Biz NSF ödül CMMI-1000724 mali destek kabul etmiş sayılırsınız.

1A. Silikon üzerinde birincil Monolayer Oluşumu <ol><li> 1cm 2 yüzeyler, toz silikon gofret kesin ve süzülmüş su ve etanol ile durulayın. </li><li> 75 º C&#39;de Nano şerit içeren bir cam tabak içinde silikon substratlar daldırarak organik kirlenme çıkarın 15 dakika sonra, her substrat deiyonize, filtre edilmiş su ile durulayın. </li><li> Yerli oksit tabakası kaldırmak için:% 5 HF çözüm (HF son derece tehlikeli bir malzeme Uyarı) her substrat yerleştirin. 5 dakika azot oksit içermeyen silikon kuru sonra </li><li> Batığın hemen 2 ml doymuş PCl 5 klorobenzen de içeren bir sintilasyon flakon her oksit içermeyen silikon parçası, bir klorlanmış substrat üretmek için. Bu çözüm, 0.2 mikron filtre edilmelidir. </li><li> Her flakon üstüne bir şişe kondenser birleştirin ve bir heatblock koyun 112 ° C &#39;de bir saat ayarlayın. </li><li> Reaksiyon tamamlandıktan sonra, şişeleri her yüzeyler tah serin ve durulamace, filtrelenmiş azot gazı altında klorobenzen ve kuru. </li><li> Propenyl-sonlandırır substrat formu için 4 ml propenyl magnezyum klorür içeren bir basınç flakon her klorlu silikon yüzeye yerleştirin. Bir heatblock her basınç flakon 130 ° C de 24 saat boyunca yerleştirin. </li><li> Her basınç flakon heatblock dışarı alın ve soğumaya bırakın. </li><li> DCM ve etanol ile hızlı bir şekilde her yüzey filtrelenmiş azot gazı altında durulayın ve kurulayın. </li></ol> 1B. Germanyum birincil Monolayer Oluşumu <ol><li> 1cm2 substratlar germanyum gofret Kes, toz ve süzülmüş su ve etanol ile durulayın. </li><li> 20 dakika boyunca bir cam tabak içeren aseton yüzeyler daldırarak organik pislikleri </li><li> 15 dakika boyunca% 10 HCl çözeltisi her yüzeye yerleştirin. Bu süreç, aynı zamanda yerli oksit tabakası ortadan kaldırır ve yüzey chlorinates. 5 dakika azot substratlar kuru sonra. </li><li> Oktil sonlandırılmış bir substrat, pla oluşturmak içince, 4 ml oktil magnezyum klorür (2 mM) içeren bir basınç flakon germanyum yüzey her klorlu. 130 ° C&#39;de 48 saat boyunca az bir heatblock her basınç flakon yerleştirin. </li><li> Her basınç flakon heatblock dışarı atın ve oda sıcaklığına kadar soğumasını bekleyin. </li><li> DCM ve etanol ile hızlı bir şekilde her yüzey filtrelenmiş azot gazı altında durulayın ve kurulayın. </li></ol> 2. Silisyum ve germanyum NHS Yüzey işlevsellik <ol><li> Karbon tetraklorür filtrelenmiş bir 0.1 M NHS diazirine çözüm hazırlayın. Uyarı: en az ışığa maruz tutun. </li><li> Metil sonlandırılır yüzeyler üzerine birkaç damla çözüm pipetleyin. Tüm yüzeye yayılmış çözüm izin verin. </li><li> (☐ = 254 nm, 0.74 inç 4400/cm2), bir UV lamba altında yüzeyler yerleştirin. Yüzeyler 30 dakika UV ışık altında tepki izin ver, sonra yüzeye daha NHS diazirine ekleyin ve ek bir 30 dakika sürmesine tepki izin. </li><li> NHS modifiye s durulayınDCM ve etanol ve filtrelenmiş azot gazı altında kuru urfaces. </li></ol> 3. Küçük Molekül işlevsellik <ol><li> React NHS değiştirilen 20 mM tert-butil carbamoyl oda sıcaklığında iki saatten diklorometan (DKMP) (Boc) etilendiamin çözüm substratlar. </li><li> Reaksiyon sonra, Boc modifiye substrat DCM ve etanol ile yıkayın. </li><li> Oda sıcaklığında bir saat DCM% 25 trifloroasetik asit (TFA) kullanarak Boc modifiye substrat korumanızı. </li><li> Filtrelenmiş azot gazı altında ortaya çıkan su ve kuru DCM ile yüzey, etanol ve% 10 (w / v) potasyum bikarbonat