Fabrication of (bio)molecular patterns with contact printing techniques

[spa] Un patrón es una colección de unidades formadoras que se repiten predeciblemente en una magnitud definida. Los investigadores han utilizado patrones para garantizar la funcionalidad y repetitividad de sus estudios. Para conseguir eso, los datos obtenidos de los estudios se comparan entre varios resultados, esperando así una correlación. Dos métodos de investigación están basados en patrones: uno requiere un sustrato con unidades repetidas localizadas en un plano cartesiano definido, obteniendo una plataforma de análisis múltiple. El segundo método utiliza localizaciones definidas con diferentes áreas de prueba, creando así una plataforma de multianálisis. La miniaturización de estas pruebas permiten reducir el costo, maximizar la eficiencia e incrementar la repetitividad de los ensayos. Los micropatrones consisten en puntos de (bio)moléculas limitados en pequeñas áreas para crear zonas de reacción múltiples. Esta tecnología fue inicialmente utilizada para crear las interacciones del ADN para estudios genómicos. La técnica evolucionó para crear patrones de proteínas y actualmente se utiliza para estudios bioquímicos a gran escala y de muy alto rendimiento. Patrones de una (bio)molécula repetida a través del sustrato son fabricados rutinariamente en muchos laboratorios utilizando técnicas de impresión por contacto, por inyección u otro métodos. El cimiento de estas técnicas es transferir una (bio)molécula de una solución a un sustrato. Esta Tesis pretende expandir los métodos de creación de micropatrones por técnicas de impresión por contacto. Inicialmente se caracterizó una máquina automatizada de impresión por microcontacto para crear patrones y estudiar las variables que afectan al momento de la impresión. Se correlacionaron la presión y el tiempo de impresión para entender la morfología del patrón resultante. Igualmente se caracterizó el posicionamiento micrométrico de los patrones para crear estructuras complejas. Posteriormente, la máquina se modificó para incluir la técnica de impresión con plumas poliméricas. Esta técnica permitió crear micropatrones en superficies minúsculas. Estos micropatrones fueron luego liberados para crear micropartículas que pueden ser personalizadas para aplicaciones diversas. Finalmente, se formuló una nueva técnica de replicación de patrones de ADN desde un patrón inicial, manteniendo la información química y espacial presente en éste.[eng] For that, the obtained data is purposely compared over and over in hope that the results are comparable. Two main research approaches are based on patterns: The initial requires a single substrate with localized and repeated units to create multiple testing sites, obtaining a repeated, multi-analysis system. The second approach uses fixed localization with different testing motifs, creating a diverse multi-analysis platform. The miniaturization of these assays provides an alternative to reduce cost, maximize efficiency, and increase repeatability. Micropatterns consist on immobilized (bio)molecular motifs constrained in small areas over a solid substrate. These fixed spots provide up to thousands of reaction sites for parallel detection. Micropatterns were first developed to study the interaction between Deoxyribonucleic acid (DNA) strands and the study of the genome. Afterwards, this technology was used to create miniaturized protein patterns. Today, this technology is essential for large-scale and high-throughput biological and biochemical studies. Single-feature microarrays are routinely reproduced at many laboratories using various contact, non-contact, or alternatively methods. The foundation is to transfer a (bio)molecule in a solution onto a solid substrate obtaining a defined feature shape. This Thesis aims to expand the current contact replication techniques for microarray fabrication. Initially, an automatized microcontact printing tool was characterized to create complex patterns on a wide range of substrates. Thiols, silanes, and various biomolecules were printed on glass, silicon oxide or gold. The printing properties were explored to create a definitive protocol for further applications. The effect of the printing force and dwell time were thoroughly studied to form a mathematical expression to understand all the variables involved during contact printing. The miniscule resolution provided by the automatized tool allowed the creation of complex micropatterns with single or multiple printings steps. This tool was later upgraded and fitted with new controllers to create smaller patterns. An alternatively contact printing technique called polymer pen lithography was used to pattern the surface of specialized substrates to create micropatterns on constricted areas. The miniaturized microarrays were later liberated to create functionalized microparticles. These microparticles can be tuned for many biochemical applications, such as protein interaction studies, drug discovery or life science. Lastly, a new contact replication method was established to fabricate DNA arrays. An initial DNA master arrays was fabricated with known contact printing techniques. Then, either hybridized or in situ synthesized strands were transported to an intermediate substrate. A second hybridization or synthesis was used to transport a replica of the master array to a new substrate, maintaining the chemical and spatial information present on the original array

Internalization and viability studies of suspended nanowire silicon chips in HeLa Cells

