Name: polymeric microneedle array fabrication by photolithography
Uploaded: 2015-11-17T13:00:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

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

1. Fotomasken Fabrication <ol><li> Reinigen einer 4 &quot;Glaswafer mit Piranha-Lösung (H 2 SO 4 / H 2 O 2 in Verhältnis 2: 1) für 20 min bei 120 ° C durch Eintauchen in einen Quarzbehälter. </li><li> Hinterlegung einer Schicht aus Chrom / Gold (30 nm Cr / 1 &amp; mgr; m Au) -Schicht auf der Glasscheibe unter Verwendung eines E-Strahl-Verdampfer 7 (1A). <ol><li> Platzieren der Scheiben in einer e-Strahl-Verdampfer. Sobald das Vakuum erreicht 5 x 10 -6 Torr, schalten Sie die Hochspannungsquelle (10 kV). Steuern Sie die Dicke von dem Monitor Bedienfeld. </li><li> Vorreinigen jedes Material für 30 Sekunden mit der E-Strahlkanone, wobei die Verschluss &quot;OFF&quot; (in der Reihenfolge, um die Ablagerung auf den Wafern zu vermeiden). </li></ol></li><li> Generieren Sie eine Cr / Au Photoresist Maskierungsschicht für tiefen Nassätzen des Glases. <ol><li> Anwenden einer 2 um dicken Photoresist durch Schleudern 5 ml der Lösung für 30 sec bei 3.000 UpM unter Verwendung eines Spin-Sprühbeschichtungsvorrichtung Systems.Vorbacken des Photoresists auf einer Heizplatte bei 100 ° C für 1,5 min. </li><li> Expose und harte Backen des Photoresist bei 120 ° C für 30 min auf einer Heizplatte. Es ist kritisch, um eine hydrophobe Oberfläche und eine starke Haftung des Photoresists auf die Metallschicht zu erzeugen. Muster der Cr / Au-Schicht unter Verwendung von Cr und Au Ätzmittel durch die Fotolackmaske 8,9. </li></ol></li><li> Zum Schutz der un-gemusterten Glasoberfläche vorübergehend verbinden die Glasscheibe auf eine Dummy-Silizium-Wafer. 9 <ol><li> Platzieren der Glaswafer auf einer heißen Platte bei 110 ° C schmilzt das Wachs auf der gegenüberliegenden Seite der Glasscheibe (in der Weise, dass die gesamte Oberfläche des Wafers mit Wachs). </li><li> Legen Sie eine Dummy-Silizium-Wafer in Kontakt mit der Glasscheibe und drücken Sie, um das überschüssige Wachs zu entfernen. Um Auslaufen des Wachses zu vermeiden, legen Sie ein sauberes Zimmer Tissue-Papier auf der Heizplatte. </li></ol></li><li> Zuführen isotropes Ätzen der Linse mit optimierten Flusssäure (49% v / v)und Salzsäure (37% v / v) Lösung (in einem Volumenverhältnis von 10: 1). mit einem magnetischen Rührer für 8,5 min 10 Anwesenheit des HCl ist kritisch bei der Erzielung einer guten Oberflächenqualität der erzeugten Linsen. <ol><li> Stellen Sie sicher, dass die Ätzrate 7 &amp; mgr; m / min; mit einem Gesamtvolumen von 200 ml Ätzlösung. Führen Ätzen in einem Kunststoffbehälter und nehmen Sie Sicherheitsmaßnahme für diesen Verarbeitungsschritt. </li><li> Reinigen des Wafers in dem deionisiertem Wasser (DI) gespült und eine weitere Trocknung bei RT. </li></ol></li><li> Nachdem der Prozess abgeschlossen, trennen Sie die Glasscheibe von der Dummy-Silizium-Wafer und erwärmen Sie das Wachs unter Verwendung einer Heizplatte bei 100 ° C für 15 Sekunden. Wie das Wachs bei dieser Temperatur schmilzt, nehmen Sie den Glaswafer von dem Dummy-Silizium-Wafer. </li><li> Entfernen restlichen Wachs, das Photoresist und die überhängenden Cr / Au-Schichten an den Rändern der Linsen mit Ultraschall für 1 Stunde unter Verwendung von N-Methyl-2-pyrrolidon als Lösungsmittel bei 80 ° C in einem Ultraschalltank.</li><li> Erstellen Sie eine PDMS-Form Replik der Mikrolinsen auf den Photomasken 11 hergestellt. </li><li> Charakterisierung der Photomaske Abmessungen (Länge und Breite) und der Mikrolinsen PDMS-Form (Tiefe und Durchmesser) Nachbildungen mit einem Rasterelektronenmikroskop und Stereo jeweils 12-14. </li></ol> 2. MN Wellen Fabrication <ol><li> Erzeugen einen Hohlraum von 2,5 cm × 0,9 cm mit den Glasobjektträger auf beiden Seiten eines Glas montiert. Die Anzahl von Objektträgern auf jeder Seite gestapelt wird die Höhe des Hohlraums, wie Abstandshalterdicke (1B) bekannt bestimmen. </li><li> Sicherung jede Schicht des Glasobjektträgers durch Aufbringen einer dünnen Schicht der Präpolymerlösung, die Poly (ethylenglycol) diacrylat (PEGDA, Mw = 258 Da) mit 0,5% w / w 2-Hydroxy-2-methyl-propiophenon (HMP) auf die Glasobjektträger, gefolgt von Bestrahlung des mit dem hochintensiven UV-Set (UV) Licht für 2 sec. </li><li> Positionieren Sie den Photomask (zuvor hergestellt) mit dem Cr / Au beschichteten Oberflächen des Inneren des Hohlraums gegenüber. Sicherzustellen, dass die Seiten der Hohlwände sind nicht verdecken die Linsen in der Fotomaske eingebettet. </li><li> Füllen des Hohlraums mit der Präpolymerlösung bis die Cr / Au-beschichtete Oberfläche in Kontakt mit der Lösung ohne sichtbare Blasen. </li><li> Bestrahlen die Einrichtung mit hoher Intensität, UV-Licht einer gewünschten Intensität für 1 sec in einem Abstand von 3,5 cm von der UV-Quelle mit der Vernetzungsstation mit einem UV-Filter-Bereich von 320-500 nm. Verwenden Sie einen Kollimator-Adapter mit dem UV-Licht-Sonde. </li><li> Messen Sie die Intensität der UV-Licht unter Verwendung eines Radiometer verwendet. </li><li> Nach der UV-Bestrahlung, entfernen Sie die Fotomaske mit dem Array von MNs. Gießen Sie das überschüssige Präpolymerlösung, die nicht in den Prozess wieder in der Originalverpackung zur Wiederverwendung polymerisiert wird. </li><li> Quantifizierung der Länge und Durchmesser der Spitze von MnS mit einem Stereomikroskop nach den Anweisungen des Herstellers. </li></ol><pclass = &quot;jove_title&quot;&gt; 3. MN Rückschicht Fabrication <ol><li> Mit einer Pinzette, legen die MNs (vorher hergestellt) auf die Photomaske in eine Vertiefung einer Platte mit 24 Vertiefungen angebracht, wie in der 1C gezeigt. </li><li> Hinzufügen eines festgelegten Volumens (300 bis 400 &amp; mgr; l) der Prepolymer-Lösung in das Bohrloch, bis die Nadeln bis zu einer gewünschten Höhe eingetaucht ist. Dieses Volumen bestimmt die Dicke der resultierenden Trägerschicht. </li><li> Bestrahlen die Einrichtung mit hoher Intensität, UV-Licht (15,1 W / cm 2), 10,5 cm von der UV-Quelle für eine Zeitdauer von 1 sec. </li><li> Trennen Sie die Rückschicht auf der MN-Array von der Photomaske mit einer scharfen Klinge. </li><li> Quantifizierung der Durchmesser der MNs Länge, Durchmesser und die Basis mit der Rückschicht mit einem Stereomikroskop nach den Anweisungen des Herstellers. </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.

