Yazarlar DFG destek için kabul etmek istiyorum.

1. Hydrophobization, manyetik nano tanecikleri <ol><li>hidrofobik manyetik parçacıklar sentezi için 0,85 g FeCl 3 hekzahidrat (3.14 mmol) ve 0.30 g FeCl 2 tetrahydrate (1,51 mmol) eklemek için 200 mL su/etanol çözüm (4:1 v/v).</li>
	<li>0,20 mL 1 H, 1 H, 2 H, 2 H-Perfluorooctyltriethoxysilane (PFOTES) bu karışıma ekleyin (5,23 mmol) manyetik karıştırıcı (500 rpm) tarafından dinç karıştırma ile. Manyetit parçacıklar ikincil oksidasyonunu önlemek için bir mantar yuvarlak alt kabı kullanarak bir asal gaz atmosferi (N 2) sentezinde gerçekleştirmek.</li>
	<li>Çözüm damla amonyum hidroksit Çözümle (1,5 M) akıllı bir pH 8 (pH pH metre tarafından belirlenen) için ayarlayın. Manyetik karıştırıcı kullanarak 24 h için çözüm karıştırın.</li>
	<li>Ayrı parçacıklar manyetik olarak çözümden bir bar şişeye koyarak mıknatıs (Neodim Mıknatıs küboid 40 x 20 x 10 mm, yapıştırıcı kuvvet 25 kg'lık). Şişeye altına yapıştırılmış mıknatıs tutarken solüsyonu dökün.</li>
	<li>Bir bar kullanırken parçacıklar üç kere su/etanol çözüm ile yıkayın 1.4 içinde açıklandığı gibi mıknatıs). 24 h (verim ca. 0.43 g) için 60 ° C'de parçacıklar kuru.</li>
	<li>Parçacıklar analiz etmek için kullanmak bir Taramalı elektron mikroskobu üretici göre &#39; s yönergeleri.</li>
</ol> 2. Microreactors imalatı <ol><li>biraz cam havaneli kullanarak kurutulmuş parçacıklar eziyet ve daha sonra tüm bunların doğrudan bir tartı tavada yerleştirin (46 × 46 × 8 mm, polistiren).</li>
	<li>Pipet 10 µL tepki çözüm (kompozisyon bölümünde tanımlandığı 5,1 olarak) tüm parçacıklar ve tartma hareket üzerine pan biraz yaklaşık 10 için dairesel bir şekilde s (parçacık kitle 10 µL microreactor için: ca. 3.2 x 10 -7 kg 33). Kalan parçacıklar (parçacıklar etrafında tepki çözüm kendi kendine topladın değil) daha da uygulamalar için oda sıcaklığında saklayın.</li>
	<li>Microreactor kişi açı ölçmek için 5 µL microreactor su ile inşa 2.2 içinde açıklandığı gibi), teflon filmde yer ve bir optik iletişim açı ölçüm cihazı üretici göre kişi açı çözümleme &#39; s yönergeleri.</li>
</ol> 3. bobin organlarının 3D baskı <ol><li>Tasarım 16 mm (bir odası), 10 mm çapında ve bir iç çapı ca. 4 mm yüksekliği ile çift bobin organları üretici göre CAD yazılımı kullanarak &#39; s yönergeleri.</li>
	<li>Yazdırma bir 3D printerlere harcama maddeler üreticiye göre coil vücutlarla &#39; polylactide filaman gibi malzemeler kullanarak s yönergeleri. Cesetleri 4.500 sargılar bir bilgisayar kontrollü Sarma makinesi kullanarak elde etmek için bir 0.08 mm bakır tel ile sarın.</li>
</ol> 4. Çalıştırma Platform imalatı bir elektrik panosu altında Peltier öğesi içeren bir matris (örneğin 3 x 3 matris) <ol><li>Yerleştir çift Kangal ve vida çift bobin üzerinde bir denetimi şerit kablo () üzerinden bağlanmak şekil 3).</li>
	<li>İstenen uygulamaya bağlı olarak, şekilde demir çekirdeğin (32 mm yükseklik, 4 mm çap) veya bir Neodim Mıknatıs (12,5 mm yükseklik, 4 mm çap, 1.035 kA m -1) daha güçlü bir manyetik alan kazanmak için bobin vücut ekleyin.</li>
	<li>Platform yer bir plaka, tercihen kuvars cam, bobin matristeki 1 mm maksimum yüksekliği ile bitirmek için.</li>
	<li>Bir microreactor platform veya çekilebilecek.</li>
	<li>Üst parçacıklar tepki eriyik--dan çekmek ve böylece bir microreactor açmak için bir bobin içinde Neodim Mıknatıs ile 4.1 içinde belirtilen denetim kullanarak etkinleştirebilirsiniz). Kapatmak için microreactor tekrar devre dışı sarmalı.</li>
	<li>Başlangıçta ca. 10 mm ayrı olan iki microreactors birleştirmek için 4.2 içinde açıklandığı gibi Neodim mıknatıslar kullanın). Ca. 25 s bir microreactor (microreactor ve mıknatıs yalan 12 mm arasında gerekli mesafe) açmak için ve diğeri aynı konuma platforma taşımak için rulolar aktive ederek gerekli bobin organlarında mıknatıs kaldırın.</li>
	<li>Reaksiyon soğumaya microreactor platform yüzeyi ve bobin sıcaklığı azaltmak için çözüm anahtarı 4.1 içinde açıklandığı gibi bobin matris altında konumlandırılmış Peltier öğesindeki).</li>
</ol> 5. Birleştirme Microreactors tarafından enzimatik reaksiyon <ol><li>erime horseradish peroksidaz 0,1 bir konsantrasyon, µg mL -1 potasyum fosfat tampon (0.1 M, pH 6,5). Belgili tanımlık substrate, 10-asetil-3,7-dihydroxyphenoxazine (dimethylsulfoxide (DMSO) 10 mM) potasyum fosfat arabelleğe (0.1 M, pH 6,5) 200 µM bir konsantrasyon ile oranında seyreltin.</li>
	Her iki microreactors 2.2 içinde açıklandığı gibi kurmak için bu çözümlerin <li>Kullanım 10 µL). İki microreactors ne demek istiyorsun manyetik kuvvetlerin birleştirme (Neodim silindir mıknatıs: 12,5 mm x 4 mm, 1,035 kA m -1) 4.6 içinde açıklandığı gibi) 25 ° C.</li>
	<li>Reaksiyon bir floresan sonda konumlandırma tarafından tespit (uyarma dalga boyu: 570 nm, emisyon dalga boyu: 585 nm) ca. birleştirme önce açılan bir microreactor üstünde 10 mm.</li>
</ol>

