Bu çalışma Gıda ve Tarım için USDA Ulusal Enstitüsü, Ulusal Rekabetçi Hibeler Programı, Ulusal Araştırma Girişimi Programı 71.1 FY 2007 tarafından kısmen finanse edilen Alerji ve Enfeksiyon Hastalıkları Hibe sayılı 2007-35503-18392 ve Ulusal Sağlık Enstitüleri, Enstitüsü gibi , R33AI94514-03.

1. İplik Dope Hazırlık <ol><li> Ve bu hesaplamalarda biopolimer tozun nem içeriği dikkate emin olun biopolimer konsantrasyonlarda bir dizi (w / v,% 30,% 0.1) araştırılmalıdır hazırlayın. Her bir konsantrasyon için, 50 ml&#39;lik bir test tüpüne biyopolimerin (nişastası veya pululan) toz tartın. Sulu dimetil sülfoksit (DMSO) çözeltisini ve bir karıştırma çubuğu ilave edin. </li><li> Bir manyetik karıştırıcı ocak üzerine sürekli karıştırılarak kaynar su içine tüp yerleştirin. </li><li> 1 saat sonra, ısı kapatın ve dispersiyon oda sıcaklığına soğumaya bırakın. Dispersiyon daha sonra, reolojik deney ve üretebilmektedir hazırdır. </li></ol> 2. Sabit Kayma Reolojisi <ol><li> Reometresi ısıtın ve 20 ° C de, kademe sıcaklığı ayarlanır. Prob (kullanılan 25 mm koni) ve sahne (plaka) arasındaki boşluğu ayarlayın. </li><li> Sahnenin merkezinde üzerine biopolimer dağılımının Yük 0.41 ml positi ayarlamak için prob düşürmek(25 mm koni için 0.053 mm boşluk) üzerinde. Dağılım eşit boşluk içinde yayılır emin olun. </li><li> Aşağıdaki deneysel parametreleri ile reolojik testi yapın: tarama modu: log, başlangıç ​​hızını: 100 sn -1, son hızı: 0.1 sn -1, on yıl başına çekiyor: 10, yapılmasından önce gecikme: 5 sn, zamanı ölçmek: 10 sn, ölçüsü başına ve tarifi: iki (saat yönünün tersine ve hem de). </li><li> Denemeler electrospinning uygun dağılım konsantrasyonları tahmin reolojik verileri analiz. <ol><li> Polimer konsantrasyonlarının bir fonksiyonu olarak kesme oranlarında karşı görünür kesme viskozitelere çizilir. 0.1 sn için görünür viskozitesi (düşük kesme hızı verileri güvenilir, düşük konsantrasyonlarda, örneğin), gerçek veya ekstrapolasyon değerleri her bir akış eğrisi, yaklaşık sıfır kesme viskoziteleri, η 0 için, 1 -. </li><li> Özgül viskozite hesaplayın: η sp = (η 0 - η s)Η s çözücü viskozitesidir / η s. </li><li> Konsantrasyonunun bir fonksiyonu olarak belirli bir viskozitelere çizilir. Semidilute dolanmamış ve dolaşmış rejimleri tanımlayın. Semidilute dolanmamış rejimi, küçük bir eğim ile, düşük konsantrasyonlu ucundan başlar ve semidilute dolaşmış rejimi dolanmamış rejimi sonrasında daha büyük bir eğime sahiptir. Her iki rejimlerde Fit güç-yasası regresyon modelleri. Güç