Die oben für die Herstellung des MNs Array beschriebenen Protokoll wurde vorgestellt, um die MNs Array von ~ 1 cm 2 herzustellen. Die Arrays können durch die Schaffung eines großen Hohlraums und durch die Verwendung eines größeren Photomaske skaliert werden. Die erhöhte Hohlraumgröße kann durch Erhöhung der Breite zwischen den Abstandshaltern auf beiden Seiten erzeugt werden. Wenn jeder Schritt die MN-Arrays im Protokoll herstellen wichtig war, waren die wichtigsten Schritte: die Fotomaske Positionie...

Transdermalen Verabreichung von Arzneimitteln bietet eine attraktive Alternative Ansatz für die Arzneimittelverabreichung, insbesondere für Biomoleküle, die fast ausschließlich von subkutanen Injektionen verabreicht werden. Jedoch Haut, insbesondere die Deckschicht (das Stratum corneum) ist eine hervorragende Barriere verhindert exogenen Molekülen von in den menschlichen Körper. Vor kurzem haben MN Geräte ermöglichen Tools, um Arzneimittel durch die Haut zu liefern entstanden. Die MN-Geräte zu erstellen temporäre Poren im Inneren der Hornschicht, damit der Durchgang von Wirkstoffmolekülen, um die gewünschte physiologische Aktivität mit verbesserte Patienten-Compliance und Convenience 1-3 zu erreichen. Es wurden verschiedene Herstellungsverfahren erlassen, um polymere MNs 4 herzustellen. Aber sie beinhalten in der Regel kompliziert und Mehrschrittverfahren für die langZeiten und / oder hohe Temperaturen, um MNs Arrays herzustellen. 4 Zur Vereinfachung des Herstellungsprozesses, einen einzigen Schritt schimmelfrei Prozess miteine Fotomaske wurde kürzlich 5,6 entwickelt. Doch mit diesem Verfahren hergestellt MNs hatten stumpfe Nadelspitzen, da kein Mechanismus vorhanden, um die ultravioletten (UV) Lichtpfad in der Photolithographie beteiligt zu ändern. In dieser Studie eingebetteten Mikrolinsen in der Fotomaske vorgeschlagen worden, um die Geometrie der MNs definieren. Das Protokoll zum Photomasken, bestehend aus eingebetteten Mikrolinsen und anschließend MN Fertigung mit scharfen Spitzen Verwendung der Fotomaske wird berichtet, zu fertigen.

