MG, MO'C and PC thank the Molecular Medicine Research Group at WSU for Research Seed Funding, as well as Michele Mason (WSU), Richard Wuhrer (Advanced Materials Characterisation Facility, AMCF, WSU) and Herv&#233; Cottet (Montpellier) for discussions.

Kitosan Filmlerin hazırlanması 1. <ol><li> ultra saf su ile, 100 ml tam buzlu asetik asit 2 g tartılır. </li><li> % 2 a / a, asetik asit sulu çözeltisi, 100 ml ekleyin, kitosan tozu üzerinden 1.7 g tartılır. ya da alüminyum folyo ile ya da karanlıkta kaplı oda sıcaklığında karıştırma çubuğu ve manyetik bir karıştırma levhası ile 5 gün için karıştırın. </li><li> 1 saat süre ile, 23 ° C&#39;de 1.076 x g&#39;de çitosan dispersiyon santrifüjleyin. bir şırınga ile süpernatant toplayın ve çökelti atın. </li><li> Her bir film, kısım, oda sıcaklığında, 9 cm plastik Petri kabı içine çitosan süspansiyonu 10 ml. en az 7 gün süreyle kurumaya örtülü filmleri bırakın. </li><li> Kullanarak makas 1 x 1 cm kare şeklinde kuru filmler kesti. Not: Deney bu aşamada durdurulmuş olabilir. </li></ol> Fosfat tamponlu tuz 2. Hazırlık (PBS) <ol><li> 8 gr sodyum klorür tartılır, 0.2 g potasyum klorür, 1.44 g disodyum hidrojen fosphate ve 0.24 g potasyum dihidrojen fosfat. </li><li> ultra saf su 800 ml bu tartıldı kimyasal çözülür ve pH 7.4 olacak şekilde derişik hidroklorik asitle titre. Not: Deney bu aşamada durdurulmuş olabilir. </li></ol> pH 9.2 75 mM sodyum borat tampon maddesi 3. hazırlanması <ol><li> borik asit 3,0915 g tartılır. ultra saf su 75 ml içinde çözülür. </li><li> 10 M veya daha yüksek bir konsantrasyonda bir sodyum hidroksit solüsyonu ile 9.2 arasında bir pH değerine, borik asit çözeltisi titre edilir. Dikkat: Konsantre sodyum hidroksit çözeltileri aşındırıcı ve eldiven ele alınmalıdır. </li><li> ultra-saf su ile komple çözelti 100 ml elde edildi. Bu pH 9.2 bir 500 mM sodyum borat tampon verir. </li><li> 75 mM sodyum borat tampon, aşırı saf su ile 500 mM sodyum borat tampon ile seyreltilir. Not: Deney bu aşamada durdurulmuş olabilir. </li></ol> Kitosan Fi 4. hazırlanmasıAşılama Reaksiyon için LMS <ol><li> Oda sıcaklığında bir Petri kabındaki 2 saat 5 ml PBS, 10 kare kitosan film (1 x 1 cm) yıkayın. </li><li> Bu süre zarfında, hazırlamak ve kapiller elektroforez cihazı (adım 5) doğrulamak. </li></ol> Kapiler Elektroforez Enstrüman 5. Hazırlanması ve Doğrulama <ol><li> 50 mikron iç çapa sahip bir 43.5 cm çıplak erimiş silis kılcal hazırlayın ile ayarlanan uzunluğunda kılcal polimer dış kaplama zayıflatarak (toplam uzunluk, algılama penceresine etkili uzunluğu 43.5 cm tipik 35 cm olduğunu) künt kap sonra kılcal yakalamaya. <ol><li> girişten 8.5 cm polimer kaplama yakmak için bir çakmak kullanarak kılcal bir pencere oluşturmak ve soğuduktan sonra etanol ile silerek temizleyin. bir çakmak ile birkaç milimetre için her iki ucunda kılcal kaplama yakmak ve soğur sonra etanol ile silerek temizleyin. </li><li> Yeri kılcal inside algılama penceresi giriş ve çıkış bölgeleri eşit boylarda yerleştirerek ve kaset iğ etrafında sararak kılcal kaset takın ve. Sonra kapiller elektroforez cihazında kaseti yükleyin. </li></ol></li><li> Her ayırma için yöntemin parametrelerini ayarlamak. Yazılım menüsünde &quot;Tüm yöntemini düzenlemek&quot; sonra &quot;yöntemi&quot; seçeneğini seçin. (Örneğin 25 ° C, 10 dakika, 30 kV) ayrılması için kullanılan sıcaklık, süre, voltaj ve küçük şişeler ayarlayın. <ol><li> (Su içinde) 1 M sodyum hidroksit (su içinde), 0.1 M sodyum hidroksit, aşırı saf su ile 5 dakika 75 mM sodyum borat tampon maddesi ile 5 dakika ile 5 dakika ile 10 dakika: Ön şartlandırma bölümünde, arka arkaya basmaları ayarlamak bir dizi analiz ilk yöntem için, pH 9.2 de. </li><li> (Su içinde) 1 M sodyum hidroksit, pH 9 75 mM sodyum borat tampon maddesi ile 5 dakika ile 1 dakika: takip eden yöntemler için, önceden ayarlanmış şartlandırma bölümünde üst üste basması ayarlayın.2. </li><li> Enjeksiyon bölümünde, tüm yöntemler için 10 saniye boyunca 30 mbar basınç hidrodinamik enjeksiyon için parametrelerini ayarlamak. Ayırma bölümünde, tüm yöntemler için 9 dakika boyunca, 25 ° C&#39;de 30 kV&#39;a ayırma koşullarını ayarlamak. NOT: