ويتم تمويل هذا العمل جزئيا من قبل المعهد الوطني للوزارة الزراعة الأميركية للأغذية والزراعة، برنامج المنح التنافسية الوطنية، البرنامج الوطني مبادرة بحوث 71.1 سنة المالية 2007 ومنحة رقم 2007-35503-18392، والمعاهد الوطنية للصحة، ومعهد للحساسية والأمراض المعدية ، R33AI94514-03.

1. الغزل تحضير المخدر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد مجموعة من تركيزات البوليمر الحيوي ليتم التحقيق (0.1٪ إلى 30٪، ث / ت) واحرص على النظر في محتوى الرطوبة من مسحوق البوليمر الحيوي في هذه الحسابات. لكل تركيز، تزن البوليمر الحيوي (النشا أو pullulan) مسحوق إلى 50 مل أنبوب اختبار. إضافة مائي ثنائي ميثيل سلفوكسيد (DMSO) حل وبقضيب. </li><li style=";text-align:right;direction:rtl"> وضع أنبوب في الماء المغلي مع التحريك المستمر على موقد النمام المغناطيسي. </li><li style=";text-align:right;direction:rtl"> بعد حوالي 1 ساعة، إيقاف الحرارة والسماح للتشتت لتبرد لدرجة حرارة الغرفة. تشتت جاهز للاختبار الانسيابية وelectrospinning ذلك الحين. </li></ol> 2. ثابت القص ريولوجيا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الاحماء مقياس غلفاني وضبط درجة الحرارة مرحلة عند 20 درجة مئوية. معايرة الفجوة بين (مم مخروط 25 المستخدمة) التحقيق ومرحلة (لوحة). </li><li style=";text-align:right;direction:rtl"> تحميل 0.41 مل من تشتت البوليمر الحيوي على مركز المسرح وخفض التحقيق لضبط positiعلى (0.053 ملم الفجوة لمدة 25 ملم مخروط). تأكد من أن تشتت ينتشر بالتساوي داخل الفجوة. </li><li style=";text-align:right;direction:rtl"> إجراء اختبار الانسيابية مع المعلمات التجريبية التالية: تسجيل، ومعدل الأولي: وضع الاجتياح 100 ثانية -1، ومعدل النهائي: 0.1 ثانية -1، يشير في العقد الواحد: 10، تأخير قبل القياس: 5 ثانية، وقياس الوقت: 10 ثانية، والاتجاهات في القياس: اثنين (سواء في اتجاه عقارب الساعة وعكس عقارب الساعة). </li><li style=";text-align:right;direction:rtl"> تحليل البيانات الريولوجية لتقدير تركيزات تشتت المناسبة لelectrospinning المحاكمات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رسم اللزوجة القص واضحة ضد معدلات القص كدالة للتركيز البوليمر. لكل منحنى التدفق، اللزوجة التقريبية الصفر القص، η 0، بواسطة القيم الفعلية أو استقراء (على سبيل المثال، في تركيزات منخفضة حيث هي بيانات معدل القص منخفضة غير موثوق بها) لاللزوجة الظاهرية في 0.1 ثانية - 1. </li><li style=";text-align:right;direction:rtl"> حساب اللزوجة محددة: س = η (η 0 - η ق)/ η ق، ق حيث η هو اللزوجة من المذيبات. </li><li style=";text-align:right;direction:rtl"> رسم اللزوجة محددة بوصفها وظيفة من التركيز. تحديد الأنظمة unentangled والمتشابكة semidilute. يبدأ النظام unentangled semidilute من نهاية تركيز منخفضة مع منحدر صغير، والنظام semidilute متشابكا لديه أكبر منحدر التالية النظام unentangled. تناسب النماذج بقانون القوة الانحدار في كلا النظامين. قيم السلطة هي المنحدرات (تركيز الاعتماد) في الأنظمة unentangled والمتشابكة semidilute على قطعة سجل سجل. تقاطع الخطين المجهزة هو تركيز التشابك، ج ه. </li></ol></li></ol> 3. Electrospinning معلمة التغيير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تجميع الإعداد electrospinning كما هو مبين في الشكل 1. تحميل المحاقن مع تشتت التكوين المناسب، على سبيل المثال، 15٪ (ث / ت) النشا أو pullulan في 100٪ DMSO، على ضخ حقنة. مقطع الأسلاك عالية الجهد (يفترضإيف) إلى إبرة. ربط حمام تخثر تحتوي على الإيثانول النقي إلى الأرض بغمر سلك الأرض (سلبي) إلى الحمام. استخدام جاك مختبر لضبط المسافة بين إبرة حقنة وحمام التخثر. تزج على شبكة معدنية في الحمام لجمع حصيرة الألياف بعد electrospinning. </li><li style=";text-align:right;direction:rtl"> تدور البوليمر الحيوي في نطاقات المعلمة التالية: معدل التغذية 0،1-0،4 مل / ساعة، وبعد المسافة الغزل من 5 الى 10 سم، والجهد 0-15 كيلو فولت. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تبدأ مسافة الغزل من 5 سم. لمعدل التغذية الأول (0.1 مل / ساعة)، المنحدر الجهد ببطء من 0 خامسا انتبه إلى شكل تشتت مقذوف في طرف الإبرة ونلاحظ عندما تتسارع تشتت يقطر ثم ممدود. </li><li style=";text-align:right;direction:rtl"> لاحظ الجهد الذي طائرة صغيرة بدأت من سطح قطرة، مشيرا electrospinnability من الحل. تسجيل الجهد الذي يبادر طائرة مستمرة، إن وجدت. </li><li style=";text-align:right;direction:rtl"> دراسة مجموعة كاملة لكل من paramete ثلاثةالتمرير ومذكرة الشروط الغزل ناجحة. جمع ألياف فقط عندما يكون هناك طائرة مستمرة من غيض. </li></ol></li><li style=";text-align:right;direction:rtl"> بعد بضع دقائق من جمع وشطف حصيرة الألياف مع الإيثانول النقي. وضع حصيرة الألياف إلى مجفف تحتوي على المجففة تحت فراغ. </li><li style=";text-align:right;direction:rtl"> كرر لكل تركيز البوليمر الحيوي لتوصيف كامل. </li></ol><img alt="الشكل 1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> الرقم 1. رسم تخطيطي من الإعداد الكهربائية الغزل الرطب. هو مقذوف وتشتت البوليمر الحيوي من ضخ حقنة. يوفر الجهد العالي DC امدادات الطاقة عالية الجهد لإبرة حادة وأسباب الحمام التخثر. الطائرة البوليمر من طرف الإبرة يسافر عبر الطريق المستقيم ثم يتطور مسار الجلد السريع (الملقب الجلد عدم الاستقرار). 4. المورفولوجية توصيف<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع قطعة من الألياف المجففة حصيرة وشل وضعها كعب SEM باستخدام شريط الكربون. </li><li style=";text-align:right;direction:rtl"> تحميل عينة كعب في الصك ووزارة شؤون المرأة والحصول على صور لتحليلها. </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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+parameters+on+nanofiber+diameter+determined+from+electrospinning+model.">Effects of parameters on nanofiber diameter determined from electrospinning model.</a> Polymer. 48 (23), 6913-6922 (2007).</li></ol>