durulayın. </li><li> Elemental kompozisyonu belirlemek için XPS tüm yüzeyleri analiz edin. </li></ol> 4. Asidik Poliüretan Akrilat Damgası (PUA) Hazırlık <ol><li> Trimethylolpropane etoksilat triacrylate B viskoziteyi azaltmak için% 30 oranında bir akrilat sulandırınız. Reaksiyon karışımı (Şekil C ve D fotobaşlatıcı ekleure 6). </li><li> 4N HCl (10 ml) dioksan bir çözüm sodyum 2-mercaptoethanesulfonate (0.2 g, 1.22 mmol) ekleyin ve oda sıcaklığında 2 dakika karıştırın. </li><li> Ince bir cam filtre ile ve daha sonra dioksan 2-mercaptoethanesulfonic asit net bir çözüm göze filtresi 0.2 μ m PTFE membran şırınga ile ilk sodyum klorür Filtresi. </li><li> Azaltılmış basınç altında dioksan buharlaşmasına </li><li> Sonuç sülfonik asit tepki poliüretan akrilat prepolymeric karışımı oda sıcaklığında 2 ml ve daha sonra vakum altında 50 ° C Hava kabarcığı karışımı tamamen ücretsiz emin olun. </li><li> Çıkan çözüm oda sıcaklığına kadar soğutulur ve iki cam mikroskop lamı veya oda sıcaklığında 2 saat süreyle UV ışınlarına maruz kalma bir cam slayt ve bir ana arasında polimerize. </li><li> Polimerizasyon sonra, master off dikkatlice damga kabuğu ve damga etanol ve su ile yıkayın ve süzülmüş nitroge kurun. </li></ol> 5. Katalitik Baskı ve SEM / AFM Analiz <ol><li> Onları bir arada tutmak için herhangi bir harici yük ile bir dakika boyunca oda sıcaklığında NHS modifiye substrat üstüne poliüretan akrilat ilgili damga yerleştirin. </li><li> Reaksiyon sonra, damga ve yüzey ayırın. </li><li> Yüzey elde edilen etil alkol, su, ve sonra filtrelenmiş azot ile kuru etanol ile durulayın. </li><li> Etil alkol, su, ve sonra filtrelenmiş azot ile kuru etanol ile damga durulayın. </li><li> Sonraki uygulamadan önce oda sıcaklığında pul tutun. </li><li> Temas modunda lateral atomik kuvvet Mikroskopi (AFM) ve taramalı elektron mikroskobu (SEM) üretilen desen analiz </li></ol> 6. Protein Desenlendirme ve Floresan Mikroskopi <ol><li> Ile durulanır sonra H20 (1:1) 1 saat süreyle oda sıcaklığında ve Lizin-N, N-diacetic asit (20 mM) ve DMF Et 3 N (100 mM) Batmak NHS desenli bifonksiyonel substratsu ve etanol. </li><li> Oda sıcaklığında 5 dakika için 50 mM NiSO4 çözüm substratlar inkübe edin. </li><li> 0 1 saat için aşırı su filtreli GFP çözüm (~ 40 μ M) ve bağlayıcı tamponu (20 mM NaP, 250 mM NaCl, 10mM imidazol, pH 7.5) ve batığın ile şelatlı substratlar durulayın ° C </li><li> PBS (pH 7.4) takip bağlayıcı tampon substratlar hemen yıkayın. </li><li> 0 ile PBS içinde yüzeylerde sulu tutmak ° C floresan mikroskopi analizi için hazır olana kadar. </li></ol> 7. Protein Desenlendirme ve Floresan Mikroskopi <ol><li> Sonra su ile durulanır H 2 0 (1:1) 1 saat süreyle oda sıcaklığında ve Lizin-N, N-diacetic asit (20 mM) ve DMF Et 3 N (100 mM) Batmak NHS desenli bifonksiyonel substrat ve etanol. </li><li> Oda sıcaklığında 5 dakika için 50 mM Niso 4 çözümü substratlar inkübe edin. </li><li> Aşırı w şelatlı substratlar durulayın0 ile 1 saat için filtrelenmiş bir GFP çözümü i. su ve bağlayıcı tampon (20 mM NaP, 250 mM NaCl, 10mM imidazol, pH 7.5) ve batığın (~ 40 mcM) ° C </li><li> PBS (pH 7.4) takip bağlayıcı tampon substratlar hemen yıkayın. </li><li> 0 ile PBS içinde yüzeylerde sulu tutmak ° C floresan mikroskopi analizi için hazır olana kadar. </li></ol> 8. Temsilcisi Sonuçlar: Yumuşak litografik katalitik nano desenleme bir örnek Şekil 7&#39;de gösterilmiştir. Bu yaklaşım, dikey olarak birbirine benzemeyen, kimyasal ve