Queremos agradecer el apoyo financiero de la concesión del NSF CMMI-1000724.

1A. Formación de una sola capa primaria en el silicio <ol><li> Corte de obleas de silicio en sustratos de 1 cm 2, el polvo y enjuague con agua filtrada y el etanol. </li><li> Eliminar la contaminación orgánica, sumergiendo los sustratos de silicio en un recipiente de vidrio que contienen nano tira a 75 º C. Después de 15 minutos, enjuague cada sustrato con agua desionizada y filtrada. </li><li> Lugar de cada sustrato en una solución al 5% de HF (Advertencia: HF es un material extremadamente peligroso) para eliminar la capa de óxido nativo. Después de 5 minutos en seco el óxido de silicio libre de nitrógeno </li><li> Para producir un sustrato de cloro, de inmediato sumergir cada pieza libre de óxido de silicio en un vial de centelleo que contiene 2 ml de grasa saturada PCl 5 en clorobenceno. Esta solución debe ser filtrado a 0,2 micras. </li><li> Montar un condensador vial en la parte superior de cada frasco y colocarlos en una heatblock conjunto a 112 ° C durante una hora. </li><li> Después de la reacción es completa, vamos a viales enfriar y enjuagar cada surface con clorobenceno y seco bajo nitrógeno filtrado. </li><li> Para formar un sustrato propenilo termina, el lugar de cada superficie de silicio tratada con cloro en un vial de presión que contiene 4 ml de cloruro de magnesio propenilo. Coloque el vial de presión en un heatblock a 130 ° C durante 24 horas. </li><li> Tome cada vial de presión de la heatblock y dejar enfriar. </li><li> Enjuague cada superficie rápidamente con DCM y el etanol y seco bajo nitrógeno filtrado. </li></ol> 1B. Formación de una sola capa primaria de germanio <ol><li> Corte de obleas de germanio en sustratos de 1cm2, el polvo y enjuague con agua filtrada y el etanol. </li><li> Eliminar la contaminación orgánica, sumergiendo la superficie en un recipiente de vidrio que contienen acetona durante 20 minutos </li><li> Coloque cada superficie en un 10% de solución de HCl durante 15 minutos. Este proceso al mismo tiempo elimina la capa de óxido nativo y clorados en la superficie. Después de 5 minutos en seco los sustratos con nitrógeno. </li><li> Para formar un sustrato octil-terminado, place cada clorados superficie de germanio en un vial de presión que contiene 4 ml de cloruro de magnesio octilo (2 mM). Coloque el vial de presión en un heatblock a 130 ° C durante 48 horas. </li><li> Tome cada vial de presión de la heatblock y deje enfriar a temperatura ambiente. </li><li> Enjuague cada superficie rápidamente con DCM y el etanol y seco bajo nitrógeno filtrado. </li></ol> 2. NHS funcionalización del sustrato en el silicio y el germanio <ol><li> Preparar un filtrado 0,1 M NHS-diazirine solución de tetracloruro de carbono. Advertencia: Mantenga la exposición a luz a un mínimo. </li><li> Pipeta unas gotas de la solución sobre la superficie de metilo terminado. Permita que la solución a extenderse por toda la superficie. </li><li> Coloque las superficies bajo una lámpara UV (☐ = 254 nm, 4400/cm2 a 0,74 pulgadas). Deje que las superficies de reaccionar a la luz ultravioleta durante 30 minutos, luego agregar más NHS-diazirine a la superficie y dejar que la reacción continúe durante otros 30 minutos. </li><li> Enjuague el NHS modificado surfaces con DCM y el etanol y seco bajo nitrógeno filtrado. </li></ol> 3. Funcionalización de moléculas pequeñas <ol><li> Reaccionar NHS modificado sustratos en 20 mM de terc-butilo carbamoil (Boc) etilendiamina solución en diclorometano (DCM) durante dos horas a temperatura ambiente. </li><li> Después de la reacción, lavar el sustrato Boc-modificado con DCM y el etanol. </li><li> Desproteger el sustrato Boc modificado con un 25% de ácido trifluoroacético (TFA) en DCM durante una hora a temperatura ambiente. </li><li> Enjuague la superficie resultante con DCM, el etanol y 10% (w / v) de bicarbonato de potasio en agua y seco bajo nitrógeno filtrado. </li><li> Analizar