Micrometer-sized silicon chips have been demonstrated to be cell-internalizable, offering the possibility of introducing in cells even smaller nanoelements for intracellular applications. On the other hand, silicon nanowires on extracellular devices have been widely studied as biosensors or drug delivery systems. Here, we propose the integration of silicon nanowires on cell-internalizable chips in order to combine the functional features of both approaches for advanced intracellular applications. As an initial fundamental study, the cellular uptake in HeLa cells of silicon 3 m 3 m nanowire-based chips with two different morphologies was investigated, and the results were compared with those of non-nanostructured silicon chips. Chip internalization without affecting cell viability was achieved in all cases; however, important cell behavior differences were observed. In particular, the first stage of cell internalization was favored by silicon nanowire interfaces with respect to bulk silicon. In addition, chips were found inside membrane vesicles, and some nanowires seemed to penetrate the cytosol, which opens the door to the development of silicon nanowire chips as future intracellular sensors and drug delivery systems

Multiple-level su-8 µtas chip transfer onto coverslips for biological applications

Trabajo presentado en MicroTAS 2021, celebrado en modalidad virtual del 10 al 14 de octubre de 2021.In this work, we present a chip-on-a-coverslip consisting of customized multiple-layer SU-8 structures transferred onto standard glass coverslips for their direct application in confocal microscopy. This approach overcomes inherent challenges with photolithography over small substrates. We demonstrate their functionality with bespoke durotaxis chips to observe the interaction between cells and buried mechanical cues

Método de obtención de un array de micropartículas planares con multiplexado molecular superficial, array obtenido y su uso

The invention relates to the controlled production of an array of planar microparticles with the multiplexing of molecules on the surface thereof, intended to function as molecular sensors and/or actuators. The invention relates to a matrix (array) of microparticles, the surface thereof being printed with all of the molecular components required to provide same with functionality. This product can be produced thanks to the design of a process in which different molecular elements are multiplexed on the surface of each particle while they are supported on a substrate by means of a structural foot engraved below same. These microparticles can be released mechanically from the support where they are produced, by means of a controlled mechanical rupture method which is not chemically aggressive and therefore does not affect the molecules previously printed on the surface. The array and the particles contained therein offer great versatility in both chemical and/or biological applications.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de BarcelonaA1 Solicitud de patente con informe sobre el estado de la técnic

Method for producing an array of planar microparticles with surface molecular multiplexing, resulting array and use thereof

Método de obtención de un array de micropartículas planares con multiplexado molecular superficial, array obtenido y su uso. La invención se refiere a la fabricación controlada de un array de micropartículas planares con multiplexado de moléculas en su superficie, cuya misión es funcionar como sensores y/o actuadores moleculares. La presente invención propone una matriz (array) de micropartículas en cuya superficie están impresos todos los componentes moleculares necesarios para dotarlas de funcionalidad. Es posible fabricar este producto gracias al diseño de un proceso en el cual los diferentes elementos moleculares son multiplexados en la superficie de cada partícula mientras están soportadas sobre un sustrato gracias al grabado de un pie debajo de ellas. Estas micropartículas pueden ser liberadas mecánicamente del soporte donde se fabrican por un método mecánico de ruptura controlada, que es un método no agresivo químicamente y por tanto no afecta a las moléculas previamente impresas en su superficie. El array y las partículas que contiene presentan una gran versatilidad en aplicaciones tanto químicas y/o biológicas.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de BarcelonaA1 Solicitud de patente con informe sobre el estado de la técnic

Method for producing an array of planar microparticles with surface molecular multiplexing, resulting array and use thereof

[EN] The invention relates to the controlled production of an array of planar microparticles with the multiplexing of molecules on the surface thereof, intended to function as molecular sensors and/or actuators. The invention relates to a matrix (array) of microparticles, the surface thereof being printed with all of the molecular components required to provide same with functionality. This product can be produced thanks to the design of a process in which different molecular elements are multiplexed on the surface of each particle while they are supported on a substrate by means of a structural foot engraved below same. These microparticles can be released mechanically from the support where they are produced, by means of a controlled mechanical rupture method which is not chemically aggressive and therefore does not affect the molecules previously printed on the surface. The array and the particles contained therein offer great versatility in both chemical and/or biological applications.[ES] Método de obtención de un array de micropartículas planares con multiplexado molecular superficial, array obtenido y su uso. La invención se refiere a la fabricación controlada de un array de micropartículas planares con multiplexado de moléculas en su superficie, cuya misión es funcionar como sensores y/o actuadores moleculares. La presente invención propone una matriz (array) de micropartículas en cuya superficie están impresos todos los componentes moleculares necesarios para dotarlas de funcionalidad. Es posible fabricar este producto gracias al diseño de un proceso en el cual los diferentes elementos moleculares son multiplexados en la superficie de cada partícula mientras están soportadas sobre un sustrato gracias al grabado de un pie debajo de ellas. Estas micropartículas pueden ser liberadas mecánicamente del soporte donde se fabrican por un método mecánico de ruptura controlada, que es un método no agresivo químicamente y por tanto no afecta a las moléculas previamente impresas en su superficie. El array y las partículas que contiene presentan una gran versatilidad en aplicaciones tanto químicas y/o biológicas.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de BarcelonaB1 Patente sin examen previ