biyolojik moieties Fonksiyonlu olabilir oksit-silisyum ve germanyum, Kemoselektif desenler oluşturur. NHS functioanlized substrat ve katalitik desenli damga arasındaki reaksiyon aktif bir NHS desenli bifonksiyonel substrat taşıyan bölgeler ve serbest karboksilik asitler alındı, konformal temas alanlarda NHS moieties hidroliz yol açar. Yaydıkları nedeniyleyöntemi iyon doğa, fotolitografi, yakın bir çözünürlük elde. Örneğin, Şekil 7, tüm silikon hızlandırıcı tabakanın yüzeyi boyunca eşit yeniden 125 nm özellikleri gösterir . Enteresandır ki, katalitik damga verimliliği kaybetmeden birden çok kez tekrar edilebilir. Biyomoleküllerin desenli yarı iletkenler Kemoselektif işlevsellik geleneksel elektronik malzemeler, algılama, tanı ve analitik araştırma alanlarında uygulamalar için son derece seçici biyolojik ürünler ile entegre umudu açılır. NHS desenli silikon seçici protein molekülleri ile Fonksiyonlu Şekil 8, böyle bir işlevsellik bir örnek gösterilmiştir. Aktive ve ücretsiz karboksilik asitler diferansiyel reaktifliğini istismar ederek, ilk NHS Fonksiyonlu bölgeler nitrilotriacetic asit-sonlandırılmış (NTA) heterobifunctional linkers yapıştırılmış ve sonra ortaya çıkanHexa-histidin etiketli GFP Şekil 8b seçici tutturulması için bir şablon olarak NTA desenli yüzey, GFP değiştirilmiş ve hidrolize ücretsiz karboksilik asit bölgeleri arasında ayırıcı floresan açıkça gösterir . Çoğaltılan özellikleri boyutu ve şekli, karbon pasifize yüzeyler olağanüstü istikrar ve damgalama yaklaşımı seçicilik teyit, NHS desenli yüzey (Şekil 8a) ve GFP modifiye yüzey (Şekil 8b) arasında tutarlı . Onun etiketli protein protokol sınırlı değildir ve DNA ve antikorlar da dahil olmak üzere diğer biyomoleküllerin desen kullanılabilir. <img src="/files/ftp_upload/3478/3478fig1.jpg" alt="Şekil 1"/> Katalitik microcontact baskı temsil Şekil 1 Genel şeması <img src="/files/ftp_upload/3478/3478fig2.jpg" alt="Şekil 2"/> Şekil 2 çift katmanlı m YapısıGe ve Si olecular sistemi. Birincil alkil tek tabaka yüzey ile istikrarlı Ge-C veya Si-C bağlar oluşturur ve temel yüzey bozulmasını koruyan bir kimyasal olarak inert ve yakın paketlenmiş sistemi sağlar. (B) İkincil overlayer birincil koruyucu bir tabaka ile istikrarlı CC bağı oluşturur ve fonksiyonel terminal sağlar gruplar <img src="/files/ftp_upload/3478/3478fig3.jpg" alt="Şekil 3"/> 3. Reaksiyon şemaları, Si (A) ve Ge birincil koruyucu mono tabakaları oluşumu temsil eden (B) Şekil <img src="/files/ftp_upload/3478/3478fig4.jpg" alt="Şekil 4"/> Şekil 4 heterobifunctional karben donör birincil koruyucu tek tabaka kimyasal işlevsellik <img src="/files/ftp_upload/3478/3478fig5.jpg" alt="Şekil 5"/> NHS Fonksiyonlu alt küçük molekül değişiklikleri gösteren Şekil 5. Tepkime planıyıllarda çok sayıda insan ve ilgili XPS spektrumları <img src="/files/ftp_upload/3478/3478fig6.jpg" alt="Şekil 6"/> Şekil 6 katalitik ön-polimer karışımı, polimerizasyon koşulları ve desenli sülfonik asit modifiye damga SEM görüntüleri Kompozisyon ve ilgili PMMA-Si master <img src="/files/ftp_upload/3478/3478fig7.jpg" alt="Şekil 7"/> Şekil 7 SEM ve AFM asidik bir damga ile Si ve Ge desenli SAMs sürtünme görüntüleri <img src="/files/ftp_upload/3478/3478fig8.jpg" alt="Şekil 8"/> Şekil 8, organik ve biyolojik molekülleri ile pasifize silikon Yumuşak litografik desenlendirme ve işlevsellik:. SEM görüntü desenli NHS değiştirilmiş substrat b: GFP modifiye substrat Floresan mikrografı .

