivity, dual tone resists) properties of the copolymer P(HEMA-co-MAAEMA), and also PMMA and PMMA/MA commercial resists, were obtained in the previous works which led to the MIP based biomimetic sensor. The second block contains the work related with the development of label−free plasmonic (bio)sensors based on nanoholes arrays fabricated in aluminium films, subject to optical interrogation and capable of detecting very small refractive index variations in the surrounding enviroment. The novelty has consisted of achieving an efficient protection of the aluminium surface against buffered aqueous solutions, thus obtaining competitive devices without the need of using gold, usually applied to similar purposes, which is thousands of times more expensive than aluminium. The performance and the stability of the devices were studied by different endurance tests, also with experiments of acquisition of the volumetric sensitivity to refractive index using solvents, and with biosensing experiments as proof of concept tests. The nanoholes arrays have been fabricated using two different substrates: glass and polycarbonate from CDs and DVDs, opening the door to the development of a technology which allows for the integration of the devices on commercial optical discs. In addition, the transfer of the metallic nanostructures fabricated on polycarbonate to common use, flexible stick tape, was investigated, as well as the refractometric sensitivity of the transferred devices. The third and last part shows the work related with the development of label−free biosensors based on SU−8 nanopillars arrays made with EBL onto an aluminium film deposited on silicon substrate, trying to improve the performance of similar architectures previously published. With this purpose, a new nanostructure consisting of truncated neiloid shape pillars, made by applying out−of−focus electron beam exposition during the lithography, has been investigated. The resultant nanopillars present a base widening, creating a resist thin film among them, and producing a guided−mode resonance effect. This effect produces an optical reflectance spectrum featuring a high quality resonance, which is very sensitive to refractive index variations of the environment in contact with the nanostructure surface. Considering both the sensitivity and the quality of the studied resonance, the developed (bio)sensors have demonstrated that the figure of merit of the proposed structure improves in two orders of magnitude comparing with previously published architectures, without adding extra cost or complexity during the manufacture process

Micro-Shaping of Nanopatterned Surfaces by Electron Beam Irradiation

We show that planar nanopatterned thin films on standard polycarbonate (PC) compact discs (CD) can be micro-shaped in a non-contact manner via direct e-beam exposure. The shape of the film can be controlled by proper selection of the e-beam parameters. As an example of application, we demonstrate a two-dimensional (2D) array of micro-lenses/reservoirs conformally covered by an Al 2D nanohole array (NHA) film on a PC CD substrate. It is also shown that such a curvilinear Al NHA layer can be easily transferred onto a flexible polymeric support. The presented technique provides a new tool for creating lab-on-CD architectures and developing multifunctional (flexible) non-planar nanostructured films and surfaces

A top-down approach for fabricating three-dimensional closed hollow nanostructures with permeable thin metal walls

We report on a top-down method for the controlled fabrication of three-dimensional (3D), closed, thin-shelled, hollow nanostructures (nanocages) on planar supports. The presented approach is based on conventional microelectronic fabrication processes and exploits the permeability of thin metal films to hollow-out polymer-filled metal nanocages through an oxygen-plasma process. The technique is used for fabricating arrays of cylindrical nanocages made of thin Al shells on silicon substrates. This hollow metal configuration features optical resonance as revealed by spectral reflectance measurements and numerical simulations. The fabricated nanocages were demonstrated as a refractometric sensor with a measured bulk sensitivity of 327 nm/refractive index unit (RIU). The pattern design flexibility and controllability offered by top-down nanofabrication techniques opens the door to the possibility of massive integration of these hollow 3D nano-objects on a chip for applications such as nanocontainers, nanoreactors, nanofluidics, nano-biosensors and photonic devices

Water-dependent photonic bandgap in silica artificial opals

Some characteristics of silica-based structuresa-like the photonic properties of artificial opals formed by silica spheresa-can be greatly affected by the presence of adsorbed water. The reversible modification of the water content of an opal is investigated here by moderate heating (below 300 °C) and measuring in situ the changes in the photonic bandgap. Due to reversible removal of interstitial water, large blueshifts of 30 nm and a bandgap narrowing of 7% are observed. The latter is particularly surprising, because water desorption increases the refractive index contrast, which should lead instead to bandgap broadening. A quantitative explanation of this experiment is provided using a simple model for water distribution in the opal that assumes a nonclose-packed fcc structure. This model further predicts that, at room temperature, about 50% of the interstitial water forms necks between nearest-neighbor spheres, which are separated by 5% of their diameter. Upon heating, dehydration predominantly occurs at the sphere surfaces (in the opal voids), so that above 65 °C the remaining water resides exclusively in the necks. A near-close-packed fcc arrangement is only achieved above 200 °C. The high sensitivity to water changes exhibited by silica opals, even under gentle heating of few degrees, must be taken into account for practical applications. Remarkably, accurate control of the distance between spheresa-from 16 to 1 nma-is obtained with temperature. In this study, novel use of the optical properties of the opal is made to infer quantitative information about water distribution within silica beads and dehydration phenomena from simple reflection spectra. Taking advantage of the well-defined opal morphology, this approach offers a simple tool for the straightforward investigation of generic adsorption-desorption phenomena, which might be extrapolated to many other fields involving capillary condensation. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.FGG was supported by the JAE Postdoctoral Program from the CSIC. This work was partially supported by EU FP7 NoE Nanophotonics4Energy grant No. 248855; the Spanish MICINN CSD2007-0046 (Nanolight.es), MAT2009-07841 (GLUSFA) and Comunidad de Madrid S2009/MAT-1756 (PHAMA) projects.Peer Reviewe

