نود أيضا أن نشكر شياو يو مياو ويلسون بن لتطوير أكثر من الأساليب المذكورة داخل. وقد تم تمويل هذا العمل من قبل المؤسسة الوطنية للعلوم (DBI 0454324) والمعهد الوطني للصحة (R21 EB005183) وPHS NRSA T32 GM07270 NIGMS من لجنة الانتخابات.

1. عشوائية صفيف الاتحاد الافريقي nanoparticle التصنيع 8،10،12،14 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم تشكيل مجموعة جسيمات متناهية الصغر من خلال إنشاء الاتحاد الافريقي لأول مرة القالب الذي يتكون من طبقة كثيفة من المجالات اللاتكس كثف عشوائيا مع قطر يعني من 454 نانومتر. ويتم تحقيق هذا عن طريق التبخير أول ذهبية على الزجاج ساترة لبسمك 20 نانومتر باستخدام الكروم والتصاق طبقة. </li><li style=";text-align:right;direction:rtl"> والبوليسترين المونولاير المجال ومن ثم تجميعها ذاتيا من خلال تعريض الركيزة المغلفة الذهب إلى خليط من 1 إيثيل - 3 - (3 - dimethylaminopropyl) carbodiimide هيدروكلوريد (EDC) ، واللثي تعليق المجال ، والمياه غير المتأينة. </li><li style=";text-align:right;direction:rtl"> ولا يسمح للعملية الامتصاص تستمر لحوالي ساعة واحدة ويتم غسلها المجالات غير استيعابها بعيدا مع كمية وفيرة من المياه. </li><li style=";text-align:right;direction:rtl"> ولا يسمح للأحادي الطبقة تشكلت في الهواء الجاف. </li><li style=";text-align:right;direction:rtl"> أخيرا ، يتم تبخير آخر 20 نانومتر من الذهب على المونولاير المجال اللاتكس لتشكيل جزيئات النانو الذهب عشوائية صفيف. </li><li style=";text-align:right;direction:rtl"> إذا كان متوفرا SEM ، يمكن أن ينظر إليه في إطار مجموعة AuNP SEM لتبدو مثل الشكل 1 ويظهر رسم بياني للعملية في الشكل 8. </li></ol> 2. إعداد العينات البيولوجية 9،11 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يظهر الآن إعداد نموذج لمحاصرة بصريا الماوس نواة الخلية. </li><li style=";text-align:right;direction:rtl"> تم الحصول على نواة الخلية 3T3 ماوس المفتاحية أكريدين الصبغة البرتقالية من مجموعة تيواري في مركز فريد هاتشينسون لأبحاث السرطان. </li><li style=";text-align:right;direction:rtl"> 10 ٪ يضاف مصل الزلال البقري (BSA) في الفوسفات مخزنة المالحة (PBS) إلى نواة الخلية الماوس في تركيز حوالي 1 : 10 (BSA : الفأر نواة الخلية). جيش صرب البوسنة يساعد على منع النواة من الالتصاق إلى الركيزة. </li><li style=";text-align:right;direction:rtl"> الحل باستخدام مزيج صوتنة. </li><li style=";text-align:right;direction:rtl"> وتودع 5 ماي من حلنا على الصفيف ساترة الألومنيوم صريف. فمن الأفضل لتنفيذ هذه الخطوة مع صريف الألومنيوم في المرحلة المجهر بحيث لم يكن لديك لنقل العينة بعد إيداع الحل. </li><li style=";text-align:right;direction:rtl"> وتستخدم اثنين من اثنين من مداخن 1 &quot;في موعد أقصاه 1&quot; لدعم coverslips ساترة الخامسة عن طريق وهو ينظر إلى العينة. </li><li style=";text-align:right;direction:rtl"> موقف العينة تحت المجهر للعرض. </li></ol> 3. طريقة لاصطياد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> هي التي شيدت في ملاقط بصرية عن طريق إرسال 35 ميغاواط ليزر الهليوم نيون من خلال محوري Imager.D1M زايس مجهزة بمجموعة 17 GFP التصفية التي يتم تعديلها للسماح لأشعة الليزر 633 نانومتر لتصل العينة. </li><li style=";text-align:right;direction:rtl"> يستخدم