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

1. ייצור Photomask <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> נקה רקיק זכוכית 4 &quot;עם פתרון פיראניה (H 2 SO 4 / H 2 O 2 ב 2: 1 יחס) במשך 20 דקות ב 120 מעלות צלזיוס על ידי טבילה בטנק קוורץ. </li><li style=";text-align:right;direction:rtl"> להפקיד רובד של כרום / זהב (30 ננומטר של Cr / 1 מיקרומטר של Au) שכבה על פרוסות סיליקון הזכוכית באמצעות מאייד 7 (איור 1 א) קורה אלקטרוני. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את הפרוסות במאייד קורה אלקטרוני. ברגע שהאבק מגיע 5 x 10 -6 Torr, להפעיל את מקור מתח הגבוה (10 קילו וולט). לשלוט בעובי של לוח בקרת צג. </li><li style=";text-align:right;direction:rtl"> טרום נקי כל חומר במשך 30 שניות באמצעות אקדח הקורה האלקטרוני, שמירה על התריס &quot;OFF&quot; (על מנת להימנע מהתצהיר על הוופלים). </li></ol></li><li style=";text-align:right;direction:rtl"> צור שכבת מיסוך photoresist Cr / Au לתחריט רטוב עמוק של הזכוכית. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> החל photoresist עבה 2 מיקרומטר ידי ספינינג 5 מיליליטר של הפתרון למשך 30 שניות בסל&quot;ד 3000 באמצעות מערכת coater ספין-ספריי.Prebake photoresist על פלטה חמה ב 100 מעלות צלזיוס במשך 1.5 דקות. </li><li style=";text-align:right;direction:rtl"> לחשוף וקשה לאפות photoresist ב 120 מעלות צלזיוס במשך 30 דקות על פלטה חשמלית. זה קריטי כדי ליצור משטח הידרופובי והדבקה חזקה של photoresist לשכבת המתכת. תבנית שכבת Cr / Au באמצעות etchants Cr וAu דרך photoresist להסוות 8,9. </li></ol></li><li style=";text-align:right;direction:rtl"> להגנה על משטח הזכוכית בדוגמת האו&quot;ם, באופן זמני אג&quot;ח רקיק הזכוכית לפרוסות סיליקון דמה. 9 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את פרוסות זכוכית על צלחת חמה ב 110 מעלות צלזיוס ולהמס את השעווה בצד השני של פרוסות סיליקון הזכוכית (באופן כזה שכל פני השטח של הרקיק מכוסה בשעווה). </li><li style=";text-align:right;direction:rtl"> מניחים פרוסות סיליקון דמה במגע עם רקיק הזכוכית ולחץ כדי להסיר את השעווה העודפת. על מנת להימנע משפיכת השעווה, למקם את נייר טישו חדר נקי על פלטה חשמלית. </li></ol></li><li style=";text-align:right;direction:rtl"> בצע תחריט איזוטרופיים של העדשה באמצעות חומצה הידרופלואורית מותאמת (49% V / V)פתרון וחומצה הידרוכלורית (37% V / V) (ביחס נפח של 10: 1). עם בוחש מגנטי עבור 8.5 דקות 10 נוכחות של HCl היא קריטית בהשגת איכות משטח טובה של עדשות שנוצרו. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא כי שיעור התחריט הוא 7 מיקרומטר / min; באמצעות נפח כולל של 200 מיליליטר של תמיסת תחריט. בצע תחריט במכל פלסטיק ולקחת אמצעי בטיחות לצעד זה עיבוד. </li><li style=";text-align:right;direction:rtl"> נקה את הרקיק במים ללא יונים (DI) על ידי שטיפה וייבוש נוסף ב RT. </li></ol></li><li style=";text-align:right;direction:rtl"> לאחר השלימה התהליך, להפריד את רקיק הזכוכית מפרוסות סיליקון דמה ולחמם את השעווה בעזרת צלחת חמה ב 100 מעלות צלזיוס במשך 15 שניות. כשעווה נמסה בטמפרטורה זו, לנתק את רקיק הזכוכית מפרוסות סיליקון דמה. </li><li style=";text-align:right;direction:rtl"> הסר שעווה שנותרה, photoresist ושכבות Cr / Au התלויים בקצוות של העדשות באמצעות ultrasonication עבור שעה 1 באמצעות -methyl-2-pyrrolidone N כממס על 80 מעלות צלזיוס בטנק קולי.