This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1. fotomáscara Fabrication <ol><li> Limpiar una oblea de 4 &quot;de vidrio con solución Piranha (H 2 SO 4 / H 2 O 2 en 2: 1 ratio) durante 20 min a 120 ° C por inmersión en un tanque de cuarzo. </li><li> Depositar una capa de cromo / oro (30 nm de Cr / 1 m de Au) capa sobre la oblea de vidrio usando un haz de electrones evaporador 7 (Figura 1A). <ol><li> Coloque las obleas en un evaporador de haz de electrones. Una vez que el vacío alcanza 5 x 10 -6 Torr, encienda la fuente de alta tensión (10 kV). Controlar el espesor del panel de control del monitor. </li><li> Pre-limpia cada material durante 30 segundos usando la pistola de haz de electrones, manteniendo el obturador &quot;OFF&quot; (con el fin de evitar la deposición sobre las obleas). </li></ol></li><li> Generar una capa de máscara fotoprotector Cr / Au de profunda grabado húmedo del vidrio. <ol><li> Aplicar un fotorresistente gruesa 2 micras por hilado 5 ml de la solución durante 30 segundos a 3.000 rpm usando un sistema recubridor de spin-pulverización.Precocción la fotoprotección en una placa caliente a 100 ° C durante 1,5 min. </li><li> Exponer y hornear dura la fotoprotección a 120 ° C durante 30 minutos en un plato caliente. Es crítico para generar una superficie hidrófoba y una fuerte adherencia de la resina fotosensible a la capa de metal. Patrón de la capa de Cr / Au usando Cr y Au reactivos de ataque a través de la máscara fotorresistente 8,9. </li></ol></li><li> Para la protección de la superficie de vidrio no-modelado, unir temporalmente la oblea de vidrio a una oblea de silicio maniquí. 9 <ol><li> Coloque la oblea de vidrio sobre una placa caliente a 110 ° C y derretir la cera en el lado opuesto de la oblea de vidrio (de tal manera que toda la superficie de la oblea se cubre con cera). </li><li> Coloque una oblea de silicio maniquí en contacto con la oblea de vidrio y presione para eliminar el exceso de cera. Con el fin de evitar el derramamiento de la cera, coloque un pañuelo de papel limpia habitación en la zona de cocción. </li></ol></li><li> Realizar ataque isotrópico de la lente utilizando ácido fluorhídrico optimizado (49% v / v)y ácido clorhídrico (37% v / v) solución (en una relación volumétrica de 10: 1). con un agitador magnético durante 8,5 min 10 Presencia del HCl es crítico en la consecución de una buena calidad de la superficie de las lentes generados. <ol><li> Asegúrese de que la velocidad de ataque es de 7 m / min; utilizando un volumen total de 200 ml de solución de ataque. Realizar el grabado en un recipiente de plástico y tomar las precauciones de seguridad para esta etapa de procesamiento. </li><li> Limpie la oblea en el (DI) agua desionizada por lavado y posterior secado a temperatura ambiente. </li></ol></li><li> Después de la terminación del proceso, separar la oblea de vidrio a partir de la oblea de silicio maniquí y calentar la cera usando una placa caliente a 100 ° C durante 15 seg. A medida que la cera se funde a esta temperatura, separar la oblea de vidrio a partir de la oblea de silicio maniquí. </li><li> Quitar la cera restante, la fotoprotección y los colgantes Cr / Au capas en los bordes de las lentes utilizando ultrasonidos durante 1 hr utilizando N-metil-2-pirrolidona como disolvente a 80 ° C en un tanque ultrasónico.