تعلن المؤلفون أن ليس لديهم ما يكشف.

الريولوجيا هي أداة أساسية لدراسة معالجة البوليمرات، بما في ذلك الألياف الغزل التقليدي وelectrospinning 13. من الدراسات ثابتة القص الريولوجية البوليمر التشكل وتفاعلاتها في مذيبات مختلفة يمكن حلها (الشكلان 2 و 3). لا بتركيزات عالية بما فيه الكفاية لجزي?...

Electrospinning هو الاسلوب الذي هي قادرة على انتاج الجزئي المستمر لألياف نانو النطاق من مجموعة واسعة من المواد. وقد اكتسب زيادة الأكاديمية والصناعية الفائدة 1. على الرغم من أن الإعداد وممارسة electrospinning تبدو واضحة، والقدرة على التنبؤ والتحكم electrospinnability خصائص الألياف لا تزال تشكل تحديا. السبب قد يكمن في حقيقة أن هناك العديد من العوامل التي تؤثر على عملية electrospinning 2 والعملية، وخاصة الطريق التي يقطعها الألياف، هو الفوضى 1. غالبا ما يستخدم هذا النهج &quot;المطبخ ونظرة&quot; التجريبية لفحص المواد electrospinnable المحتملة. ومع ذلك، للسيطرة أفضل على عملية electrospinning وخصائص الألياف الناتجة، فهم أكثر اكتمالا لآليات التي تحكم electrospinnability هو مطلوب. وقد وجد الباحثون أن العديد من التورط الجزيئي للبوليمرات في مخدر الغزل هو ضروريهل شرطا مسبقا لنجاح electrospinning 3- 5. الريولوجيا هو أداة قوية للبحث في التشكل الجزيئي والتفاعل في التفرق البوليمر. على سبيل المثال، ماكي وآخرون. التحقيق في التشكل الجزيئي للبولي الخطية وتشعبت (الاثيلين تيريفثاليت-CO-isophthalate الإثيلين) بوليمرات في المذيبات التي تحتوي على الكلوروفورم / تيريفثالات ثنائي ميثيل (7/3، V / V)، وقرر أن تركيز البوليمر كان لا بد 2-2.5x تركيز التشابك لelectrospinning ناجح 4. هناك اهتمام متجدد حاليا في الألياف من البوليمرات الحيوية بسبب مزاياها في التحلل البيولوجي، توافق مع الحياة، والتجديد وجها لوجه نظرائهم الاصطناعية. بعد الممارسين مواجهة العديد من التحديات التي تنشأ عادة من تعقيدها الهيكلي، وصعوبة في المعالجة الحرارية والخواص الميكانيكية رديئة. النشا، وجدت في الأنسجة النباتية، هو آمونز والبوليمرات الحيوية الأكثر وفرة وغير مكلفة على وجه الأرض. ملفقة الألياف النشا النقي باستخدام الأجهزة الكهربائية الرطب الغزل وصفت مؤخرا 6. Pullulan هو السكاريد خطي إنتاج خارج الخلية بواسطة بكتيريا معينة. التناوب المنتظم لل(1 → 4) و (1 → 6) ويعتقد أن السندات glucosidic أن تكون مسؤولة عن العديد من الخصائص المميزة لpullulan، بما في ذلك الألياف الممتاز / فيلم تشكيل قدرة 7،8. تم الإبلاغ عن Electrospinning من الألياف pullulan من تشتت مائي من قبل عدد من الباحثين 9،10. في منشوراتنا السابقة، electrospinnability اثنين من البوليمرات الحيوية والنشا 11 و 12 pullulan، تم مناقشتها. يركز هذا التقرير على إثبات بروتوكول لاستخدام مبادئ الريولوجية في التحقيق في electrospinnability هذه البوليمرات الحيوية اثنين.