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft-Lithographic+Approach+to+Functionalization+and+Nanopatterning+Oxide-Free+Silicon.">Soft-Lithographic Approach to Functionalization and Nanopatterning Oxide-Free Silicon.</a> Langmuir. 27 (10), 6478-6485 (2011).</li></ol>

Biz ifşa etmek başka bir şey var

Sunulan protokol evrensel basit sipariş mono tabakaları destekleme yeteneğine sahip herhangi bir yüzeye uygulanabilir bir form veya mürekkepsiz microcontact baskı. Bu yöntemde, damga immobilize katalizör, ilgili fonksiyonel grupları taşıyan bir yüzey bir model aktarır. Süreci, geleneksel ve reaktif μCP uzadıya çözünürlük sınırlaması yüzey damga mürekkep transferi temel almadığından, nano ölçekli nesneleri rutin üretim izin, ötelendi. Bir ilköğretim sipariş yüksek moleküler sistemi dahil, oksidasyon hasarı altta yatan yarı iletken tam koruma sağlar. Yöntem, aynı zamanda ikincil reaktif overlayer kullanan hantal reaktif gruplar immobilizasyon destekler; birlikte sistem hem koruma ve hem de işlevsellik elde. Bu teknik, kimyasal olarak inert Primar için izin kararlı karbon yüzey bağları oluşumu ile başlary tek tabakalı oksit oluşumuna etkin bir bariyer görevi görür. Ikincil reaktif overlayer oluşumu, kimyasal ve biyolojik moieties çeşitliliği için bağlantı noktaları olarak hizmet terminali NHS fonksiyonel gruplar sağlar. Bu istikrarlı bilayered moleküler sistemi, daha sonra katalitik μCP yaklaşım kullanarak desenli. Bu çalışmada sunulan yaklaşım, organik ve biyolojik malzemeler geniş bir yelpazesi ile desenlendirme yarı iletken yüzeyler için genel bir yöntem sunuyor. Pahalı, karmaşık enstrümantasyon olmadan desenli organik yarı iletken arayüzleri oluşturma yeteneği, elektronik, nanoteknoloji, biyokimya, biyofizik gibi alanlarda sayısız fırsatlar sunmaktadır.