Método de obtención de un array de micropartículas planares con multiplexado molecular superficial, array obtenido y su uso

[EN] The invention relates to the controlled production of an array of planar microparticles with the multiplexing of molecules on the surface thereof, intended to function as molecular sensors and/or actuators. The invention relates to a matrix (array) of microparticles, the surface thereof being printed with all of the molecular components required to provide same with functionality. This product can be produced thanks to the design of a process in which different molecular elements are multiplexed on the surface of each particle while they are supported on a substrate by means of a structural foot engraved below same. These microparticles can be released mechanically from the support where they are produced, by means of a controlled mechanical rupture method which is not chemically aggressive and therefore does not affect the molecules previously printed on the surface. The array and the particles contained therein offer great versatility in both chemical and/or biological applications.[ES] La invención se refiere a la fabricación controlada de un array de micropartículas planares con multiplexado de moléculas en su superficie, cuya misión es funcionar como sensores y/o actuadores moleculares. La presente invención propone una matriz (array) de micropartículas en cuya superficie están impresos todos los componentes moleculares necesarios para dotarlas de funcionalidad. Es posible fabricar este producto gracias al diseño de un proceso en el cual los diferentes elementos moleculares son multiplexados en la superficie de cada partícula mientras están soportadas sobre un sustrato gracias al grabado de un pie debajo de ellas. Estas micropartículas pueden ser liberadas mecánicamente del soporte donde se fabrican por un método mecánico de ruptura controlada, que es un método no agresivo químicamente y por tanto no afecta a las moléculas previamente impresas en su superficie. El array y las partículas que contiene presentan una gran versatilidad en aplicaciones tanto químicas y/o biológicas.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de BarcelonaA1 Solicitud de patente con informe sobre el estado de la técnic

Método de obtención de un array de micropartículas planares con multiplexado molecular superficial, array obtenido y su uso

The invention relates to the controlled production of an array of planar microparticles with the multiplexing of molecules on the surface thereof, intended to function as molecular sensors and/or actuators. The invention relates to a matrix (array) of microparticles, the surface thereof being printed with all of the molecular components required to provide same with functionality. This product can be produced thanks to the design of a process in which different molecular elements are multiplexed on the surface of each particle while they are supported on a substrate by means of a structural foot engraved below same. These microparticles can be released mechanically from the support where they are produced, by means of a controlled mechanical rupture method which is not chemically aggressive and therefore does not affect the molecules previously printed on the surface. The array and the particles contained therein offer great versatility in both chemical and/or biological applications.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de BarcelonaA1 Solicitud de patente con informe sobre el estado de la técnic

Internalization and Viability Studies of Suspended Nanowire Silicon Chips in HeLa Cells

© 2020 by the authors.Micrometer-sized silicon chips have been demonstrated to be cell-internalizable, offering the possibility of introducing in cells even smaller nanoelements for intracellular applications. On the other hand, silicon nanowires on extracellular devices have been widely studied as biosensors or drug delivery systems. Here, we propose the integration of silicon nanowires on cell-internalizable chips in order to combine the functional features of both approaches for advanced intracellular applications. As an initial fundamental study, the cellular uptake in HeLa cells of silicon 3 µm × 3 µm nanowire-based chips with two different morphologies was investigated, and the results were compared with those of non-nanostructured silicon chips. Chip internalization without affecting cell viability was achieved in all cases; however, important cell behavior differences were observed. In particular, the first stage of cell internalization was favored by silicon nanowire interfaces with respect to bulk silicon. In addition, chips were found inside membrane vesicles, and some nanowires seemed to penetrate the cytosol, which opens the door to the development of silicon nanowire chips as future intracellular sensors and drug delivery systems.This research was financed by the Spanish government through projects MINAHE6 (TEC2017-85059-C3) with FEDER funding and partially by FORCEFORFUTURE (CSD2010-00024).Peer reviewe

Suspended Planar-Array Chips for Molecular Multiplexing at the Microscale

Suspended planar-array (SPA) chips embody millions of individual miniaturized arrays to work in extremely small volumes. Here, the basis of a robust methodology for the fabrication of SPA silicon chips with on-demand physical and chemical anisotropies is demonstrated. Specifically, physical traits are defined during the fabrication process with special focus on the aspect ratio, branching, faceting, and size gradient of the final chips. Additionally, the chemical attributes augment the functionality of the chips with the inclusion of complete coverage or patterns of selected biomolecules on the surface of the chips with contact printing techniques, offering an extremely high versatility, not only with the choice of the pattern shape and distribution but also in the choice of biomolecular inks to pattern. This approach increases the miniaturization of printed arrays in 3D structures by two orders of magnitude compared to those previously demonstrated. Finally, functional micrometric and sub-micrometric patterned features are demonstrated with an antibody binding assay with the recognition of the printed spots with labeled antibodies from solution. The selective addition of physical and chemical attributes on the suspended chips represents the basis for future biomedical assays performed within extremely small volumes