Qualitative and quantitative analysis of crystallographic defects present in 2D colloidal sphere arrays

In this work, we present a study of the typical spontaneous defects present in self-assembled colloidal monolayers grown from polystyrene and silica microspheres. The quality of two-dimensional crystals from different colloidal suspensions of beads around 1 μm in diameter has been studied qualitatively and quantitatively, evaluated in 2D hexagonal arrays at different scales through Fourier analysis of SEM images and optical characterization. The crystallographic defects are identified to better understand their origin and their effects on the crystal quality, as well as to find the best conditions colloidal suspensions must fulfill to achieve optimal quality samples. © 2011 American Chemical Society.This work was partially supported by EU FP7 NoE Nanophotonics 4 Energy grant no. 248855; the Spanish MICINN CSD2007-0046 (Nanolight.es), MAT2009-07841 (GLUSFA), CSIC PIF08-016 and Comunidad de Madrid S2009/MAT-1756 (PHAMA) projects.Peer Reviewe

Aluminum Nanohole Arrays Fabricated on Polycarbonate for Compact Disc-Based Label-Free Optical Biosensing

[EN] Al nanohole array plasmonic biosensors have been fabricated on polycarbonate (PC) substrates from conventional compact discs (CD). Standard micro and nanofabrication processes have been used and optimized to be PC compatible. The viability of this CD-based plasmonic platform for label-free optical biosensing has been demonstrated through a competitive bioassay for biotin analysis using biotin-functionalized dextran-lipase conjugates immobilized on the transducer surface.This work was funded by Projects: TEC2010-10804-E (MICINN, Spain), TEC2012-31145 and CTQ2012-37573-C02-02 (MINECO, Spain), FEDERCTQ2010-15943 (CICYT, Spain) and GVA PROMETEO 2010/008. The Spanish MEC provided MAO with a PhD studies grant.Angulo Barrios, C.; Canalejas Tejero, V.; Herranz, S.; Moreno Bondi, M.; Avella-Oliver, M.; Puchades, R.; Maquieira Catala, Á. (2014). Aluminum Nanohole Arrays Fabricated on Polycarbonate for Compact Disc-Based Label-Free Optical Biosensing. Plasmonics. 9(3):645-649. https://doi.org/10.1007/s11468-014-9676-5S64564993Tanious FA, Nguyen B, Wilson WD (2008) Biosensor-surface plasmon resonance methods for quantitative analysis of biomolecular interactions. In: Methods in cell biology. biophysical tools for biologists. Edit. Correia JJ and Detrich HW, III. Volume 84, Chapter 3, pp 53–77Schasfoort RBM, Tudos AJ (2008) Handbook of surface plasmon resonance. The Royal Society of Chemistry, CambridgeLindquist NC, Nagpal P, McPeak KM, Norris DJ, Oh SH (2012) Engineering metallic nanostructures for plasmonics and nanophotonics. Rep Prog Phys 75:036501Ebbesen TW, Lezec HJ, Ghaemi HF, Thio T, Wolff PA (1998) Extraordinary optical transmission through sub-wavelength hole arrays. Nature 391:667–669Brolo AG, Gordon R, Leathem B, Kavanagh KL (2004) Surface plasmon sensor based on the enhanced light transmission through arrays of nanoholes in gold films. Langmuir 20:4813–4815Dahlin A, Zäch M, Rindzevicius T, Käll M, Sutherland DS, Höök F (2005) Localized surface plasmon resonance sensing of lipid-membrane-mediated biorecognition events. J Am Chem Soc 127:5043–5048Yang JC, Ji J, Hogle JM, Larson DN (2009) Multiplexed plasmonic sensing based on small-dimension nanohole arrays. Biosens Bioelectron 24:2334–2338Erickson JS, Ligler FS (2008) Analytical chemistry: home diagnostic to music. Nature 456:178–179Bañuls MJ, González-Pedro V, Puchades R, Maquieira A (2007) PMMA isocyonate modified digital discs as a support for oligonucleotide-based assays. Bioconjugate Chem 18:1408–1414Challener WA, Ollmann RR, Kam KK (1999) A surface plasmon resonance gas sensor in a ‘compact disc’ format. Sensors Actuators B 54:254–258Dou X, Phillips BM, Chung PY, Jiang P (2012) High surface plasmon resonance sensitivity enabled by optical disks. Opt Lett 37:3681–3683Herranz S, Marciello M, Olea D, Hernández M, Domingo C, Vélez M, Gheber LA, Guisán JM, Moreno-Bondi MC (2013) Dextran-lipase conjugates as tools for low molecular weight ligand immobilization in microarray development. Anal Chem 85:7060–7068Rodrigo SG, García-Vidal FJ, Martín-Moreno L (2008) Influence of material properties on extraordinary optical transmission through hole arrays. Phys Rev B 77:075401Canalejas-Tejero V, Herranz S, Bellingham A, Moreno-Bondi MC, Barrios CA (2014) Passivated aluminum nanohole arrays for label-free biosensing applications. ACS Appl Mater Interfaces 6:1005–1010Foquet M, Samiee KT, Kong X, Chauduri BP, Lundquist PM, Turner SW, Freudenthal J, Roitman DB (2008) Improved fabrication of zero-mode waveguides for single-molecule detection. J Appl Phys 103:034301Chen Q, Martin C, Cumming DRS (2012) Transfer printing of nanoplasmonic devices onto flexible polymer substrates from a rigid stamp. Plasmonics 7:755–761Fang Z, Lin C, Ma R, Huang S, Zhu X (2010) Planar plasmonic focusing and optical transport using CdS nanoribbon. ACS Nano 4:75–82Fang Z, Thongrattanasiri S, Schlather A, Liu Z, Ma L, Wang Y, Ajayan PM, Nordlander P, Halas NJ, de García Abajo FJ (2013) Gated tunability and hybridization of localized plasmons in nanostructured graphene. ACS Nano 7:2388–239