NEOFLUAR 50x صورة موضوعية لنواة الخلية التي ما يقرب من 5 ميكرون في القطر -- ألف دينار زايس EC Epiplan. </li><li style=";text-align:right;direction:rtl"> بعد يتم وضع العينة في إطار الهدف ، والتركيز المجهر على صفيف الذهب جسيمات متناهية الصغر أو مقضب الحيود. </li><li style=";text-align:right;direction:rtl"> ترجمة المجهر عموديا حتى يتحقق التركيز على النواة التي كنت ترغب في الفخ. </li><li style=";text-align:right;direction:rtl"> وينبغي أن موقف فخ بقعة الليزر أكثر من الجسيمات والجسيمات في المحافظة ومن ثم وضعها في بقعة الليزر حتى عندما تتم ترجمة هذه المرحلة. </li></ol> 4. ممثل النتائج : ينبغي للجسيمات متناهية الصغر إجراءات الذهب مجموعة عشوائية من إيداع المونولاير AuNP والتي يمكن عرضها في إطار SEM لتبدو مثل الشكل 1. يمكن محاصرة القوة التي أوجدتها هذه plasmonic ملاقط يكون 10-20 أضعاف القوة التي تم إنشاؤها بواسطة ملاقط بصرية القياسية. وتظهر شدة الدنيا المطلوبة لتحقيق plasmonic ملاقط الحبس الجسيمات عن جسيمات ذات أحجام مختلفة في الشكل 4. 9،10 صريف حيود تحقيق المواءمة والملائمة مع كفاءة أعلى 20 مرة من محاصرة nanodots الذهب ، ويمكن تحقيق الملائمة مع اقل كما يسكونس 17 / ام 2 (الشكل 7). 11 <img alt="الشكل 1" src="/files/ftp_upload/3390/3390fig1.jpg" /> الشكل 1 10 (أ) صورة مجهرية SEM من جزيئات الذهب الذاتي تجميعها. القطر من جزيئات الذهب الفرد حوالي 450 نانومتر. (ب) صورة NSOM الركيزة plasmonic حيث توزيع متناثر جسيمات متناهية الصغر ، والتي تبين الأشعة القريبة من الميدان. الطول الموجي لليزر الإثارة هو 633 نانومتر. (ج) عرض التكبير السامي للمنطقة مع وضع علامة حمراء في مربع (ب). (د) كفاءة الطيف نثر الركيزة plasmonic ، والتي تبين الذروة في 624 نانومتر. (ه) الطيف كفاءة امتصاص الركيزة plasmonic ، والتي تبين الذروة في 668 نانومتر. <img alt="الشكل 2" src="/files/ftp_upload/3390/3390fig2.jpg" /> الشكل 2 13 (أ) الاتحاد الافريقي nanospheres موزعة بشكل عشوائي على نطاق 2D 1 × 1 ميكرومتر 2. كل نقطة زرقاء تمثل مركز nanosphere (أ = 60 نانومتر). وتظهر التوزيعات مجال تناثر على طائرات المراقبة التي هي موازية لمجموعة nanosphere عشوائي في الفقرة (ب) -- (ه). غير مضاءة بشكل موحد الصفيف nanosphere موجة الطائرة في الطول الموجي من 540 نانومتر. معامل الانكسار من الوسط المحيط هو 1.33. وبولاالاتجاه rization من النقاط على طول موجة الطائرة المحور السيني (الأفقي في الفقرة (أ)). ويفترض أن حجم الحقل الكهربائي الحادث لكي تكون 1 في الحساب. يتم تعريف الفاصل بين الطائرة والمراقبة الصفيف nanosphere كما ح ب) ح = أ. ج) ح = 2A. د) ح = λ. ه) ح = 2λ. <img alt="الشكل 3" src="/files/ftp_upload/3390/3390fig3.jpg" /> الرقم 3 9 تخطيطي لتكوين مخصص المجهر مضان بما في ذلك تصفية الإثارة وتجاوز مزدوج اللون استبدال الحزم الخائن. هذا هو التكوين تستخدم لمحاصرة في وقت واحد والتصوير مضان. <img alt="الشكل 4" src="/files/ftp_upload/3390/3390fig4.jpg" /> الرقم 4 10 كثافة الليزر للحفاظ على الحد الأدنى في فخ بوصفها وظيفة من معدل تدفق السوائل المحيطة الاستفادة من محاصرة plasmonic. ويتم قياس جميع الكثافات الضوئية على الطائرة العينة تحت المجهر الهدف. (أ) -- (و) تظهر نتائج القياس للالخرز البوليسترين واحد بقطر 7.3 و 6.3 (غير كروي) ، 5.0 ، 3.9 ، 2.5 و 1.1 ميكرومتر ، على التوالي. وinsets عرض الصور المجهرية المقابلة من الجسيمات. أشرطة النطاق في كل الصور تمثل 5 ميكرون في الطول. <img alt="الشكل 5" src="/files/ftp_upload/3390/3390fig5.jpg" /> الشكل 5 10 المنحدر من خط تركيبها من خلال الأصل في الشكل. 