<table border="1"><tbody><tr><th> Reaktifi Adı </th><th> Şirket / model </th></tr><tr><td> XPS spektrometresi </td><td> Kratos Eksen Ultra </td></tr><tr><td> Atomik kuvvet mikroskobu </td><td> Veeco D3100 </td></tr><tr><td> SEM-FEG mikroskop </td><td> FEI XL30 </td></tr><tr><td> Floresan mikroskop </td><td> Zeiss Axio Imager </td></tr><tr><td> Heatblock </td><td> VWR </td></tr><tr><td> Vakum pompası </td><td> Boc Edwards </td></tr><tr><td> Su arıtma sistemi </td><td> Millipore </td></tr><tr><td> Esp silikon sondalar </td><td> Veeco </td></tr><tr><td> Silikon </td><td></td></tr><tr><td> Basınç şişeler </td><td> Chemglass </td></tr><tr><td> Vakum manifoldu </td><td> Chemglass </td></tr><tr><td> UV Lambası </td><td> UVP </td></tr><tr><td> Malzeme Damgası </td><td> 20 ve 18 referans </td></tr><tr><td> PFTE şırınga filtreleri </td><td> VWR </td></tr><tr><td> Nano Strip </td><td> Cyantek </td></tr><tr><td> HCl </td><td> Sigma </td></tr><tr><td> Etanol </td><td> Sigma </td></tr><tr><td> Aseton </td><td> Sigma </td></tr><tr><td> HF </td><td> Sigma </td></tr><tr><td> Klorobenzen </td><td> Sigma </td></tr><tr><td> PCL5 </td><td> Sigma </td></tr><tr><td> Propenyl Magnezyum Klorür </td><td> Sigma </td></tr><tr><td> Oktil Magnezyum klorür </td><td> Sigma </td></tr><tr><td> Karbon tetraklorür </td><td> Sigma </td></tr><tr><td> Boc korumalı etilendiamin </td><td> Sigma </td></tr><tr><td> TFA </td><td> Sigma </td></tr><tr><td> Sodyum 2-mercaptoethanesulfonate </td><td> Sigma </td></tr><tr><td> Dioksan içinde 4N HCl çözeltisi </td><td> Sigma </td></tr><tr><td> Lizin-N, N-diacetic asit </td><td> Sigma </td></tr><tr><td> Et 3 N </td><td> Sigma </td></tr><tr><td> DMF </td><td> Sigma </td></tr><tr><td> Niso 4 </td><td> Sigma </td></tr><tr><td> NaP </td><td> Sigma </td></tr><tr><td> NaCl </td><td> Sigma </td></tr><tr><td> imidazol </td><td> Sigma </td></tr><tr><td> PBS </td&gt; <td> Sigma </td></tr></tbody></table>

soft lithographic functionalization and patterning oxide-free silicon and germanium

Hibrid elektronik cihazların geliştirilmesi, verimli elektron taşıma izin verir ve oksidatif bozulma temel substratlar korur istikrarlı bir arayüz üzerinden (biyo), organik maddeler ve inorganik yarı iletkenler entegrasyonu büyük ölçüde güvenir. Grup IV yarıiletkenler etkili bir şekilde, hem organik ve sulu çözümler vurdumduymaz engeller olarak hareket basit alkil zincirleri oluşur yüksek sipariş kendi kendine monte mono tabakaları (SAMs) ile korunabilir. Basit alkil SAMs Ancak, etkisiz ve geleneksel desenlendirme teknikleri için uygun değildir. Yarıiletkenlerin organik moleküler sistemlerin hareketsizleştirir için motivasyon yanı sıra kimyasal ve biyolojik aktivitesi, optik, elektronik ve mekanik fonksiyon sağlayabilir yüzeyine yeni işlevler vermek. Microcontact baskı (μ CP) sayısız yüzeyler üzerinde desenlendirme SAMs için litografik yumuşak bir tekniktir. 1-9, simp rağmenlicity ve çok yönlülüğü, bu yaklaşım büyük oranda soy metal yüzeylere sınırlı olmuştur ve oksit-silisyum ve germanyum gibi teknolojik önemli yüzeye desen transferi için geliştirilen olmamıştır. Ayrıca, bu tekniğin elastomer yüzeye desen transfer etmek için mürekkep difüzyon dayanmaktadır çünkü, gibi geleneksel baskı çözünürlüğü aslında 10-16 m. 1 μ yakın sınırlıdır. Geleneksel baskı aksine, mürekkepsiz μ CP desenlendirme bir yüzeye immobilize substrat ve damga bağlı katalizör arasında belirli bir reaksiyon dayanmaktadır. Tekniği uzadıya SAM oluşumu güvenmek değil, çünkü patternable yüzeylerin çeşitliliği önemli ölçüde genişletir. Buna ek olarak, çok küçük (&lt;200 nm) özellikleri çoğaltma kolaylaştırmak mürekkepsiz tekniği, moleküler difüzyon dayattığı özellik boyut sınırlamaları ortadan kaldırır. 17-23 Ancak, şimdiye kadar, mürekkepsiz μ CP bozulmadan altta yatan yüzeyleri korumak desenlendirme nispeten düzensiz bir moleküler sistemleri, ağırlıklı olarak kullanılmıştır. Burada, silisyum ve germanyum pasifize reaktif organik mono tabakaları ile desenlendirme için basit, güvenilir, yüksek verimli bir yöntem raporu ve küçük moleküller ve proteinlerin iki desenli yüzeylerin seçici işlevsellik göstermek. Teknik oksit-silisyum ve germanyum, önceden oluşturulmuş bir NHS-reaktif bilayered sistemi kullanır. NHS benzer parçaları NHS aktif ve ücretsiz karboksilik asitler kimyasal olarak farklı desenler üretmek için sülfonik asit modifiye akrilat damga ile desen belirli bir şekilde hidrolize olur. Birçok μ CP teknikleri çözümü için önemli bir sınırlama mekanik sertliği, yüksek sadakat transferi için gerekli yoksun PDMS malzeme kullanımı . Bu sınırlama hafifletmek için poliüretan akrilat polimer, nispeten sert bir malzeme kullanılmıştır.farklı organik moieties kolayca Fonksiyonlu. Bizim desenlendirme yaklaşım tamamen kimyasal oksidasyon hem silikon ve germanyum korur desenli özellikleri, şekli ve büyüklüğü üzerinde hassas kontrol sağlar, ve, daha organik ve biyolojik molekülleri ile Fonksiyonlu olabilir kimyasal ayrımcılık desen hazır erişim sağlar. Bu yaklaşım, genel ve teknolojik ilgili diğer yüzeylere uygulanabilir.