değerleri log-log arsa üzerinde semidilute dolanmamış ve dolaşmış rejimlerde yamaçları (konsantrasyon bağımlılık) vardır. Yerleştirilmiş iki tane çizginin kesişimi dolanması yoğunluğu C * e. </li></ol></li></ol> 3. Elektroeğirme Parametre Değişimi <ol><li> Örneğin,% 15, Şekil 1 &#39;de gösterildiği gibi. Elektro setup monte uygun bir bileşim dağılımı ile şırınga yükleyin (a / h)% 100 DMSO içinde, bir şırınga pompası üzerine nişastası veya pululan. Pozisyon (yüksek gerilim teli bağlayınıziğne ive). Banyoya topraklama kablosunu (negatif) daldırarak toprağa saf etanol içeren pıhtılaşma banyosu bağlayın. Şırınga iğnesi ve pıhtılaşma banyosu arasındaki mesafeyi ayarlamak için bir laboratuar kriko kullanın. Electrospinning sonra elyaf yüzey toplamak için banyoda bir metal örgü bırakın. </li><li> Aşağıdaki parametre aralıkları içinde biyopolimerin Spin: besleme oranı 0.1 0.4 ml / saat, 5 ila 10 cm arasında iplik mesafe ve gerilim 0 ile 15 kV. <ol><li> 5 cm&#39;lik bir mesafede eğirme ile başlayın. Birinci besleme hızı (0.1 ml / saat) için, iğne ucu sıkıp dispersiyonun şekline yavaş yavaş 0 V dikkate alınız voltajı kadar rampa ve damlatma dispersiyon hızlandırılmış ve daha sonra uzun zaman not edin. </li><li> Küçük bir püskürtme çözeltisi electrospinnability gösteren damla yüzeyinden başlamasina voltajı dikkat edin. Bir sürekli bir biçimde jetle varsa başlatılır, burada voltajı kaydedin. </li><li> Üç Paramete her biri için komple bir aralığı incelemekrs ve not başarılı eğirme koşulları. Ucundan sürekli bir püskürtme olduğunda yalnızca lifleri toplamak. </li></ol></li><li> Toplamaya mahsus bir kaç dakika sonra, saf etanol ile bir elyaf yüzey yıkayın. Vakum altında kurutucu madde ihtiva eden bir desikatör içine elyaf yüzey yerleştirin. </li><li> Tam karakterizasyonu için her biopolimer konsantrasyon için tekrarlayın. </li></ol><img alt="Şekil 1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> Elektro-ıslak eğirme kurulum Şekil 1 şematik çizimi. Biyopolimer dispersiyon, bir şırınga pompası sıkılır. Bir yüksek gerilim DC güç kaynağı künt iğne ve zemin koagülasyon banyosu yüksek voltaj sağlar. Iğne ucundan polimer jet düz bir yolda geçecek ve daha sonra (aka istikrarsızlığı kırbaçlanan) hızlı çırpma yolu geliştirir. 4. Morfolojik Karakterizasyonu<ol><li> Kurutulmuş elyaf mat bir parça kesin ve karbon bandı kullanarak bir SEM kütüğü üzerine hareketsiz. </li><li> SEM aracı haline numune saplama yükleyin ve analiz için görüntüler elde. </li></ol>