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Yazarlar ifşa gerek yok.

Mikrosıvısal teknolojilerin başarılı kullanımı için Biyoteknolojik sentezi ve analizleri gereksinimlerine karşılık gelen tepki hareket önemlidir. Burada sunulan çalıştırma platformu Manyetik kuvvet tarafından mikrosıvısal damlacıkları taşımayı mümkün kılan. Hareket serbestçe bir tepki platformu düzlemsel bir yüzeye iki boyutta bir manyetik superhydrophobic kabuk ile sıvı damla içine alarak gerçekleştirilebilir. Böylece önceden tanımlanmış mikrosıvısal kanalları ile karmaşık ak...

Mikro reaksiyonlar ile teknik uygulamaları önceden tanımlanmış microchannel fiş ağırlıklı olarak devam etmektedir. Bu sistemleri yaygın olarak kurulan ve kapsamlı bir şekilde literatürde açıklanan (Inter alia 1,2,3). 2011 yılında 6,2 milyar euro 4mikrosıvısal teknolojileri dünya çapında cirosunun buldu. Buna ek olarak, özgürce hareketli mikro reaktör bölmeleri kullanımı daha önce sadece muayene ve sınırlı bir ölçüde yayınlandı. Sulu mikro damlacıkları taşımak için en yaygın yöntem electrowetting 5' tir. Damla yüzeylerde hareket için diğer yöntemleri elektrik alanlar 6, Manyetik kuvvet 7 veya akustik çalıştırma 8üzerinde temel alır. Hacim oranı onların olumsuz yüzeyine nedeniyle, bu microreactor damlacık tabanlı sistemler güçlü buharlaşma etkilere maruz kalır. Böylece, açılan hareket genellikle üst aşama buharlaşma sulu faz koruma yüksek kaynama noktasına sahip olduğu sıvı iki aşamalı bir sistem olarak kuruldu. Bununla birlikte, bu yaklaşım tepki damlacık tarafından kontrol edilmeyen Difüzyon kirletici yüksek risk içerir. Bu bahsedilen sistemlerinin teknik kurulması için önemli bir engeldir.
Son iş yapışık olmayan sıvı-katı faz geçişleri ile ilgilidir. Superhydrophobic yüzeyler, küresel sulu damlacıkları oluşumunu sağlayan kullanımı son derece etkili bir yaklaşımdır. Mikro tepki bölmeleri bir superhydrophobic yüzey veya kabuk, politetrafloroetilin (PTFE) parçacıklar 9örneğin oluşabilir kullanımı bu reaksiyon kavramı bir uzantısıdır. Kişi kendi açıları yüzeylerde genellikle (bağlı olarak yüzey pürüzlülüğü) 160 ° arasındadır. Küresel bölmeleri böylece bir yüzey üzerinde hareket en az direnç sağlamak ve aynı anda su buharlaşma karşı koruma sağlar.
Mikro ölçekli PTFE parçacıkları ile kaplı sulu damla çapı yaklaşık 2 mm kadar küresel kendi şeklini korumak. Daha yüksek birimleri hidrofobik kabuk genellikle tamamen artık 10kapalı değil. Diğer kabuk malzeme etkisi ve nonpolar çözücüler sıvı mermer uygulama alanının genişlemesi Gao ve McCarthy tarafından iyonik sıvılar 12kullanarak uygulanmıştır. Hidrofobik parçacık dayanarak kabuklarını oluşumu için ana kadar açıklanan 11,14,16parçacık çapı 10 nm-30 µm boyutlarda oldu. Yeni çalışmalar kabuk malzeme olarak hidrofobik nano tanecikleri microparticles 13bundan daha iyi kullanım olduğunu gösterdi. İlk istikrar çalışmalar doğruladı bir artış istikrar üzerinden parçacık boyutu küçüldüğünde ca. 600 nm ca. 100 nm için. Bu olası sonuçları sulu küre 15çevresinde yoğun parçacık dağılımından.