</li><li style=";text-align:right;direction:rtl"> צור העתק עובש PDMS של microlenses המפוברק על photomasks 11. </li><li style=";text-align:right;direction:rtl"> לאפיין את ממדי photomask (אורך ורוחב) ועובש PDMS microlenses (עומק וקוטר) העתקים באמצעות מיקרוסקופ אלקטרונים סורק וסטראו בהתאמה. 12-14 </li></ol> ייצור 2. גלים MN <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור חלל של 2.5 סנטימטר × 0.9 סנטימטר באמצעות שקופיות הזכוכית רכובה על שני צדדים של זכוכית. מספר שקופיות הזכוכית שנערמו על שני הצדדים יקבע את הגובה של החלל הידוע כעובי spacer (איור 1). </li><li style=";text-align:right;direction:rtl"> Secure כל שכבה של שקופיות זכוכית על ידי יישום שכבה דקה של פתרון prepolymer המכיל פולי (אתילן גליקול) diacrylate (PEGDA, MW = 258 Da) עם 0.5% w / w 2-2-מתיל-propiophenone הידרוקסי (HMP) על גבי שקופיות זכוכית ואחרי הקרנה של האור להגדיר עם אולטרה סגול בעוצמה הגבוהה (UV) למשך 2 שניות. </li><li style=";text-align:right;direction:rtl"> מקם את photoma<html> SK (מפוברק בעבר) עם משטחי Cr / Au המצופה מול הפנים של החלל. ודא שהצדדים של קירות החלל לא מסתירים את העדשות משובצות בphotomask. </li><li style=";text-align:right;direction:rtl"> למלא את החלל עם פתרון prepolymer עד המשטח המצופה כרום / Au הוא במגע עם הפתרון ללא כל בועות גלויות. </li><li style=";text-align:right;direction:rtl"> מכשיר את ההתקנה עם אור UV בעוצמה גבוהה של אינטנסיביות רצויה עבור 1 שניות במרחק של 3.5 סנטימטרים ממקור UV באמצעות תחנת ריפוי UV עם מגוון מסנן UV של 320-500 ננומטר. השתמש במתאם collimating עם חללית אור UV. </li><li style=";text-align:right;direction:rtl"> מדוד את עוצמת אור UV משמשת באמצעות מד קרינה. </li><li style=";text-align:right;direction:rtl"> בעקבות חשיפה לקרינת UV, להסיר את photomask עם המערך של MNS. יוצקים את פתרון prepolymer העודף שאינו polymerized בתהליך חזרה למכל המקורי שלה לשימוש חוזר. </li><li style=";text-align:right;direction:rtl"> לכמת את קוטר אורך וקצה MNS באמצעות סטראו לפי הוראות יצרן. </li></ol>class = &quot;jove_title&quot;&gt; 3. ייצור שכבת גיבוי MN <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עם מלקחיים, למקם את MNS (מפוברק בעבר) מצורף אל photomask בבאר של צלחת 24 גם כפי שמוצג באיור 1 ג. </li><li style=";text-align:right;direction:rtl"> הוספת נפח שצוין (300-400 μl) של פתרון prepolymer לתוך הבאר עד המחטים שקועות עד לגובה רצוי. כרך זה קובע את עובי שכבת גיבוי התוצאה. </li><li style=";text-align:right;direction:rtl"> מכשיר את ההתקנה עם אור בעוצמה גבוהה UV (15.1 W / 2 סנטימטר), 10.5 סנטימטרים ממקור UV למשך תקופה של 1 שניות. </li><li style=";text-align:right;direction:rtl"> הפרד את שכבת הגיבוי על מערך MN מphotomask באמצעות סכין חד. </li><li style=";text-align:right;direction:rtl"> לכמת את קוטר אורך, קוטר קצה ובסיס של MNS עם שכבת הגיבוי באמצעות סטראו לפי הוראות יצרן. </li></ol>