4 مقابل حجم الجسيمات لمحاصرة plasmonic. أشرطة الخطأ تظهر الانحرافات المعيارية ليناسب الخطية. المنحدر من خط المجهزة (النسبة بين شدة العتبة البصرية ومعدل التدفق) في الشكل. 4 وجود علاقة خطية تقريبا مع حجم الجسيمات كما هو مبين في هذا الشكل ، مما يدل على الاستفادة من محاصرة plasmonic وخاصة بالنسبة للجسيمات أصغر. <img alt="الشكل 6" src="/files/ftp_upload/3390/3390fig6.jpg" /> الشكل 6 11 (أ) رسم تخطيطي لمحاصرة تعزيز الاستفادة من النانو البصرية الدوري 1 - D. شعاع الحادث diffracted من البنية النانوية الدوري في الميدان حتى الآن. (ب) توزيع شدة الضوء مع اثنين من البنية النانوية متعامد الاستقطابات في حقل بعيد. (ب) توزيع شدة الضوء مع اثنين من الاستقطاب المتعامد على السطح من صريف الألومنيوم مع فترة 417 نانومتر تم الحصول عليها باستخدام المحاكاة FDTD. هو تطبيع توزيعها على كثافة على سطح الألومنيوم مسطح. (ج) و (د) إمكانية ملائمة للجسيمات مباشرة فوق سطح صريف مقابل موقع للجسيمات لالبوليسترين ألف (ج) 350 نانومتر حبة و (د) والبوليسترين 1 ميكرومتر حبة. الدوائر البيضاء توضيح أحجام الجسيمات. Insets تظهر إمكانات محاصرة فوق سطح الالومنيوم شقة لحجم الجسيمات نفس المقارنات. تم تطبيع القيم لكل حجم الجسيمات. لجميع الأرقام محاكاة FDTD مجال الرؤية هو 10 × 8 2 ميكرون. <img alt="الشكل 7" src="/files/ftp_upload/3390/3390fig7.jpg" /> الشكل 7 11 (أ) الكفاءة والحد الأدنى من شدة فخ نصب الفخاخ لقياس الخرز البوليسترين من مختلف الأحجام مع عمودي الاستقطاب شعاع لخطوط صريف. أقحم يظهر التفاوت في فخ الكفاءة الملائمة لترجمة والبوليسترين 3.87 عمودي أم حبة ومتوازية لقواعد صريف. يتم الحصول على خط الصلبة (التباين الكبير) مع عمودي الاستقطاب الحادث الضوء على صريف ، ويتم الحصول على خط شرطة (عدم التماثل الصغيرة) مع الحادث الضوء المستقطب موازية لصريف. وحدة في (PN [ميغاواط / ميكرون 2] -1). (ب) -- (د) مظاهرة ملائمة لالفلورسنت 590 نانومتر حبة البوليسترين. دائرة حمراء تدل على موقف بقعة الليزر ضوء الليزر كما كانت خافتة جدا بحيث لا يمكن رؤيتها. في البداية محصورة الجسيمات داخل بقعة في أعلى السلطة ، كما هو خفض قوة الحركة البراونية للجسيمات يتغلب على قوة محاصرة ، والسماح للجسيمات من الفرار. (ه) -- (ز) مظاهرة ملائمة من نواة الخلية السرطانية المبيض الفلورسنت. كانت كثافة الحد الأدنى المطلوب لبدء محاصرة μW 16 / 2 ميكرون تم الحصول عليها باستخدام عدسة الهدف 20X. <img alt="الشكل 8" src="/files/ftp_upload/3390/3390fig8.jpg" /> الشكل 8 التصنيع الداخلي 14 من جزيئات الذهب على شكل قبعة : أ التبخر) من الكروم والاتحاد الافريقي طبقة رقيقة على الزجاج ساترة. ب) التعرض للتعليق البوليسترين مجال من المجالات ، وامتصاص لمدة 1 ساعة. ج) إزالة المجالات البوليسترين غير كثف وتجفيف السطح. د) التبخر من طبقة أخرى من الاتحاد الافريقي على رأس مجالات القالب. ه) تخطيطي للصفيف سقف على شكل جسيمات متناهية الصغر الاتحاد الافريقي ، حيث لا يغطي سوى الاتحاد الافريقي في الجانب العلوي من المجالات القالب.