Sulu tepki bölmeleri sıvı mermerler olarak kendi tayin ve hidrofobik kabuğu korunması ilk Aussillous vd ve Mahadevan vd. 2001 yılında tanımlanmıştır 17 , 18. o zamandan beri bu tanımlanmış tepki bölmeleri birkaç uygulama tarif edilmistir. Örneğin, sıvı mermerler 19 ve tabanlı bir optik nitel olarak su kirliliği için bir algılama yöntemi dayalı bir gaz sensörü gelişmiş 20olmuştur. Yazarlar yüksek tepki oranları avantajları ve kimyasallar mikro tepki sistemlerinin düşük tüketim farkı. Son yayınlar pH duyarlı sıvı mermerler 16 üretimi veya 'Janus parçacıklar' temsil farklı işlevler iki farklı kaplamalar ile uğraşmak. Örneğin, Bormashenko ve ark. Teflon ve yarıiletken karbon siyah 21kabukları ile bir microreactor sentez. Ayrıca microreactors verimli olabilir gösterilmiştir ve uygun olarak comonomer den geçirgen gaz-sıvı arayüzey 24dış oksijen emerek polyperoxides sentez. Başka bir yaklaşım sıvı mermerler silis-parçacık tabanlı kabuk klasik gümüş ayna tepki 26düzenleyen reaktif substrat yüzeyler sağlar. Araştırma ve geliştirme alanında hidrofilik-core-hidrofobik-kabuk damlacıkları için geçerli sorunları parçacık boyutu ayarlama, monodisperse damlacıkları tekrarlanabilir üretimi, yüzeylerin wettability ve ikinci bir etkisi vardır hidrofilik kabuk damlacık yörüngeler, örneğin sürekli microPCR-sistemleri 4geliştirilmesi için bir daha iyi kontrol yanı sıra mikro tepki bölmeleri 22.
Bu microreactors manyetik bir çalıştırma ve gücü iyi bir seçicilik ve nispeten yüksek hareket aralıkları yararlanmak biyokimyasal sistemlerinde çalışırken sunmaktadır. Hidrofobik manyetit parçacıkları kullanırken, hem Manyetik kuvvet iletim microreactors hareketine işlevinin yanı sıra hidrofobik bir kabuk işlevi yerine getirmek. Lehmann ve ark. tarafından 2006 yılında ilk kez bir damlacık manyetik partikülleri ile damlacıkları manyetik hareket öne 23 ve Shikida vd. Kim el ile kullanılan 25, Kalıcı mıknatıslar aktüatörler tek bir damlacık seferberlik için alındı. Küçük bir miktar sıvı taşımak için başka bir yaklaşım hidrofobik Fe3O4 parçacıklar manyetik kabuk olarak kullanılan Zhao vd.tarafından gerçekleştirildi. Manyetik sıvı mermer kabuk damla üst tarafında dikey ters manyetik alan 27tarafından açıldı. Bu kavram üzerinde bağlı olarak, Xue vd. yüzey gerilimi 20,1 dyne cm– 1 28ile microreactor oluşturan parçacıklar geliştirmek mümkün. Lin ve ark. manyetik sıvı damlacık ulaşım ve manipülasyon 31için Tanrı istikrar sağlayan roman selüloz tabanlı Mikro/nano hiyerarşik küreler hem superparamagnetism hem de superhydrophobicity ile imal edilmiştir. Bir kanıtı-of-ilke eğitim ve herhangi bir uygulama için kullanılacak değil gibi bu defa sadece yayımlanmıştır. Sıvı mermerler manyetik ve elektrik kontrollerinin şu anda ilk yaklaşımlar takip etti. Zhao ve ark. 2010 15 ve Zhang vd. 2012 29 Daimi Mıknatıs çekirdeği-kabuk damlacıkları altında manuel (elle işletilen) hareketi tarafından bir damlacık işleme geliştirmek başardık. Bormashenko vd. 11 ferromanyetik sıvı mermeri 25 cm s-1 hız hızlanma Neodim Mıknatıs yaklaşan tarafından elde. Çalışmalar sadece küçük bir Daimi Mıknatıs manuel hareketi tarafından yürütülen yukarıda belirtilen ilke. Bir sonraki gelişim adım olarak, Zhao ve ark. son zamanlarda manyetik sıvı mermer hareketi için gerekli manyetik akı yoğunluğu Daimi Mıknatıs 30mesafesini değiştirerek tahmin edebilirsiniz. Ortak laboratuvar-on-a-chip sistemleri karşılaştırılabilir bir reaksiyon denetim için ayrı sıvı v otomatik kontrol anlamına gelir sağlamak için kaçınılmaz görünüyorolumes. Bu ihtiyacı karşılamak için sabitleştirmek, hareket ve manyetik microreactors açmak için değişken alan degradeler dayalı yeni bir kontrol sistemi geliştirdi.