</li><li> Crear un molde réplica de PDMS de las microlentes fabricados en las fotomáscaras 11. </li><li> Caracterizar las dimensiones fotomáscaras (longitud y anchura) y el molde microlentes PDMS (profundidad y el diámetro) réplicas utilizando un microscopio electrónico de barrido y microscopio estereoscópico respectivamente 12-14. </li></ol> 2. Ejes MN Fabrication <ol><li> Crear una cavidad de 2,5 cm x 0,9 cm utilizando los portaobjetos de vidrio montadas en ambos lados de un vaso. El número de los portaobjetos de vidrio apiladas en ambos lados determinará la altura de la cavidad conocida como espesor del espaciador (Figura 1B). </li><li> Asegure cada capa de la lámina de vidrio mediante la aplicación de una capa delgada de la solución de prepolímero que contiene poli (etilenglicol) diacrilato (PEGDA, MW = 258 Da) con 0,5% w / w 2-hidroxi-2-metil-propiofenona (HMP) en el portaobjetos de vidrio seguido de irradiación de la luz creado con la alta intensidad ultravioleta (UV) durante 2 segundos. </li><li> Coloque el Photomask (previamente fabricado) con las superficies de Cr / Au recubiertas que enfrenta el interior de la cavidad. Asegúrese de que los lados de las paredes de la cavidad no están ocultando las lentes incrustadas en la fotomáscara. </li><li> Llenar la cavidad con la solución de prepolímero hasta que la superficie revestida Cr / Au se encuentra en contacto con la solución sin burbujas visibles. </li><li> Irradiar la configuración con alta intensidad de luz UV de una intensidad deseada durante 1 segundo a una distancia de 3,5 cm desde la fuente de UV utilizando la estación de curado UV con una gama de filtros UV de 320-500 nm. Utilice un adaptador de colimación con la sonda de la luz UV. </li><li> Medir la intensidad de la luz UV se utiliza mediante un radiómetro. </li><li> Después de la exposición UV, retire la fotomáscara con el conjunto de los MNs. Vierta la solución de prepolímero exceso que no se polimeriza en el proceso de nuevo en su envase original para reutilizarlo. </li><li> Cuantificar la longitud y el diámetro de la punta el MNS utilizando un estereomicroscopio de acuerdo con las instrucciones del fabricante. </li></ol><pclass = &quot;jove_title&quot;&gt; 3. MN capa Copia de Fabricación <ol><li> Con unas pinzas, coloque el MNS (previamente fabricado) conectados a la fotomáscara en un pocillo de una placa de 24 pocillos como se muestra en la Figura 1C. </li><li> Añadir un volumen especificado (300 - 400 l) de la solución de prepolímero en el pozo hasta que las agujas están sumergidos a una altura deseada. Este volumen determina el espesor de la capa de soporte resultante. </li><li> Irradiar la instalación con luz de alta intensidad UV (15,1 W / cm 2), 10,5 cm de distancia de la fuente de UV con una duración de 1 seg. </li><li> Separar la capa de soporte de la matriz de MN de la fotomáscara usando una cuchilla afilada. </li><li> Cuantificar la longitud, el diámetro de la punta y la base de diámetro de los los MNs con la capa de respaldo utilizando un estereomicroscopio de acuerdo con las instrucciones del fabricante. </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preshaping+photoresist+for+refractive+microlens+fabrication.">Preshaping photoresist for refractive microlens fabrication.</a> P Soc Photo-Opt Ins. 1992, 275-282 (1993).</li><li>Friedman, G. B., Sandhu, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+Spherical+Aberration+of+a+Thin+Lens.">Longitudinal Spherical Aberration of a Thin Lens.</a> Am J Phys. 35, 628 (1967).</li><li>Xu, Q. A., Li, J., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collimated+the+laser+diode+beam+by+the+focus+lens.">Collimated the laser diode beam by the focus lens.</a> Semiconductor Lasers and Applications IV. 7844, (2010).</li><li>Lin, T. W., Chen, C. 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The authors declare no conflict of Interest.