<ol>
	<li>Greiner, A., Wendorff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+self-assembled+nanofibers+by+electrospinning.">Functional self-assembled nanofibers by electrospinning.</a> Self-aseembled nanomaterials. 1, 107-171 (2008).</li><li>Ramakrishna, S., Fujihara, K., Teo, W. E., Lim, T. C., Ma, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Electrospinning and Nanofibers. , (2005).</li><li>Klossner, R. R., Queen, H. A., Coughlin, A. J., Krause, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+Chitosan&#8217;s+Rheological+Properties+and+Its+Ability+to+Electrospin.">Correlation of Chitosan&#8217;s Rheological Properties and Its Ability to Electrospin.</a> Biomacromolecules. 9 (10), 2947-2953 (2008).</li><li>McKee, M. G., Wilkes, G. L., Colby, R. H., Long, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlations+of+Solution+Rheology+with+Electrospun+Fiber+Formation+of+Linear+and+Branched+Polyesters.">Correlations of Solution Rheology with Electrospun Fiber Formation of Linear and Branched Polyesters.</a> Macromolecules. 37 (5), 1760-1767 (2004).</li><li>McKee, M. G., Hunley, M. T., Layman, J. M., Long, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+rheological+behavior+and+electrospinning+of+cationic+polyelectrolytes.">Solution rheological behavior and electrospinning of cationic polyelectrolytes.</a> Macromolecules. 39 (2), 575-583 (2006).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+pure+starch+fibers+by+electrospinning.">Fabrication of pure starch fibers by electrospinning.</a> Food Hydrocolloids. 36, 20-25 (2014).</li><li>Singh, R. S., Saini, G. K., Kennedy, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pullulan:+microbial+sources,+production+and+applications.">Pullulan: microbial sources, production and applications.</a> Carbohydrate Polymers. 73 (4), 515-531 (2008).</li><li>Leathers, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotechnological+production+and+applications+of+pullulan.">Biotechnological production and applications of pullulan.</a> Applied Microbiology and Biotechnology. 62 (5), 468-473 (2003).</li><li>Karim, M. R., Lee, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+electrospun+pullulan/montmorillonite+nanofiber+mats+in+aqueous+solution.">Preparation and characterization of electrospun pullulan/montmorillonite nanofiber mats in aqueous solution.</a> Carbohydrate Polymers. 78 (2), 336-342 (2009).</li><li>Stijnman, A. C., Bodnar, I., Hans Tromp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+food-grade+polysaccharides.">Electrospinning of food-grade polysaccharides.</a> Food Hydrocolloids. 25 (5), 1393-1398 (2011).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+molecular+entanglements+in+starch+fiber+formation+by+electrospinning.">Role of molecular entanglements in starch fiber formation by electrospinning.</a> Biomacromolecules. 13 (8), 2247-2253 (2012).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+aspects+in+fabricating+pullulan+fibers+by+electro-wet-spinning.">Rheological aspects in fabricating pullulan fibers by electro-wet-spinning.</a> Food Hydrocolloids. 38, 220-226 (2014).</li><li>Han, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fiber Spinning Rheology and Processing of Polymeric Materials: Volume 2: Polymer Processing. , 257-304 (2007).</li><li>Morris, E. R., Cutler, A. N., Ross-Murphy, S. B., Rees, D. A., Price, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+shear+rate+dependence+of+viscosity+in+random+coil+polysaccharide+solutions.">Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions.</a> Carbohydrate Polymers. 1 (1), 5-21 (1981).</li><li>Thompson, C. J., Chase, G. G., Yarin, A. L., Reneker, D. 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Yazarlar ifşa hiçbir şey olmadığını beyan ederim.