<table border="0" cellpadding="0" cellspacing="0" width="903">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">3D-printer</td>
			<td style="width:163px;">FelixPrinters</td>
			<td style="width:211px;">Pro1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red)</td>
			<td>Life Technologies</td>
			<td>A12222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium hydroxide</td>
			<td style="width:163px;">TU-KL</td>
			<td>1072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CAD software</td>
			<td style="width:163px;">Siemens</td>
			<td style="width:211px;">Soled edge</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Contact angle measuring device</td>
			<td style="width:163px;">Dataphysics</td>
			<td style="width:211px;">OCA 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Cylinder magnet</td>
			<td style="width:163px;">Webcraft GmbH</td>
			<td style="width:211px;">S-04-13-N</td>
			<td>https://www.supermagnete.de/stabmagnete-neodym-rund/stabmagnet-durchmesser-4mm-hoehe-12.5mm-neodym-n42-vernickelt_S-04-13-N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dipotassium phosphate</td>
			<td>Bernd Kraft</td>
			<td>7758-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drying oven</td>
			<td>Binder</td>
			<td>FD 115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>68-17-5</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">FeCl2 tetrahydrate</td>
			<td style="width:163px;">TU-KL</td>
			<td>1625</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">FeCl3 hexahydrate</td>
			<td style="width:163px;">TU-KL</td>
			<td>1622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence probe</td>
			<td style="width:163px;">PerkinElmer</td>
			<td style="width:211px;">LS 55</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horseradish peroxidase</td>
			<td>Carl Roth</td>
			<td>9003-99-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen peroxide</td>
			<td>Th.Geyer GmbH &#38; Co</td>
			<td>7722-84-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monopotassium phosphate</td>
			<td>Bernd Kraft</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peltier element</td>
			<td style="width:163px;">Conrad&#160;</td>
			<td style="width:211px;">193569</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfluoroctyltriethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>51851-37-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanning Electron Microscope</td>
			<td style="width:163px;">FEI</td>
			<td style="width:211px;">Helios NanoLab 650 DualBeam</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Separation bar magnet</td>
			<td style="width:163px;">Webcraft GmbH</td>
			<td style="width:211px;">Q-40-20-10-N,&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Winding machine</td>
			<td style="width:163px;">IWT GmbH</td>
			<td style="width:211px;">FW122</td>
			<td></td>
		</tr>
	</tbody>
</table>

aqueous droplets used as enzymatic microreactors and their electromagnetic actuation