todas las superficies por XPS para determinar la composición elemental. </li></ol> 4. Ácidos de poliuretano acrilato Stamp (PUA) Preparación <ol><li> Diluir acrilato A un 30% con B triacrilato de trimetilolpropano etoxilado para reducir la viscosidad. Añadir fotoiniciadores C y D a la mezcla de reacción (Fig.Ure 6). </li><li> Añadir sodio 2-mercaptoethanesulfonate (0,2 g, 1,22 mmol) a una solución 4N HCl en dioxano (10 ml) y se agita a temperatura ambiente durante 2 minutos. </li><li> Filtrar el cloruro de sodio por un filtro de cristal y luego a través de una membrana de 0,2 μ m jeringa filtro PTFE para proporcionar una solución clara de 2-ácido mercaptoethanesulfonic en dioxano. </li><li> Se evaporan bajo presión reducida dioxano </li><li> Reaccionar el ácido sulfónico resultante con 2 ml de la mezcla de poliuretano-acrilato prepolymeric a temperatura ambiente y luego al vacío a 50 º C. Asegúrate de liberar por completo la mezcla de burbujas de aire atrapadas. </li><li> Enfriar la solución resultante a temperatura ambiente y se polimerizan entre dos portaobjetos de vidrio o una lámina de vidrio y un maestro por la exposición a los rayos UV durante 2 horas a temperatura ambiente. </li><li> Después de la polimerización, pelar cuidadosamente el sello de la maestra y lavar el sello con etanol y agua y seque con nitroge filtradan. </li></ol> 5. Impresión de catalizador y análisis SEM / AFM <ol><li> Lugar correspondiente de poliuretano-acrilato sello en la parte superior del sustrato NHS-modificado a temperatura ambiente durante un minuto sin carga externa para mantenerlos juntos. </li><li> Después de la reacción, por separado el sello y el sustrato. </li><li> Enjuague la superficie con etanol, agua, etanol y luego seca con nitrógeno filtrado. </li><li> Enjuague el sello con el etanol, agua, etanol y luego seca con nitrógeno filtrado. </li><li> Mantenga sellos a temperatura ambiente antes de la próxima aplicación. </li><li> Analizar el patrón de producción con el modo de contacto lateral microsopy de fuerza atómica (AFM) y microscopía electrónica de barrido (SEM) </li></ol> 6. Patrones de proteínas y microscopía de fluorescencia <ol><li> Sumerja el sustrato NHS-patrón bifuncional en lisina-N, N-diacético (20 mM) y Et3N (100 mM) en DMF: H20 (1:1) a temperatura ambiente durante 1 hora y luego enjuaga conagua y etanol. </li><li> Incubar los sustratos en 50 mM NiSO4 solución durante 5 minutos a temperatura ambiente. </li><li> Enjuague los sustratos quelados con agua abundante y tampón de unión (20 NaP mM, 250 mM NaCl, 10 mM imidazol, pH 7,5) y se sumergen en una solución de filtrado GFP (~ 40 μ M) durante 1 hora a 0 ° C. </li><li> Lavar inmediatamente los sustratos con tampón de unión seguida de PBS (pH 7,4). </li><li> Mantener hidratado sustratos en PBS a 0 º C hasta que estuvieran listos para el análisis de microscopía de fluorescencia. </li></ol> 7. Patrones de proteínas y microscopía de fluorescencia <ol><li> Sumerja el sustrato NHS-patrón bifuncional en lisina-N, N-diacético (20 mM) y Et3N (100 mM) en DMF: H 2 0 (1:1) a temperatura ambiente durante 1 hora y luego enjuaga con agua y el etanol. </li><li> Incubar los sustratos en 50 mM NiSO4 solución durante 5 minutos a temperatura ambiente. </li><li> Enjuague los sustratos quelados excesivamente wagua ITH y tampón de unión (20 NaP mM, 250 mM NaCl, 10 mM imidazol, pH 7,5) y se sumergen en una solución de filtrado GFP (~ 40 M) durante 1 hora a 0 ° C. </li><li> Lavar inmediatamente los sustratos con tampón de unión seguida de PBS (pH 7,4). </li><li> Mantener hidratado sustratos en PBS a 0 º C hasta que estuvieran listos para el análisis de microscopía de fluorescencia. </li></ol> 8. Los resultados representativos: Un ejemplo de soft-litográfica patrón nano catalizador se muestra en la Figura 7. El enfoque crea patrones en quimioselectiva libre de óxido de silicio y germanio, que puede ser ortogonalmente funcionalizados con diferentes productos