Reolojisi geleneksel fiber sıkma dahil olmak üzere ve 13 electrospinning polimerlerin işlenmesi incelemek için önemli bir araçtır. Devamlı kesme reolojik çalışmaları, konformasyon ve polimer, farklı çözücüler içinde etkileşimlerinde kaynaktan çözülebilir (Şekil 2 ve 3). Biyopolimeri molekülleri birbirleriyle örtüşen için yeterli değil, yüksek konsantrasyonlarda, konsantrasyon bağımlılığı iyi çözücü 3,4 diğer polimerler bildi...

Elektro malzemeler çok çeşitli nano-ölçekli elyaf sürekli mikroskopik olarak üretme kabiliyetine sahip olan bir tekniktir. Bu akademik ve endüstriyel ilgi 1 artan kazanmıştır. Kurulum ve Elektrospinning uygulaması basit gibi görünse de, elyaf özelliklerini electrospinnability tahmin ve kontrol yeteneği bir sorun olmaya devam etmektedir. Nedeni Elektroeğirme işlemini 2 ve süreci, fiber tarafından seyahat, özellikle yolu, 1 kaotik etkileyen pek çok faktör vardır ki aslında yalan olabilir. Genellikle ampirik &quot;bakmak cook-ve-&quot; yaklaşımı potansiyel electrospinnable malzemelerin taranması için kullanılır. Bununla birlikte, elektro işlemi üzerinde daha iyi kontrol ve elde edilen elyaf özelliklerinin gereklidir electrospinnability yöneten mekanizmaların daha iyi anlaşılmasını sağlamak için. Bir dizi araştırmacı, iplik dop içinde polimerlerin molekül dolanması bir bizim için esastır buldukBaşarılı Elektrospinning 3 5 l önkoşuldur. Reolojisi polimer dağılımlarına moleküler yapısını ve etkileşimi araştırmak için güçlü bir araçtır. Örneğin, McKee ve diğ. kloroform / dimetil tereftalat (7/3, h / h) içeren bir çözelti içinde lineer moleküler yapıya ve dallanmış poli (etilen tereftalat-ko-etilen izoftalat) kopolimerleri araştırılmıştır ve polimer konsantrasyonu 2-2.5x olması gerektiğini tespit Başarılı üretebilmektedir 4 dolanması konsantrasyonu. Çünkü biyobozunurluk, biyouyumluluk ve vis-à-vis onların sentetik meslektaşları yenilenebirlik kendi avantajları biyopolimerlerden lifleri şu anda yenilenen ilgi var. Ancak uygulayıcılar ısıl işlem ve alt mekanik özelliklerinde yapısal karmaşıklık, zorluk genellikle kaynaklanan birçok sorunlarla karşı karşıya. Bitki dokularında bulunan nişasta, amon olduğug yeryüzünde en bol ve ucuz biopolimerlerden. Saf nişasta lifleri bir elektro-ıslak-bükme aparatı son 6 tarif edildi kullanılarak imal. Pullulan bazı bakteriler tarafından hücre dışı bir lineer polisakkarittir. (1 → 4) ve (1 → 6) düzenli almaşı glukosidik bağlar yeteneği 7,8 şekillendirme mükemmel lif / film dahil pullulan birkaç ayırıcı özelliklerinin, sorumlu olduğuna inanılmaktadır. Sulu dispersiyondan pullulan liflerin elektro araştırmacılar 9,10 bir dizi bildirilmiştir. Daha önceki yayınlarda iki biyopolimer, nişasta 11 ve pullulan 12 electrospinnability, tartışılmıştır. Bu rapor, bu iki Biyopolimerlerin electrospinnability soruşturmasında reolojik ilkelerini kullanan için protokol gösterilmesine yöneliktir.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:253px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelose 80 starch</td>
			<td style="width:253px;">Ingredion</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pullulan</td>
			<td style="width:253px;">Hayashibara Co. Ltd</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide</td>
			<td style="width:253px;">BDH Chemicals</td>
			<td style="width:119px;">BDH1115-4LP</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Ethanol</td>
			<td style="width:253px;">VWR International</td>
			<td style="width:119px;">89125-172</td>
			<td>200 proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:253px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rheometer</td>
			<td style="width:253px;">TA Instruments</td>
			<td style="width:119px;">ARES&#160;</td>
			<td>50 mm cone and plate geometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe (10 mL)</td>
			<td style="width:253px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">309604</td>
			<td>Syringe with Luer-Lok&#174; Tip</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">High voltage generator</td>
			<td style="width:253px;">Gamma High Voltage Research, Inc.</td>
			<td style="width:119px;">ES40P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe pump</td>
			<td style="width:253px;">Hamilton Company</td>
			<td style="width:119px;">81620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">FEI Company</td>
			<td style="width:119px;">Quanta 200</td>
			<td>for starch fibers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">Phenom-World</td>
			<td style="width:119px;">Phenom G2 Pro</td>
			<td>for pullulan fibers</td>
		</tr>
	</tbody>
</table>