Ultrasensitive non-chemically amplified low-contrast negative electron beam lithography resist with dual-tone behaviour

A non-chemically amplified negative-tone electron-beam resist with an extremely high sensitivity is presented in this work. The resist, poly(2-hydroxyethyl methacrylate-co-2-methacrylamidoethyl methacrylate) (P(HEMA-co-MAAEMA)), has been synthesized using free radical polymerization of 2-hydroxyethyl methacrylate and 2-aminoethyl methacrylate, and exhibits a crosslinking threshold dose as low as 0.5 μC cm−2. Exposed resist patterns show good adherence to silicon substrates without the assistance of adhesion promoters or thermal treatments and are shown to be adequate for use as a mask for both wet and dry etching of Si. A low contrast value of 1.2 has been measured, indicating that the synthesized polymeric mixture is particularly suitable for achieving grey (3D) lithography. Other relevant properties of the new e-beam resist are optical transparency, visible photoluminescence when crosslinked at low electronic doses, and dose-dependent dual-tone behaviour.The authors gratefully acknowledge financial support from MICINN (TEC2010-10804-E, CTQ2009-14565-C03-03 and TEC2008-06574-C03-03), and the Moncloa Campus of International Excellence (CEI).Peer Reviewe

A Proof-of-Concept of Label-Free Biosensing System for Food Allergy Diagnostics in Biophotonic Sensing Cells: Performance Comparison with ImmunoCAP

Food allergy is a common disease worldwide with over 6% of the population (200–250 million people) suffering from any food allergy nowadays. The most dramatic increase seems to be happening in children and young people. Therefore, improvements in the diagnosis efficiency of these diseases are needed. Immunoglobulin type E (IgE) biomarker determination in human serum is a typical in vitro test for allergy identification. In this work, we used a novel biosensor based on label-free photonic transducers called BICELLs (Biophotonic Sensing Cells) for IgE detection. These BICELLs have a thin film of nitrocellulose over the sensing surface, they can be vertical optically interrogated, and are suitable for being integrated on a chip. The BICELLs sensing surface sizes used were 100 and 800 µm in diameter. We obtained calibration curves with IgE standards by immobilizating anti-IgE antibodies and identified with standard IgE calibrators in minute sample amounts (3 µL). The results, in similar assay format, were compared with commercially available ImmunoCAP®. The versatility of the interferometric nitrocellulose-based sensing surface was demonstrated since the limit of detections for BICELLs and ImmunoCAP® were 0.7 and 0.35 kU/L, respectively