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

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

Burada, küçük moleküller ve proteinlerin desenli yüzeylerin desenlendirme oksit-silisyum ve germanyum ve reaktif organik mono tabakaları göstermek işlevsellik için basit bir yöntem açıklanmaktadır. Bu yaklaşım tamamen kimyasal oksidasyon yüzeyleri korur özelliği morfolojisi üzerinde hassas kontrol sağlar, ve kimyasal ayrımcılık desenleri hazır erişim sağlar.

Yumuşak Litografik işlevsellik ve Desenlendirme Oksit Silisyum ve germanyum

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

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

Research

JoVE Journal

Bioengineering

14.6K Görüntüleme.  Duke University. Burada, küçük moleküller ve proteinlerin desenli yüzeylerin desenlendirme oksit-silisyum ve germanyum ve reaktif organik mono tabakaları göstermek işlevsellik için basit bir yöntem açıklanmaktadır. Bu yaklaşım tamamen kimyasal oksidasyon yüzeyleri korur özelliği morfolojisi üzerinde hassas kontrol sağlar, ve kimyasal ayrımcılık desenleri hazır erişim sağlar.

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

Plasma Lithography Surface Patterning for Creation of Cell Networks

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

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

Plazma Litografi Yüzey Desenlendirme Hücre Ağları Oluşturma

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

Biyomühendislik

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

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

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

Polianhidrid Nanopartiküller Yüksek throughput Karbonhidrat sentezi ve işlevsel

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

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

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

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

Mikroorganizmalarda Cortex Organizasyon ve Dinamiği Görselleştirme, Toplam İç Yansıma Floresan Mikroskobu kullanılarak

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

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

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

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

Hücre Kültürü Uygulamaları için Hidrojeller Işık aracılı Oluşumu ve Desenlendirme

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

A Versatile Method of Patterning Proteins and Cells

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

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

Desenlendirme Proteinlerin ve Hücreleri Çok Yönlü Yöntemi

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

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

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

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

Görünüm-bağlantı noktaları görünür ve kızılötesi ışık şeffaf plastik mikrosıvısal cihazlar üretmek için yumuşak tekniğinde yordamı

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

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

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

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

Gözenekli silisyum filmleri sinir büyüme faktörü taşıyıcı olarak tasarlama

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

Bioinspired Soft Robot with Incorporated Microelectrodes

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

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

Anonim Mikroelektrotlu Bioinspired Yumuşak Robot

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

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

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

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

Silikon Nanowires ve Optik Stimülasyon İç-ve Hücrelerarası Elektrik Kaplin İncelemeler için

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

Simple, Affordable, and Modular Patterning of Cells using DNA

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

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

DNA Kullanarak Hücrelerin Basit, Uygun Fiyatlı ve Modüler Deseni

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