El protocolo descrito anteriormente para la fabricación de la matriz de los MNs se ha presentado para la fabricación de la matriz de los MNs ~ 1 cm 2. Las matrices se pueden ampliar mediante la creación de una cavidad de tamaño grande y mediante el uso de una fotomáscara más grande. El aumento del tamaño de la cavidad se puede crear mediante el aumento de la anchura entre los espaciadores a cada lado. Aunque cada paso para fabricar las matrices MN en el protocolo era importante, los pasos más importan...

Administración transdérmica de fármacos ofrece un enfoque alternativo atractivo para la administración de fármacos, especialmente para las biomoléculas, que se administran casi exclusivamente por inyecciones hipodérmicas. Sin embargo, la piel, especialmente la capa superior (el estrato córneo), es una barrera formidable prevención de moléculas exógenas de entrar en el cuerpo humano. Recientemente, los dispositivos MN se han convertido en herramientas que permite entregar medicamentos a través de la piel. Los dispositivos MN crean poros temporales dentro de la capa córnea para permitir el paso de moléculas de fármaco para conseguir la actividad fisiológica deseada con un mejor cumplimiento y conveniencia 1 a 3 paciente. Varios métodos de fabricación se han adoptado para fabricar los MNs poliméricos 4. Sin embargo, por lo general implican procesos de pasos complicados y múltiples que requieren tiempos largos y / o altas temperaturas para fabricar matrices Mns. 4 Para simplificar el proceso de fabricación, un solo proceso libre de moho paso utilizandouna fotomáscara fue desarrollado recientemente 5,6. Sin embargo, con este método, inventado los MNs tenían puntas de las agujas romas, como ningún mecanismo estaba en su lugar de modificar la trayectoria ultravioleta (UV) que participan en la fotolitografía. En este estudio, se han propuesto microlentes incrustadas en la fotomáscara para definir la geometría de los MNs. El protocolo para fabricar máscaras que constan de microlentes incrustadas y posteriormente MN fabricación con puntas afiladas utilizando el fotomáscara se reportan.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td>
			<td>SIGMA&#160;</td>
			<td>475629-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-hydroxy-2-methyl-propiophenone (HMP)</td>
			<td>SIGMA&#160;</td>
			<td>405655-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine collagen type 1, FITC conjugate&#160;</td>
			<td>SIGMA&#160;</td>
			<td>C4361</td>
			<td></td>
		</tr>
		<tr>
			<td>UV curing station&#160;&#160;&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>OmniCure S200-XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating Adaptor&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>EXFO 810-00042</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ 1500 stereomicroscope&#160;</td>
			<td>Nikon, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dillon GL-500 digital force gauge&#160;</td>
			<td>Dillon, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A-1R confocal microscope&#160;</td>
			<td>Nikon, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
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			<td></td>
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		</tr>
	</tbody>
</table>

polymeric microneedle array fabrication by photolithography

Este manuscrito describe la fabricación de microagujas polimérico (MN) matrices mediante fotolitografía. Se trata de un proceso libre de moho sencilla utilizando una fotomáscara que consta de microlentes incrustadas. No se encontraron microlentes incrustadas para influir en la geometría MN (nitidez). MN matrices sólidas con diámetros de punta que oscilan entre 41,5 micras ± 8,4 micras y 71,6 micras ± 13,7 m, con dos longitudes diferentes (1336 m ± 193 micras y 957 micras ± 171 micras) se fabricó. Estas matrices MN pueden proporcionar aplicaciones potenciales en el suministro de agentes terapéuticos de bajo peso molecular y macromolecular través de la piel.

La geometría de los los MNs puede verse afectada de manera significativa por las características fotomáscaras y microlentes incrustadas. El grado de refracción afecta la ruta de transmisión de los rayos UV, que influyeron en la geometría MN (Figura 2). Cada microlente se encontró que tenía un diámetro de 350 micras, 130 micras un aplanados superficie convexa, y un 62,3 micras de profundidad (Figura 2B-D). Usando el teorema de Pitágoras, el radio de curvatura de la primera supe...

Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

Polymeric Microneedle Array Fabrication by Photolithography

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

Fabrication polimérico matriz de microagujas por fotolitografía

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

polymeric-microneedle-array-fabrication-by-photolithography

Research

JoVE Journal

Bioengineering

11.9K vistas.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

Video: Polymeric Microneedle Array Fabrication by Photolithography

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabricación de hidrogeles micropatterned para sistemas de cultivo neuronal usando fotolitografía dinámica de proyección Máscara

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Investigación

Bioingeniería

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Formulación de nanopartículas poliméricas mediante la técnica de dibloque Nanoprecipitation

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

Hollow Microneedle basado en sensores para la detección electroquímica multiplexado transdérmica

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

La evaluación de la administración de genes nanopartículas poliméricas por nanopartículas de análisis de seguimiento y de alto rendimiento Citometría de Flujo

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabricación Microfluídica De Fibras Poliméricas Y Biohíbridas Con Tamaño y Forma Prediseñados

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrana Fabrication por la bicapa lipídica (SALB) Método-Solvente asistida

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Fabricación superhidrófobas materiales poliméricos para aplicaciones biomédicas

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Múltiples pasos Fotolitografia variable Altura de múltiples capas con válvula dispositivos de microfluidos

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabricación de una Matriz microambiente celular Artificial multiplexados

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.