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The authors declare no conflict of Interest.

הפרוטוקול שתואר לעיל עבור ייצור של מערך MNS הוצג לפברק מערך MNS של ~ 1 סנטימטר 2. ניתן לשנותם על ידי יצירת מערכי חלל גודל גדול ובאמצעות photomask גדול יותר. גודל החלל המוגבר ניתן ליצור על ידי הגדלת הרוחב בין מפרידי שני צדדים. למרות שכל צעד לפברק מערכי MN בפרוטוקול היה חשוב, ...

העברת תרופות דרך העור מציעה גישה חלופית אטרקטיבית למתן תרופה, במיוחד למולקולות ביולוגיות, המנוהלים באופן כמעט בלעדי על ידי זריקות מזרק. עם זאת, העור, במיוחד את השכבה העליונה (השכבה הקרנית), הוא מחסום אדיר מניעת מולקולות אקסוגניים מלהיכנס לגוף האדם. לאחרונה, מכשירי MN צמחו כמאפשרים כלים כדי לספק תרופות דרך עור. מכשירי MN ליצור נקבוביות זמני בתוך השכבה הקרנית לאפשר המעבר של מולקולות תרופה כדי להשיג את הפעילות הפיזיולוגית הרצויה עם תאימות מטופל השתפרה ונוחות 1-3. שיטות ייצור שונות אומצו לפברק MNS פולימריים 4. עם זאת, הם בדרך כלל כרוכים בתהליכים מסובכים ומרובים צעד דורשים פעמים רבות ו / או טמפרטורות גבוהות לפברק מערכי MNS. 4 כדי לפשט את תהליך הייצור, באמצעות תהליך ללא עובש צעד אחדphotomask פותח לאחרונה 5,6. עם זאת, בשיטה זו, מפוברק היה MNS טיפים מחט בוטים, כפי שאין מנגנון היה במקום לשנות את נתיב אור אולטרה סגול (UV) המעורב בphotolithography. במחקר זה, microlenses המוטבע בphotomask הוצעו להגדיר את הגיאומטריה של MNS. הפרוטוקול לפברק photomasks בהיקף של microlenses המשובץ ולאחר מכן MN ייצור עם טיפים חדים באמצעות photomask מדווחים.

<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

כתב יד זה מתאר את הייצור של מערכי microneedle פולימרים (MN) על ידי photolithography. זה כרוך בתהליך ללא עובש פשוט באמצעות photomask המורכב ממייקרו-עדשות מוטבעות. מיקרו-עדשות משובצות נמצאו להשפיע גיאומטריה MN (חדות). מערכי MN חזקים בקטרים ​​הנעים בין קצה 41.5 מיקרומטר ± 8.4 מיקרומטר ו71.6 מיקרומטר ± 13.7 מיקרומטר, עם שני אורכים שונים (1,336 מיקרומטר ± 193 מיקרומטר ומיקרומטר 957 ± 171 מיקרומטר) היו מפוברקים. מערכי MN אלה עשויים לספק יישומים פוטנציאליים באספקה ​​של תרופות מולקולריות וmacromolecular נמוכות דרך עור.

הגיאומטריה של MNS יכולה להיות מושפעת באופן משמעותי על ידי מאפייני photomask וmicrolens המוטבעים. התואר של שבירה משפיע על נתיב העברת קרני UV, שהשפיעה על הגיאומטריה MN (איור 2 א). כל microlens נמצא כי קוטר 350 מיקרומטר, 130 מיקרומטר שיטח משטח קמור, ו62.3 מיקרומטר עומק (איור 2-D).</strong...

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.

ייצור מערך microneedle פולימרית יתוגרפיה

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 Views.  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

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 ליתוגרפיה לייצור מיקרו submicron דפוסי פולימר על מצעים

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

Engineering

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.

המצאה של הידרוג micropatterned למערכות התרבות עצבית באמצעות photolithography דינמי הקרנה מסכת

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

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.

Microarrays פולימר לגילוי תפוקה גבוהה של Biomaterials

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

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.

חיישן הולו Microneedle מבוסס על מרובב דרך העור אלקטרוכימי חישה

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.

נימי חיל יתוגרפיה להנדסת רקמות לב

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

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

בצפיפות גבוהה DNA ו-RNA מיקרומערכים-פוטוליתוגרפיה סינתזה, היברידיזציה והכנת ספריות גרעין גדול חומצות

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Genetics

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

העצרת ומעקב אחר פיתוח הקהילה מיקרוביאלית בתוך פלטפורמת מערך Microell

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

ננוליתוגרפיה בדיקה סריקת שטח גדול בהנחייתם של יישור אוטומטי ויישומו המצע פבריקציה נוספת ללימודי תרבות תא

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

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.

ייצור של מערך מרובבת MicroEnvironment הסלולר מלאכותי

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.

ייצור של אלקטרודות בסדר על הקצה של מחט Hypodermic באמצעות תרסיס Photoresist ציפוי גמיש Photomask ביו יישומים

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 ...