químicos y restos biológicos. La reacción entre el sustrato NHS-functioanlized y el sello estampado catalizador conduce a la hidrólisis de los restos del SNS en las áreas de contacto de conformación, produciendo un patrón regiones bifuncional teniendo sustrato de NHS activa y libre de ácidos carboxílicos. Debido a la diffusla naturaleza de iones libres de nuestro método, conseguir una resolución similar a la de la fotolitografía. Por ejemplo, la figura 7 muestra las características 125 nm, que se reproducen de manera uniforme en la superficie de silicio sustrato. Sorprendentemente, el sello de catalizador puede ser reutilizado varias veces sin perder eficiencia. Funcionalización quimioselectiva de semiconductores con dibujos de biomoléculas abre la perspectiva de la integración de los materiales tradicionales de electrónica con sustratos biológicos altamente selectiva para aplicaciones en sensores, diagnósticos, y las áreas de análisis de la investigación. Un ejemplo de funcionalización como se muestra en la Figura 8, donde el NHS patrón de silicio se funcionalizados selectivamente con las moléculas de proteínas. Mediante la explotación de la reactividad diferencial de los ácidos carboxílicos activados y libre, en primer lugar fijado nitrilotriacético terminado ácido (NTA) enlazadores heterobifuncionales a las regiones NHS-funcionalizados, y luego se usa el resultadoNTA-con dibujos de superficie como una plantilla para la fijación selectiva de 8b hexa-histidina de etiquetado GFP. Figura muestra claramente la intensidad de fluorescencia diferencial entre GFP-modificado y se hidroliza regiones libres de ácido carboxílico. El tamaño y la forma de replicar las características son consistentes entre ambos NHS superficie modelada (Figura 8) y GFP-modificado la superficie (Figura 8b), lo que confirma la notable estabilidad de pasivado de carbono-las superficies y la selectividad del método de estampado. El protocolo no se limita a Su-etiquetados proteínas, y puede ser utilizado para otros biomoléculas como el ADN patrón y los anticuerpos. <img src="/files/ftp_upload/3478/3478fig1.jpg" alt="Figura 1"/> Figura 1. Esquema general que representa la impresión microcontacto catalítico <img src="/files/ftp_upload/3478/3478fig2.jpg" alt="Figura 2"/> Figura 2. Estructura de la bi-capa msistema olecular en Ge y el Si. Primaria monocapa alquilo formas estables Ge-C o C-Si los bonos con el sustrato y ofrece un sistema químicamente inerte y lleno de cierre que protege la superficie subyacente de la degradación. (B) sobrecapa secundaria forma enlaces estables CC con capa de protección primaria y proporciona terminales funcionales grupos <img src="/files/ftp_upload/3478/3478fig3.jpg" alt="Figura 3"/> Figura 3. Esquemas de reacción que representa la formación de monocapas de primaria de protección sobre el Si (A) y Ge (B) <img src="/files/ftp_upload/3478/3478fig4.jpg" alt="Figura 4"/> Figura 4. Funcionalización química de la monocapa de protección primaria con un donante carbeno heterobifuncionales <img src="/files/ftp_upload/3478/3478fig5.jpg" alt="Figura 5"/> Esquema de la figura 5. Reacción demuestra modificaciones de moléculas pequeñas de NHS-funcionalizados subsustratos y los correspondientes espectros de XPS <img src="/files/ftp_upload/3478/3478fig6.jpg" alt="Figura 6"/> Figura 6. Composición del catalizador pre-mezcla polimérica, condiciones de polimerización, y las imágenes de SEM de los patrones de ácido sulfónico modificada sello y el maestro correspondiente de PMMA-Si <img src="/files/ftp_upload/3478/3478fig7.jpg" alt="Figura 7"/> Figura 7. SEM y AFM imágenes de fricción de SAMs patrón de Si y Ge, con un sello de ácido <img src="/files/ftp_upload/3478/3478fig8.jpg" alt="Figura 8"/> Figura 8 Soft-litográfica patrones y funcionalización de silicio pasivado con moléculas orgánicas y biológicas a:.. La imagen SEM de la NHS patrón modificado con sustrato b:. Micrografía fluorescente GFP de sustrato modificado.