üreticileri arasında değişebilir CE cihazı çalıştırmak için prosedür olarak özel CE cihazının kullanım kılavuzuna başvurun. günde 1 M sodyum hidroksit solüsyonu hazırlayın. </li></ol></li><li> Enjekte edilir ve (75 mM sodyum borat tampon maddesi 450 ul seyreltilmiş, su içinde% 10 h / h dimetilsülfoksit 10 ul (DMSO)), nötr bir iç standart ayırın. Sonra enjekte ve aynı şekilde bir oligoacrylate standardı ayrı, kılcal geçerliliğini kontrol etmek (10 g ∙ L -1 de ultra saf suda çözünmüş Malzeme listesi bakınız). aşılama reaksiyonu başlatmak için hazır olana kadar burada dizisi Pause. </li></ol> Kitosan Film üzerine RGDS 6. Tırnak <ol><li> peptid tarttın (1 mg RGDS)ve bağlama maddeleri (3 mg EDC-HCl ve 2 mg NHS). </li><li> 2 saat PBS içinde çitosan filmi ıslatma başlamasından sonra peptit ve PBS 5 ml bağlama maddeleri çözülür. <ol><li> Bu çözeltinin 50 ul tablet alın. kısım için bir iç standart olarak nötral su içinde% 10 h / h DMSO 2 ul ekle. CE kısım analiz (adım 7). </li></ol></li><li> 5 ml PBS Petri kabı kitosan filmler durulanması için kullanılmıştır çıkarın. çitosan filmler içeren Petri kabı peptidi ve birleştirme ajanlarının 5 mi solüsyonu ilave. </li><li> parafın film Petri kabı kapak ve oda sıcaklığında bir orbital çalkalayıcı üzerine yerleştirin. set zamanlarda reaksiyon ortamı 50 ul alikotları alın. Not: CE toplam analiz süresi 15 dakika olan ve böylece bir alikosu her 15 dakikada bir (veya iki reaksiyonları paralel olarak izlenmektedir halinde her 30 dakika, vs.) alınabilir. <ol><li> Her al bir iç standart olarak nötral su içinde% 10 h / h DMSO 2 ul ekleiquot. NOT: Alikot en kısa sürede de alınır gibi CE analiz edilmelidir (adım 7). </li></ol></li><li> çalkalama ve bir alikot alımından 4 saat sonra, karıştırıcı Petri çanağı çıkarın. Petri kabı reaksiyon ortamı çıkarın. kitosan filmlerin durulama PBS 5 ml ekleyin. </li><li> , Petri kabı PBS çıkarın ultra saf su ile kitosan filmi durulayın ve onları gece boyunca kurumaya bırakın. ultra saf su çıkarın ve bir plastik Petri kabındaki -20 ° C&#39;de filmleri saklayın. </li></ol> Tırnak Reaksiyon 7. İzleme CE kullanma <ol><li> Enjekte edilir ve derhal bölüm 5.2 olarak analiz koşullar kullanılarak Petri kabı çıkarıldıktan sonra reaksiyon ortamının ayrı alikotları. </li><li> ayrılmaların tamamlandıktan sonra 10 dakika süre ile, aşırı saf su ile kılcal yıkayın. 10 dakika boyunca boş bir şişede (hava) ile bir flaş boyunca kurutun. NOT: Deney bu aşamada durdurulmuş olabilir. </li></ol> 8. Veri Trea<html> CE tment <ol><li> ayrılması sırasında akım ve (bu durumda DMSO) elektroozmotik hareketlilik marker göç zamanı hem oligoacrylate standart ayrılması için gözlenen benzeyen olduğunu kontrol ederek, her ayrılık geçerliliğini kontrol edin. NOT: varyasyon yaklaşık 50 uA ve 1.3 dakika göç zaman değerinin beklenen cari değeri kabul edilebilir% 10-15 kadar (daha yüksek bir tekrarlanabilirlik gerekiyorsa elektroforetik mobilite değerleri yerine göç kez kullanılmalıdır). </li><li> Her başarılı ayrılması için, sağ ihracat tıklayıp uygun bir sinyali seçmek, belirli bir veri kümesi seçerek kapiller elektroforez yazılımından ham veri ihracat. </li><li> CE tarafından kaydedilen ham veri dönüştürme (göç, zamanın bir fonksiyonu olarak, UV absorbans ile gösterilmiştir). Denklem 1 Aşağıdaki bir elektroforetik mobilite u içine X ekseni (göç zamanı t m) dönüştürmek: n 1 &quot;src =&quot; / files / ftp_upload / 54549 / 54549eq1.jpg &quot;/&gt; (1) L D detektöre uzunluğudur, L T kılcal toplam uzunluğu, V gerilim olduğu ve T EO nötr tür olan (bu durumda DMSO iç standart) 27 göç zamandır. 28: Denklem 2 Aşağıdaki elektroforetik hareketlilik W (μ) bir dağılımına ham veri (au absorbans) Y-ekseni dönüştürmek <img alt="denklem 2" src="/files/ftp_upload/54549/54549eq2.jpg" /> (2) </li></ol> Peptid-aşılı Film 18 9. Ek Karakterizasyonu <ol><li> 4 mm solid-state NMR rotor kendi etrafında haddelenmiş peptid-aşılı kitosan filmlerin, yerleştirin. filmler şişer ve rotor kapatmak için, fosfat-tamponlu tuzlu su ile rotor doldurun. Birkaç saat bekleyin. </li><li> 13 filmi analiz &lt;/ sup&gt; C NMR spektroskopisi 18. </li></ol>