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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

Electrospinning هي تقنية رائعة لافتعال الجزئي لألياف نانو النطاق من مجموعة واسعة من المواد. لالبوليمرات الحيوية، وجد التورط الجزيئي للبوليمرات المكونة في الغزل منشطات أن يكون شرطا أساسيا لنجاح electrospinning. الريولوجيا هو أداة قوية للتحقيق في التشكل الجزيئي والتفاعل من البوليمرات الحيوية. في هذا التقرير، علينا أن نظهر بروتوكول لاستخدام الريولوجيا لتقييم electrospinnability اثنين من البوليمرات الحيوية والنشا وpullulan، من هم ثنائي ميثيل سلفوكسيد (DMSO) / التفرق المياه. تم الحصول بشكل جيد النشا والألياف pullulan مع متوسط ​​أقطار في submicron على نطاق ميكرون. تم تقييم Electrospinnability عن طريق الملاحظة البصرية ومجهرية من الألياف تشكيلها. خلال ربط الخصائص الريولوجية من التفرق إلى electrospinnability، وعلينا أن نبرهن على التشكل الجزيئي، التشابك الجزيئي، واللزوجة القص كلها تؤثر المنتخبrospinning. الريولوجيا ليست مفيدة فقط في اختيار نظام المذيبات وعملية التحسين، ولكن أيضا في فهم آلية تشكيل الألياف على المستوى الجزيئي.

تم الحصول على منحنيات تدفق التفرق البوليمر الحيوي كدالة للتركيز البوليمر الحيوي وتركيز DMSO في المذيبات. تظهر اثنين من الشخصيات التمثيلية منحنيات تدفق النشا (الشكل 2A) وpullulan (الشكل 2B) كدالة للتركيز في المذيب النقي DMSO. وقد تآمر اللزوجة محددة ضد تركيز ال...

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

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning هي تقنية رائعة المستخدمة لصنع الجزئي لألياف نانو النطاق من مجموعة واسعة من المواد. التورط الجزيئي للبوليمرات المكونة في مخدر الغزل ضروري لelectrospinning ناجحة. نقدم بروتوكول لاستخدام الريولوجيا لتقييم electrospinnability اثنين من البوليمرات الحيوية والنشا وpullulan.

تشابك الجزيئية وElectrospinnability من البوليمرات الحيوية

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

13.6K Views.  Pennsylvania State University.  Electrospinning هي تقنية رائعة المستخدمة لصنع الجزئي لألياف نانو النطاق من مجموعة واسعة من المواد. التورط الجزيئي للبوليمرات المكونة في مخدر الغزل ضروري لelectrospinning ناجحة. نقدم بروتوكول لاستخدام الريولوجيا لتقييم electrospinnability اثنين من البوليمرات ال...

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.

مضان التصوير عمر الدوار الجزيئية في الخلايا الحية

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

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.

الجمعية في المختبر من نصف اصطناعية آلة الجزيئية واستخدامها للكشف عن الحمض النووي من التلف

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.

بروتين منخفض الوزن الجزيئي تخصيب اليورانيوم على أفلام السيليكا Mesoporous رقيقة لاكتشاف العلامات البيولوجية

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.

قياسات جمود العاطفة من البوليمرات الحيوية باستخدام فحوصات الانزلاق

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

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

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.

أأليسيا إعداد Ganglia للتحليلات الكهربية والجزيئية للخلايا العصبية المفردة

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.

الميكروسكيل Electroporator لتوصيل متسلسل الجزيئية بمساعدة دوامة

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.

Biofunctionalized البروسية الأزرق النانوية الجزيئية لالمتعدد الوسائط تطبيقات التصوير

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.

الاعتراف الجزيئية والهندسة مع البوليمرات الحيوية المحاكاة على واحدة المسورة الكربون الأنابيب النانومترية

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.