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No tenemos nada que revelar

El protocolo presentado es una forma o de impresión por microcontacto sin tinta que puede aplicarse universalmente a cualquier sustrato capaz de soportar simples y bien ordenado monocapas. En este método, un sello inmovilizada catalizador de transferencia de un patrón para una superficie de apoyo correspondientes grupos funcionales. Debido a que el proceso no se basa en la transferencia de la tinta de sello a la superficie de la limitación de resolución difusivo de μCP tradicionales y reactivos se obvia, lo que permite la fabricación de rutina de los objetos a nanoescala. La incorporación de una primaria muy ordenado sistema molecular proporciona una protección completa de los semiconductores subyacente de los daños de oxidación. Al mismo tiempo, el método es compatible con la inmovilización de los voluminosos grupos reactivos mediante la utilización de una sobrecapa reactiva secundaria, que en conjunto el sistema alcanza la protección y funcionalización. La técnica comienza con la formación de la superficie estable enlaces carbono-que permite químicamente inerte Primarmonocapa y que sirve como una barrera efectiva a la formación de óxido. Formación de una sobrecapa reactiva secundaria ofrece grupos terminales NHS funcionales que sirven como puntos de anclaje para una variedad de grupos químicos y biológicos. Esta estabilidad del sistema molecular de dos capas es posteriormente modelado utilizando nuestro enfoque μCP catalítico. El enfoque presentado en este estudio ofrece un método general para sustratos semiconductores patrón con una amplia gama de materiales orgánicos y biológicos. La capacidad para crear patrones orgánicos semiconductores interfaces sin instrumentación costoso, complejo ofrece numerosas oportunidades en campos como la electrónica, la nanotecnología, la bioquímica y la biofísica.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa / modelo </th></tr><tr><td> XPS espectrómetro </td><td> Kratos eje ultra </td></tr><tr><td> Microscopio de fuerza atómica </td><td> Veeco D3100 </td></tr><tr><td> SEM-FEG microscopio </td><td> FEI XL30 </td></tr><tr><td> Microscopio de fluorescencia </td><td> Zeiss Axio Imager </td></tr><tr><td> Heatblock </td><td> VWR </td></tr><tr><td> Bomba de vacío </td><td> Boc Edwards </td></tr><tr><td> Sistema de purificación de agua </td><td> Millipore </td></tr><tr><td> TESP sondas de silicio </td><td> Veeco </td></tr><tr><td> Silicio </td><td></td></tr><tr><td> Los viales de presión </td><td> Chemglass </td></tr><tr><td> Vacío en el colector </td><td> Chemglass </td></tr><tr><td> Lámpara UV </td><td> UVP </td></tr><tr><td> Material del sello </td><td> Véanse las referencias 20 y 18 </td></tr><tr><td> PFTE filtros de jeringa </td><td> VWR </td></tr><tr><td> Nano Franja </td><td> Cyantek </td></tr><tr><td> HCl </td><td> Sigma </td></tr><tr><td> Etanol </td><td> Sigma </td></tr><tr><td> Acetona </td><td> Sigma </td></tr><tr><td> HF </td><td> Sigma </td></tr><tr><td> Clorobenceno </td><td> Sigma </td></tr><tr><td> PCl5 </td><td> Sigma </td></tr><tr><td> Propenil Cloruro de Magnesio </td><td> Sigma </td></tr><tr><td> Cloruro de Magnesio octilo </td><td> Sigma </td></tr><tr><td> Tetracloruro de carbono </td><td> Sigma </td></tr><tr><td> Boc protegido etilendiamina </td><td> Sigma </td></tr><tr><td> TFA </td><td> Sigma </td></tr><tr><td> De sodio 2-mercaptoethanesulfonate </td><td> Sigma </td></tr><tr><td> 4N una solución de HCl en dioxano </td><td> Sigma </td></tr><tr><td> Lisina-N, N-diacético </td><td> Sigma </td></tr><tr><td> Et 3 N </td><td> Sigma </td></tr><tr><td> DMF </td><td> Sigma </td></tr><tr><td> NiSO4 </td><td> Sigma </td></tr><tr><td> NaP </td><td> Sigma </td></tr><tr><td> NaCl </td><td> Sigma </td></tr><tr><td> imidazol </td><td> Sigma </td></tr><tr><td> PBS </td&gt; <td> Sigma </td></tr></tbody></table>

soft lithographic functionalization and patterning oxide-free silicon and germanium