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Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

Burada açıklanan protokol sadeliği bu ideal yaygın uygulamaya uygun hale getirir. Ancak, özellikle dikkat aşağıdaki kilit adımların ödenecek gerekmektedir. Uygun CE enstrüman hazırlama Gün kılcal ve aletin geçerliliğini kontrol etmek için bilinmeyen numunelerin ayrılması hemen önce bilinen bir standart (aynı zamanda da ayrılmaların dizisinin sonu) ayırmak önemlidir. Bu standart, bir oligoacrylate 27 veya göç...

Ücretsiz çözüm kılcal elektroforezi (CE) kendi yük-sürtünme oranı 1,2 dayalı çözümler bileşikleri ayıran bir tekniktir. Şarj-boyutlu oranı genellikle literatürde açıklanan, ancak bu sadeleştirme bu çalışmada polipeptitleri dahil olmak üzere, polielektrolitler için geçerli değildir, ve aynı zamanda küçük organik moleküller 3 için uygun değildir gördü. CE bir durağan fazı, bir arka plan elektrolit (genellikle bir tampon) yok diğer ayırma teknikleri farklıdır. Bu teknik, 5, fermantasyon Brews 6 sentetik polimerler 7, gıda numuneleri 8 ve sıkıcı numune hazırlık yapmadan pek çözünür peptidler 9 ve üzerine aşılama bitki lifleri gibi karmaşık matrisler 4 örneklerin geniş bir ürün yelpazesi analiz kabiliyeti güçlü olmasını sağlar saflaştırılması. Bu çözünme sorunları var kompleks polielektrolitler (ler için özellikle önemlidirUCH nedenle kitosan 10 ve gellan sakızı 11) olarak birleştirilmiş ya da çözelti içinde çökeltilmiş ana kadar başarıyla örnek, süzme olmadan, analiz edilmiştir. Dahası, kahvaltılık tahıllar şekerlerin analizi suda 8 çöktürülmüş kahvaltı gevreği örneklerinin parçacıkları ile örnekleri enjekte çıkıyor. Bu aynı zamanda da dallı polielektrolitler veya kopolimerler 12,13 analizi için de geçerlidir. Kapsamlı çalışmalar da, özellikle proteomik 14, doğal ya da sentetik peptidler, 15 ve protein ve peptid 16 mikroçip ayırmaların şiral ayrımı için proteinlerin analizi CE tekniklerinin geliştirilmesi tamamlanmıştır. Ayırma ve analiz kılcal gerçekleşecek bu yana, sadece küçük örnek hacimleri ve çözücüler kromatografi 5,6,17 dahil olmak üzere diğer ayırma tekniklerinden daha düşük çalışma maliyeti için CE sağlayan kullanılmaktadır. CE tarafından ayırma hızlı olduğundan, monito veriyorReaksiyon kinetiğinin halka. Bu iyileştirilmiş hücre yapışması için 18 kitosan filmleri üzerine peptidlerin aşılama durumunda gösterilmiştir. Kitosan kitin N -deacetylation türetilmiş bir polisakkarittir. Nedeniyle çitosan en biyouyumluluk 21, 18,20 19 biyo yapışkanlar ve hücre büyümesi substratlar Chitosan filmler gibi çeşitli biyomedikal uygulamalar için kullanılabilir. Fibronektin, kolajenler ve laminin gibi özel hücre