<table><tbody><tr><td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td><td>SIGMA&amp;nbsp;</td><td>475629-500ML</td><td></td></tr><tr><td>2-hydroxy-2-methyl-propiophenone (HMP)</td><td>SIGMA&amp;nbsp;</td><td>405655-50ML</td><td></td></tr><tr><td>Bovine collagen type 1, FITC conjugate&amp;nbsp;</td><td>SIGMA&amp;nbsp;</td><td>C4361</td><td></td></tr><tr><td>UV curing station&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>OmniCure S2000-XL</td><td></td></tr><tr><td>Collimating Adaptor&amp;nbsp;</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&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr><tr><td>Dillon GL-500 digital force gauge&amp;nbsp;</td><td>Dillon, USA</td><td></td><td></td></tr><tr><td>A-1R confocal microscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr></tbody></table>

polymeric microneedle array fabrication by photolithography

Diese Handschrift beschreibt die Herstellung von polymeren Mikronadel (MN) Arrays durch Photolithographie. Es geht um eine einfache schimmelfrei Prozesses durch Verwendung einer Photomaske, bestehend aus eingebetteten Mikrolinsen. Eingebettete Mikrolinsen wurden gefunden, um MN-Geometrie (Schärfe) zu beeinflussen. Robusten MN Arrays mit Spitzendurchmessern im Bereich zwischen 41,5 &amp; mgr; m ± 8,4 &amp; mgr; m und 71,6 ± 13,7 &amp; mgr; m &amp; mgr; m, mit zwei verschiedenen Längen (1,336 &amp; mgr; m ± 193 &amp; mgr; m und 957 &amp; mgr; m ± 171 &amp; mgr; m) wurden hergestellt. Diese MN Arrays können potenzielle Anwendungen in der Lieferung von niedermolekularen und makromolekularen therapeutischen Wirkstoffen durch die Haut zu schaffen.

Die Geometrie der MNs können durch die Photomaske Eigenschaften und eingebettete Mikrolinsen erheblich beeinflusst werden. Der Brechungsgrad beeinflusst den Übertragungsweg der UV-Strahlen, die den MN-Geometrie (2A) beeinflusst. Jede Mikrolinse hatte einen Durchmesser von 350 um, 130 um eine abgeflachte konvexe Oberfläche und eine 62,3 &amp; mgr; m Tiefe (2B-D). Verwendung des Satz des Pythagoras, wurde der Krümmungsradius der ersten Fläche festgestellt, 272.89 um sein. Die Brennwe...

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.

Polymeren Mikronadelanordnung Fabrication durch Photolithographie

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

12.0K Aufrufe.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

Serie: Polymeric Microneedle Array Fabrication by Photolithography

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

Micropunching Lithographie zur Erzeugung von Mikro-und Submikron-Muster auf Polymersubstrate

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of ...

Forschung

Ingenieurwesen

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.

Herstellung von mikrostrukturierten Hydrogele für Neural Kultur-Systeme mit Dynamic Mask Projektion Photolithographie

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

Biotechnik

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Polymer Microarrays für High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

Vorgeschriebene 3-D direkte Schreiben von Hänge Micron / Sub-Mikrometer-Maßstab Faserstrukturen über eine Robotic Dosiersystem

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

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.

Hohle Mikronadel-basierten Sensor für Multiplex Transdermale elektrochemische Nachweis

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

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Kapillar-Force-Lithographie für Herz-Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Einfache Lithographie-freie Einzelzell-Mikromusterung mit Laser-Cut Schablonen

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Entwicklungsbiologie

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.

Herstellung eines Multiplex künstliche zellulären Mikroumgebung Arrays

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

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

Herstellung von feinen Elektroden an der Spitze der Injektionsnadel mit Fotolack Spritzen und Flexible Fotomaske für biomedizinische Anwendungen

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

Neurowissenschaften

Anwendungen des 3D-Drucks für mikroskalige neuronale Zellkulturgeräte

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Autor im Rampenlicht: Modulare neuronale Netze zur Analyse von Gehirnfunktionen

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Zwei-Photonen-Polymerisation 3D-Druck von neuronalen Zellkulturgeräten im Mikromaßstab

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...