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المغزى من هذه الأساليب لأنها محاصرة انخفاض كثافة الضوئية اللازمة لمحاصرة مستمرة من مكان ما على ترتيب مكان للμW 10 3 / 2 ميكرون بناء على أمر من μW 10 / 2 ميكرون. 10،11 والقيود المفروضة على هذه التقنيات هي أن جسيمات متناهية الصغر مجموعة الذهب الخبرات الت...

<table border="1" style=";text-align:right;direction:rtl"><tr><td> اسم المادة </td><td> نوع </td><td> شركة </td><td> رقم كتالوج </td><td> تعليق </td></tr><tr><td> تصوير محوري مجهر </td><td> D1M </td><td> زايس </td><td> D1M </td><td> زايس محوري Imager.D1M </td></tr><tr><td> الهدف المجهر </td><td> 50x/0.55 </td><td> زايس </td><td></td><td> LD EC Epiplan -- NEOFLUAR 50x/0.55 HD مدينة دبي للإنترنت </td></tr><tr><td> زايس مجهر الكاميرا </td><td> AxioCam MRC </td><td> زايس </td><td></td><td></td></tr><tr><td> ليزر الهليوم نيون </td><td> 35 ميغاواط </td><td> البحوث الكهربائية والبصريات </td><td></td><td></td></tr><tr><td> متغير المخفف </td><td> متغير باستمرار ND </td><td> ThorLabs </td><td> NDC - 100C - 4M </td><td> لضبط شدة المجهر </td></tr><tr><td> تصفية مجموعة زايس </td><td> تصفية مجموعة # 17 </td><td> زايس </td><td> 488017-9901-000 </td><td> تصفية مجموعة # 17 </td></tr><tr><td> المجهر الشرائح </td><td> سماكة 0.5 مم </td><td> VWR </td><td></td><td></td></tr><tr><td> 3T3 نواة الخلية الماوس </td><td></td><td colspan="2"> فريد هاتشينسون لبحوث السرطان مركز </td><td> مثل البرد ممكن مخزن </td></tr><tr><td> أكريدين صبغ برتقالي </td><td></td><td colspan="2"> فريد هاتشينسون لبحوث السرطان مركز </td><td></td></tr><tr><td> الأبقار مصل الزلال </td><td> 1-10 حصة في برنامج تلفزيوني </td><td colspan="2"> فريد هاتشينسون لبحوث السرطان مركز </td><td></td></tr><tr><td> 454 نانومتر المجالات اللاتكس البوليسترين </td><td></td><td> Polysciences ، وشركة </td><td></td><td></td></tr><tr><td> carbodiimide هيدروكلوريد (EDC) </td><td> 1 إيثيل - 3 - (3 - dimethylaminopropyl) </td><td> G - العلوم البيولوجية </td><td> BC25 - 1 </td><td></td></tr><tr><td> الذهب (لترسب) </td><td></td><td></td><td></td><td></td></tr><tr><td> تعكس مقضب الحيود حكمت </td><td></td><td> ادموند بصريات </td><td></td><td></td></tr><tr><td> التخزين المؤقت المالحة الفوسفات (PBS) </td><td> Dulbecco والفوسفات مخزنة المالحة (D - PBS) (1X) </td><td> Invitrogen </td><td> 14190-144 </td><td></td></tr></table>

utilization of plasmonic and photonic crystal nanostructures for enhanced micro- and nanoparticle manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Watch this Scientific Journal Video about Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation at JoVE.com

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

وترد وملاقط Plasmonic النانو الكريستال الضوئية لإنتاج تحسينات مفيدة في السيطرة والتوجيه من الكفاءة محاصرة بصريا المتناهية الصغر ومتناهية الصغر.

الاستفادة من النانو كريستال Plasmonic والضوئية لتعزيز التلاعب الصغرى ، والجسيمات النانوية

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

utilization-plasmonic-photonic-crystal-nanostructures-for-enhanced

Research

JoVE Journal

Bioengineering

12.2K Views.  University of Washington. وترد وملاقط Plasmonic النانو الكريستال الضوئية لإنتاج تحسينات مفيدة في السيطرة والتوجيه من الكفاءة محاصرة بصريا المتناهية الصغر ومتناهية الصغر.

Video: Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

مستو وثلاثي الابعاد من أحبار الطباعة موصل

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

التوليف ، والجمعية ، وتوصيف أحادي الطبقة المحمية الجسيمات النانوية الذهبية للأفلام كهربية البروتين أحادي الطبقة

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

تقييم توصيل الجينات البوليمرية الجسيمات النانوية من تحليل تتبع جسيمات متناهية الصغر وعالية الإنتاجية قياس التدفق الخلوي

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

الجسيمات الدقيقة Velocimetry صورة لسرعة الشخصي قياسات يتدفق الدم مايكرو

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

ألياف النانو البروتين ECM والنانو المهندسة طريق الجمعية بمبادرة السطحية

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

إعداد Monodomain الكريستال السائل اللدائن والكريستال السائل المرنة Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

تقنيات لتطور قوية خميرة تخمر البنتوز للالتحويل البيولوجي من مادة الخشب إلى إيثانول

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

ميكروفلويديك تبادل العازلة لمايكرو / الجسيمات النانوية هندسة الخلايا خالية من التدخل

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

استهدفت البلازما غشاء تسليم ومسعور الشحن مغلف في الكريستال السائل الجسيمات النانوية الناقل

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

توليف حيويا الكريستال السائل المرنة كسوات كما السقالات الخليوي لزراعات الخلايا 3D المكانية

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...