dışı matris proteinlerine Hücre bağlanması, doğrudan hücre 22 hayatta kalma ile bağlantılıdır. Özellikle, farklı hücre tipleri çoğu zaman hayatta kalma ve uygun işlev için, farklı hücre dışı matris proteinlerine eki gerektirir. Kitosan filmlerin Hücre eki fibronektin 23 aşılama yoluyla geliştirilmiş olduğu gösterilmiştir; Bununla birlikte, terkip, saflaştırma ve bu büyük proteinlerin aşılama ekonomik açıdan uygun değildir. Alternatif olarak, küçük peptidlerin bir serisi havE, büyük hücre dışı matris proteinlerinin özelliklerini taklit etmek mümkün olduğu gösterilmiştir. Örneğin, bu gibi fibronektin mimetikleri RGD peptitler (arjinin - glisin - aspartik asit) ve RGDS (arjinin - glisin - aspartik asit - serin) kolaylaştırmak ve hücre eki 24 geliştirmek için kullanılmıştır. Kitosan filmlerin üzerine RGDS kovalent aşılama vivo 18 fibronektin takmak için bilinen hücreler için gelişmiş hücre eki sonuçlandı. Daha büyük protein ikamesi aynı işlevselliği önemli bir maliyet düşüşü sağlamaktadır sahip küçük peptitler ile fibronektin seviyor. Daha önce 18 yayınlanan Burada, kitosan için aşılama peptid yapıldı. Daha önce gösterildiği gibi, bu yaklaşım için RGDS karboksilik asit işlevselleştirilmesi bağlama maddeleri EDC-HCL (1-etil-3- (3-dimetilaminopropil) karbodiimid) ve NHS (N-hidroksisukinimid) kullanarak, basit ve etkili bir aşılama sağlar üzerine aşılanmışkitosan filmi. Bu aşılama yöntemi önemli avantajı, çitosan veya peptit herhangi bir değişiklik gerektirmez, ve gelecekteki hücre kültür uygulamaları 18,20 ile uyumluluğu arttırmak için, sulu bir ortam içinde yürütülmektedir. bağlama maddeleri ve peptit şarj edilebilir CE reaksiyon kinetiği analizi için uygun bir yöntemdir. Önemlisi, CE aracılığıyla reaksiyon kinetiği analizi aşılama reaksiyonu gerçek zamanlı izlenmesini sağlar ve böylece hem optimize ve aşılama derecesinin miktarını sağlar. Rutin gerekli olmasa da, CE analizi sonuçları kovalent aşılama göstermek için katı hal NMR (Nükleer Manyetik Rezonans) Spektroskopisi 25,26 kullanılarak kitosan filmler üzerine aşılama peptidin doğrudan ölçülmesi ile off-line doğrulanabilir filmin 18 üzerine peptid. Bununla birlikte, katı hal NMR spektroskopisi ile karşılaştırıldığında, gerçek zamanlı analizi ile teminCE gerçek zamanlı olarak peptit tüketimi ve reaksiyonun kinetiği değerlendirmek, böylece yeteneğinin ölçümü sağlar. Yukarıda belirtilen yöntem basit ve aşılama ölçüde dolaylı ölçümü ile kitosan filmlerin üzerine aşılama peptid gerçek zamanlı analiz sağlar. gösterilen yöntem, farklı kimyasal reaksiyonlar sürece reaktanlar ya da şarj edilebilir analiz edilecek ürünlerin gerçek zamanlı kantitatif değerlendirmesi uzatılabilir.