PCR ve Elektroforez, gibi mikrosıvısal tepki sistemleri başarılı uygulanması için küçük sıvı birimleri hareketi esastır. Geleneksel laboratuvar-Tarih-a-chip-platformlarında, çözücüler ve örnekleri karmaşık akış denetimi yüklemelerinin ile tanımlanmış mikrosıvısal kanallar aracılığıyla geçirilir. Burada sunulan damlacık çalıştırma platformu gelecek vaat eden bir alternatiftir. Bununla beraber, sıvı damla (microreactor) bir tepki platformu (laboratuvar-içinde-a-damla) düzlemsel bir yüzeye taşımak mümkündür. Microreactors platformu hidrofobik yüzey üzerinde çalıştırma manyetik kuvvetler superhydrophobic manyetit parçacıklar ince bir tabaka halinde yapılan Dış kabuğun sıvı damla üzerinde hareket kullanımı temel alır. Platformun hidrofobik yüzey sıvı çekirdek ve microreactor düzgün bir hareket sağlamak için yüzey arasında herhangi bir temas önlemek için gereklidir. Platformda, bir veya daha fazla microreactors 10 µL hacimleri ile konumlandırılmış ve aynı anda taşındı. Platformu kendisi Neodim veya demir çekirdeği yerleştirmek elektrik çift bobin ise ışını bir 3 x 3 matris oluşur. Manyetik alan degradeler otomatik olarak kontrol edilir. Manyetik alan degradeler varyasyon tarafından microreactors manyetik hidrofobik kabuk otomatik olarak microreactor taşımak veya kabuk geri dönülebilir olarak açmak için manipüle edilebilir. Yüzeylerde ve ilgili enzimler reaksiyonları microreactors birleştirilmesi veya temas yüzey immobilize katalizörler duruma getirmeden tarafından başlatılabilir.

Kabuk parçacıklar çapı yaklaşık 640 var nm. Bu fluorosilane kabuk parçacıklar içine magnetizable nano tanecikleri çapları arasında 22 Aralık var nm ve 37 nm. 5 µL microreactor bir sıvı çekirdek olarak su ile temas açısı yaklaşık 160 ° vardı.
Yukarıda açıklandığı gibi bir 10 µL microreactor var. 1,34 ± 0,08 µN taşımak için gerekli kuvvet şekil 1 gösterir bir bobi...

Watch this Scientific Journal Video about Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation at JoVE.com

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

Laboratuvar içinde bırak tepki sistemleri karmaşık tepkiler çok yönlü uygulanması bir mikrosıvısal ölçeğinde izin verir. Elektromanyetik bobinler bir 3 x 3 matris oluşan bir otomatik çalıştırma platformu geliştirilmiştir ve başarıyla birleştirme iki 10 µL microreactors için kullanılan ve böylece elde edilen sıvı mermer bir enzimatik reaksiyon başlatabilir.

Sulu damlacıkları enzimatik Microreactors ve onların elektromanyetik çalıştırma kullanılan

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

aqueous-droplets-used-as-enzymatic-microreactors-their

Research

JoVE Journal

Bioengineering

5.3K Görüntüleme.  University of Kaiserslautern. Laboratuvar içinde bırak tepki sistemleri karmaşık tepkiler çok yönlü uygulanması bir mikrosıvısal ölçeğinde izin verir. Elektromanyetik bobinler bir 3 x 3 matris oluşan bir otomatik çalıştırma platformu geliştirilmiştir ve başarıyla birleştirme iki 10 µL microreactors için kullanılan ve böylece elde edilen sıvı mermer bir enzimatik reaksiyon başlatabilir.

Video: Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Yüksek Sıralı Nanosheets Peptoid Polimerler ve Self-Meclis Solid-faz Submonomer Sentezi

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Biyomühendislik

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

Sulu Ortamlarda Hidrokarbon Dönüşüm Enable A Toolkit

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Sulu İki fazlı sistemler kullanma Hücre Co-kültür desenlendirme

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Gözenek Gradiyentler Polielektrolitler ve Bunların Dolaylı İzleme ile Metalik İmplant Çok Ölçekli Modifikasyon<em&gt; In vivo</em

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

Için Mikrometrik Sondalar uzaktan manyetik uyarılı<em&gt; In situ</emBakteriyel biyofilm Fiziksel Özelliklerinin&gt; 3D Mapping

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Hücresel Lipid damlacıklar İzolasyonu: Maya Hücreleri ve insan plasentası itibaren iki Arıtma Teknikleri

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Bir<em&gt; İn Vitro</em&gt; Enzimatik Deneyi Galyum (III) ve H Transkripsiyon İnhibisyonunu Tedbir<sub&gt; 3</sub&gt; 5,10,15-tris (pentaflüorofenil) corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

TiO kullanarak sulu bir ortamda Proteinler ve Hücreler photopatterning<sub&gt; 2</sub&gt; Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

Bir akış oranı mikroreaktör MS Algılama Proteinlerin hızlı enzimatik işlenmesi

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Sulu damlacıkları enzimatik Microreactors ve onların elektromanyetik çalıştırma kullanılan

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