molecular entanglement and electrospinnability of biopolymers

Elektro malzemeler çok çeşitli nano-ölçekli liflere mikro imal etmek için ilginç bir tekniktir. Biyopolimerler için, eğirme dop içindeki kurucu polimerlerin molekül dolanması başarılı bir şekilde üretebilmektedir için önemli bir ön-koşul olduğu bulunmuştur. Reolojisi Biyopolimerlerin moleküler yapısını ve etkileşimi araştırmak için güçlü bir araçtır. Bu raporda, dimetil sülfoksit (DMSO) / su dispersiyonlan gelen iki biyopolimer, nişasta ve pullulan ve electrospinnability değerlendirmek için reoloji kullanılması için protokol göstermektedir. Mikron aralığı için mikron altından ortalama çapları ile iyi bir şekilde oluşturulmuş nişasta ve pullulan lifler elde edilmiştir. Electrospinnability oluşan liflerin görsel olarak veya mikroskopik gözlem ile değerlendirilmiştir. Bunların electrospinnability için dispersiyonların reolojik özelliklerini ilişkilendirerek, tüm seçecek etkiler moleküler konformasyon, moleküler dolanması ve kayma viskozitesi göstermektedirrospinning. Reoloji sadece çözücü sistemi seçimi ve işlem optimizasyonu faydalı değil, aynı zamanda moleküler seviyede lif oluşumu mekanizmasının anlaşılması değildir.

Biyopolimer konsantrasyonu ve çözücü DMSO konsantrasyonunun bir fonksiyonu olarak biyopolimer dispersiyonların akış eğrileri elde edilmiştir. İki temsili verileri saf DMSO çözücüsü içindeki konsantrasyonunun bir fonksiyonu olarak nişasta (Şekil 2A) ve pullulan (Şekil 2B) akış eğrilerini göstermektedir. Belirli viskoziteler, biyopolimer konsantrasyonu nişasta (Şekil 3A ve Şekil 3B pullulan) karşı grafik halinde gösterilmişti...

Watch this Scientific Journal Video about Molecular Entanglement and Electrospinnability of Biopolymers at JoVE.com

Molecular Entanglement and Electrospinnability of Biopolymers

Elektro malzemeler çok çeşitli nano-ölçekli liflere mikroskopik olarak imal etmek için kullanılan bir ilginç bir tekniktir. Eğirme dop içindeki kurucu polimerlerin moleküler dolanması başarılı bir şekilde üretebilmektedir için gereklidir. Biz iki Biyopolimerlerin, nişasta ve pullulan bir electrospinnability değerlendirmek reolojisini kullanan bir protokol mevcut.

Moleküler Dolaşıklık Biyopolimerlerin Electrospinnability

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

molecular-entanglement-and-electrospinnability-of-biopolymers

Research

JoVE Journal

Bioengineering

13.7K Görüntüleme.  Pennsylvania State University.  Elektro malzemeler çok çeşitli nano-ölçekli liflere mikroskopik olarak imal etmek için kullanılan bir ilginç bir tekniktir. Eğirme dop içindeki kurucu polimerlerin moleküler dolanması başarılı bir şekilde üretebilmektedir için gereklidir. Biz iki Biyopolimerlerin, nişasta ve pullulan bir electrospinnability değerlendirmek reolojisini kullanan bir protokol mevcut. 

Video: Molecular Entanglement and Electrospinnability of Biopolymers

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Yaşayan Hücreler Moleküler Rotorlar arasında Flüoresans Ömrü Görüntüleme

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Biyomühendislik

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Yarı yapay Moleküler Makine ve Kullanımı DNA Hasar Tespiti için in vitro Meclisi

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

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 ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Kayma Tahliller kullanma Biyopolimerler eğilme Katılığı Ölçümleri

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Aplysia Tek Nöronların Elektrofizyolojik ve Moleküler Analizleri için Gangliyon Hazırlama

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Microscale Vortex destekli Sıralı Moleküler Teslimat için Electroporator

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Modlu Moleküler Görüntüleme Uygulamaları için Biofunctionalized Prusya Mavisi Nanopartikülleri

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Tek Karbon Nanotüp Bio-mimetik Polimerler ile Mühendislik Moleküler Tanıma

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Kurutma Hava-LC Arayüz üzerinde Megamolecular Biyopolimerlerin Kendinden entegrasyonu için Yöntemler

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

İnsan Nötrofil Swarming in vitro Kemotaktik ve Moleküler Analizi için Biyopartikül Mikrodizileri

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...