<table><tbody><tr><td>Water</td><td>Millipore</td><td></td><td>All water used in the experiment has to be of Milli-Q quality</td></tr><tr><td>Chitosan powder (medium molecular weight)</td><td>Sigma-Aldrich</td><td>448877</td><td>lot MKBH1108V was used. Significant batch-to-batch variations occur with natural products such as polysaccharides</td></tr><tr><td>Acetic acid - Unilab</td><td>Ajax Finechem</td><td>2-2.5L GL</td><td>laboratory reagent</td></tr><tr><td>Dimethylsulfoxide</td><td>Sigma-Aldrich</td><td>D4540</td><td>laboratory reagent, slightly hazardous to skin, hazardous if ingested</td></tr><tr><td>Sodium hydroxide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>221465&amp;nbsp;</td><td>laboratory reagent, corrosive&amp;nbsp;</td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl)carbodiimide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D80002</td><td>Irritant to skin&amp;nbsp;</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-hydroxysuccinimide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>130672</td><td>Irritant to skin</td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Ajax Finechem</td><td>466-500G</td><td>laboratory reagent</td></tr><tr><td>Potassium chloride - Univar</td><td>Ajax Finechem</td><td>384-500G</td><td>analytical reagent, slight skin irritant</td></tr><tr><td>Disodium hydrogen phosphate - Unilab</td><td>Ajax Finechem</td><td>1234-500G</td><td>laboratory reagent, slight skin irritant</td></tr><tr><td>Potassium dihydrogen phosphate - Univar</td><td>Ajax Finechem</td><td>4745-500G</td><td>analytical reagent, slight skin irritant</td></tr><tr><td>Oligoacrylate standard</td><td>custom made</td><td></td><td>See reference for synthetic protocol: Castignolles, P.; Gaborieau, M.; Hilder, E. F.; Sprong, E.; Ferguson, C. J.; Gilbert, R. G. &lt;em&gt;Macromol. Rapid Commun.&lt;/em&gt; 2006, &lt;strong&gt;27&lt;/strong&gt;, 42-46</td></tr><tr><td>Boric acid&amp;nbsp;</td><td>BDH AnalR, Merck Pty Ltd</td><td>10058</td><td>Corrosive</td></tr><tr><td>Hydrochloric acid - Unilab</td><td>Ajax Finechem</td><td>A1367-2.5L</td><td>laboratory reagent, corrosivie</td></tr><tr><td>Fused silica tubing</td><td>Polymicro (Molex)</td><td>TSP050375</td><td>Flexible fused silica capillary tubing with standard polyimide coating, 50 &amp;micro;m internal diameter, 363 &amp;micro;m outer diameter&amp;nbsp;</td></tr><tr><td>Agilent 7100 CE</td><td>Agilent Technologies</td><td>G7100CE</td><td>Capillary electrophoresis instrument</td></tr><tr><td>Orbital shaker&amp;nbsp;</td><td>IKA</td><td>KS260</td><td></td></tr><tr><td>Electronic balance</td><td>Mettler Toledo</td><td>MS204S</td><td></td></tr><tr><td>Milli-Q Synthesis&amp;nbsp;</td><td>Millipore</td><td>ZMQS5VF01</td><td>Ultrapure water filtration system</td></tr><tr><td>Parafilm&amp;nbsp;</td><td>Labtek</td><td>PM966</td><td>Parrafin wax</td></tr></tbody></table>

capillary electrophoresis to monitor peptide grafting onto chitosan films in real time