El desarrollo de híbridos de dispositivos electrónicos se basa en gran parte de la integración de la (bio) materiales orgánicos e inorgánicos semiconductores a través de una interfaz estable que permita el transporte de electrones eficiente y protege sustratos subyacentes de la degradación oxidativa. Grupo IV semiconductores pueden ser efectivamente protegido con muy ordenada monocapas auto-ensambladas (SAMs), compuesto por simples cadenas de alquilo que actúan como barreras impermeables a las soluciones orgánicas y acuosas. Simples SAM alquilo, sin embargo, son inertes y no susceptibles a las técnicas de modelado tradicionales. La motivación para la inmovilización de los sistemas orgánicos moleculares en los semiconductores es la transmisión de una nueva funcionalidad a la superficie que puede proporcionar una función óptica, electrónica y mecánica, así como la actividad química y biológica. Impresión por microcontacto (CP μ) es una técnica suave litográfica para SAMs patrones en las superficies múltiples. 1-9 A pesar de su simpinformación y publicidad y la versatilidad, el enfoque se ha limitado a las superficies de metal noble y no ha sido bien desarrollada para la transferencia de modelo a los sustratos tecnológicos importantes, como libre de óxido de silicio y el germanio. Además, dado que esta técnica se basa en la difusión de tinta para transferir patrones de elastómero de sustrato, la resolución de la impresión tradicional, se limita esencialmente a cerca de 1 μ m. 10.16 En contraste con la impresión tradicional, patrones sin tinta CP μ se basa en una reacción específica entre una superficie inmovilizada sustrato y un catalizador de marca de ruedas. Porque la técnica no se basa en la formación por difusión SAM, se amplía significativamente la diversidad de superficies patternable. Además, la técnica sin tinta evita las limitaciones de tamaño de la característica impuesta por la difusión molecular, lo que facilita la replicación de características muy pequeñas (&lt;200 nm). 17-23 Sin embargo, hasta ahora, sin tinta μ CP se ha utilizado principalmente para modelar sistemas moleculares relativamente desordenada, que no protegen las superficies subyacentes de la degradación. En este sentido, un informe simple, fiable y de alto rendimiento método para modelar pasivado de silicio y germanio con reactivos orgánicos monocapas y demostrar funcionalización selectiva de los sustratos con dibujos de ambas moléculas pequeñas y proteínas. La técnica utiliza un preformado NHS reactiva el sistema de dos capas de óxido libre de silicio y germanio. La fracción del NHS se hidroliza en forma de patrones específicos con un ácido sulfónico modificada acrilato sello para producir patrones químicamente distintas de NHS-activa y libre de ácidos carboxílicos. Una limitación significativa a la resolución de muchas de las técnicas μ CP es el uso de material de PDMS que carece de la rigidez mecánica necesaria para la transferencia de alta fidelidad. Para paliar esta limitación se utilizó un polímero de acrilato de poliuretano, un material relativamente rígido que se puedefácilmente funcionalizadas con diferentes restos orgánicos. Nuestro enfoque de modelado completamente protege tanto de silicio y germanio a partir de la oxidación química, proporciona un control preciso sobre la forma y el tamaño de las características de modelado, y le da acceso inmediato a los patrones químicos que pueden ser discriminados más funcionalizados con moléculas orgánicas y biológicas. El enfoque es general y aplicable a otras superficies tecnológicamente relevantes.

Watch this Scientific Journal Video about Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium at JoVE.com

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

A continuación se describe un método sencillo para modelar funcionalización libres de óxido de silicio y germanio con reactivos orgánicos monocapas y demostrar de los sustratos con dibujos de pequeñas moléculas y proteínas. El enfoque completamente protege las superficies de oxidación química, proporciona un control preciso sobre la morfología característica, y proporciona acceso rápido a los patrones discriminar químicamente.

Funcionalización suaves litográfica y Patrones libre de óxido de silicio y el germanio

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

soft-lithographic-functionalization-patterning-oxide-free-silicon

Research

JoVE Journal

Bioengineering

14.6K vistas.  Duke University. A continuación se describe un método sencillo para modelar funcionalización libres de óxido de silicio y germanio con reactivos orgánicos monocapas y demostrar de los sustratos con dibujos de pequeñas moléculas y proteínas. El enfoque completamente protege las superficies de oxidación química, proporciona un control preciso sobre la morfología característica, y proporciona acceso rápido a los patrones discriminar químicamente.

Video: Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

La litografía de plasma superficial Patrones de Creación de redes de telefonía

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Investigación

Bioingeniería

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

De alto rendimiento de síntesis de carbohidratos y funcionalización de nanopartículas polianhidrido

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

La visualización de la corteza organización y dinámica de los microorganismos, con total Microscopía de fluorescencia de reflexión interna

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formación luz mediada y el patrón de hidrogeles para aplicaciones de cultivo celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Un método versátil de Patterning proteínas y células

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Procedimiento suave litográfica para la producción de dispositivos microfluídicos plástico con puertos de vista transparentes a luz Visible e infrarroja

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Diseño de películas de silicio poroso como portadores de Factor de crecimiento nervioso

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Robot suave bioinspirado con microelectrodos incorporados

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Nanohilos de silicio y estimulación óptica para investigaciones de acoplamiento eléctrico intra e intercelular

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

Patrones simples, asequibles y modulares de células utilizando ADN

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...