Özgür-çözelti kılcal elektroforezi (CE), bir elektrik alanının uygulanmasıyla çözeltisi analitlerin genellikle şarj bileşikleri ayırır. kromatografi gibi diğer analitik ayırma teknikleri ile karşılaştırıldığında, CE, sağlam, ucuz ve etkili bir şekilde (karmaşık, doğal matrisler ya da polimerik bir numune sayısı için) herhangi bir numune hazırlamasını gerektirir. CE hızlı ve ayrılan bileşikler için gözlenen sinyal çözeltisi içindeki miktarı ile doğru orantılı olarak, gerçek zamanlı olarak (örneğin, kimyasal reaksiyon kinetiği) karışımların evrimi takip etmek için kullanılabilir. Burada, CE verimliliği sonraki biyomedikal uygulamalar için kitosan filmlerin üzerine peptidlerin kovalent aşılama izlemek için gösterilmiştir. Kitosan antimikrobiyel ve biyouyumlu özellikleri böyle hücresi büyüme yüzeylerde gibi biyomedikal uygulamalarda çekici bir malzeme yapmak. Kovalent peptid RGDS (arjinin aşılama - glisin -aspartik asit - serin) kitosan filmlerin yüzeyine hücre eki geliştirmeyi amaçlamaktadır. Tarihsel olarak, kromatografi ve amino asit analizi aşılanmış peptidin miktarı doğrudan ölçümünü sağlamak için kullanılmaktadır. Ancak, CE tarafından sağlanan örnek hazırlama hızlı ayrılması ve yokluğu peptid aşılama sürecinin eşit doğru ama gerçek zamanlı izlenmesini sağlar. CE ayrılması ve reaksiyon karışımının farklı bileşenlerinin ölçülmesi mümkün: (non-aşılı) peptid ve kimyasal ilave maddeleri içerebilir. Bu şekilde, CE kullanımı alt uygulamaları için geliştirilmiş filmler ile sonuçlanır. kitosan filmler katı hal NMR (Nükleer Manyetik Rezonans) Spektroskopisi vasıtasıyla karakterize edildi. Bu teknik daha zaman alıcı ve gerçek zamanlı olarak uygulanabilir, ancak peptit direkt ölçümü verir ve bu nedenle CE tekniğini doğrular.

CE kitosan filmleri üzerine peptidler (ör RGDS) aşılanmasını takip için uygundur. Müsait bağlama maddeleri kitosan üzerine aşılanabilir (Şekil 1) peptidi aktive EDC-HCl ve NHS bulunmaktadır. CE reaksiyon ortamından farklı ilgi molekülleri ayırabilmektedir. elektroferogramdaki üzerinde doruklarına atamak için, saf RGDS, EDC-HCl ve NHS, çözünmüş enjekte ve ayrı ayrı ayrıldı. Pik belirlemesinden sonra, reaksiyon ortamı enjekte edilmi?...

Watch this Scientific Journal Video about Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time at JoVE.com

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Kapiler Elektroforez Real Time Kitosan Films üzerine Peptit Aşılama Monitör

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

capillary-electrophoresis-to-monitor-peptide-grafting-onto-chitosan

Research

JoVE Journal

Chemistry

9.0K Görüntüleme.  Western Sydney University. Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Video: Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Biyomarker Discovery için Mezogözenekli Silika İnce Filmler üzerine düşük molekül ağırlıklı Protein Zenginleştirme

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Biyomühendislik

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Biofabrication ile Bio-Elektronik Arayüz Bridging

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Yoğunluk Gradient Katmanlı Polimerizasyonu (DGMP): Multi-bölmesi oluşturma için A Novel Tekniği, Doku Mühendisliği için Özelleştirilebilir İskelesi

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

Floresan peptid Zymography tarafından proteaz aktivitesinin algılama

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Kanser Araştırmaları

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Birleştiren QD FRET Mikroakiskan Real-Time DNA Nanocomplex Self-Meclis Monitör

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Biyoloji

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Biyomoleküllerin Geçici olarak Kontrollü Release için Yüzey Polielektrolit Elyaf Kompozit Yapı iskeleleri

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Doku Mühendisliği Aljinat mikropartiküllerin ile Thermogelling Poli (N-izopropil) -graft-kondroitin Sülfat Kompozitlerin Sentezi

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Sistein Thiols rekombinant proteinler Vitro siteye özgü Glikozilasyon için hedefleme

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

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A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 &#176;C, -20 &#176;C, -80 &#176;C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

Crosslinking Ajanlar ve Diflunisal Salınım Çalışmaları olmadan Chitosan-Poly(vinil alkol) Hydrogels hazırlamak için Bir Freeze-Eritme Yöntemi

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Doku Mühendisliği Uygulamaları için Boyut Kontrollü ve Emülsiyonsuz Kitosan-Genipin Mikrojellerinin İmalatı

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

Kapiler Elektroforez Real Time Kitosan Films üzerine Peptit Aşılama Monitör

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