فإن الكتاب أود أن أشكر العلوم الطبيعية والهندسة مجلس البحوث كندا، كندا برنامج كراسي البحث والمعاهد الكندية لأبحاث الصحة، وزارة أونتاريو للبحوث والابتكار من خلال بهم الباحثون المبكر برنامج جائزة للحصول على الدعم المالي.

1. ضوئيه للماجستير تصنيع القالب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يستحم لمدة 3 بوصة رقاقة السيليكون في الأسيتون لمدة 30 ثانية ثم الميثانول لمدة 30 ثانية. شطف مع DH 2 0 الماء لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> يجف سطح الرقاقة في التعامل مع البندقية ضربة N2. تسخين رقاقة على طبق ساخن عند 140 درجة مئوية لمدة 2 دقيقة. </li><li style=";text-align:right;direction:rtl"> البلازما أكسدة سطح رقاقة السيليكون (1 دقيقة، 50 W). </li><li style=";text-align:right;direction:rtl"> تدور معطف سطح الرقاقة مع 3 مل SU-8 100 مقاوم الضوء (40 ثانية؛ 1،750 دورة في الدقيقة). </li><li style=";text-align:right;direction:rtl"> قبل خبز الرقاقة المغلفة على طبق ساخن عند 65 درجة مئوية لمدة 10 دقيقة، ثم المنحدر درجة حرارة تصل إلى 95 درجة مئوية أكثر من 2 دقيقة. الحفاظ على هذا الإعداد ل1 ساعة إضافية. </li><li style=";text-align:right;direction:rtl"> محاذاة الضوئية التي تحتوي على تصميم القناة المطلوبة. فضح مقاومة إلى 550-600 ميغا جول / سم 2 من ضوء الأشعة فوق البنفسجية (350-400 نانومتر). ويمكن تصميم Photomasks في أوتوكاد وطباعتها على الشفافية مع طباعة عالية القرار. </li><li style=";text-align:right;direction:rtl"> بعد خبز الرقاقة على طبق ساخن عند 65 درجة مئوية لمدة 1 دقيقة و 95 درجة مئوية لمدة 10 دقيقة، التعلية رانه درجة الحرارة كما كان من قبل. </li><li style=";text-align:right;direction:rtl"> تزج الرقاقة في SU-8 حل المطور لمدة 10-15 دقيقة. تحقق لاستكمال التطوير من قبل الشطف مع الأيزوبروبانول. إذا ظهر راسب أبيض، مواصلة تطوير. يظهر القالب الرئيسي في الشكل 2A. </li></ol> 2. الطباعة الحجرية الناعمة لتصنيع متناهية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خلط 35 مل polydimethylsiloxane (PDMS) قاعدة المطاط الصناعي مع 3.5 مل PDMS كيل علاج. </li><li style=";text-align:right;direction:rtl"> وضع القالب الرئيسي ملفقة (خطة مواجهة) ورقاقة السيليكون فارغ في أطباق بتري واصطف مع رقائق الألومنيوم. </li><li style=";text-align:right;direction:rtl"> صب 20 مل PDMS سابق البلمرة في القالب طبق رئيسي و 15 مل في طبق الثاني. القضاء على جيوب الهواء تحت رقائق طريق الضغط بلطف عليهم مع قضيب خشبي القابل للتصرف. </li><li style=";text-align:right;direction:rtl"> تشمل كلا من أطباق ويوضع جانبا لمدة يوم لعلاج. بدلا من ذلك، لسرعة المعالجة، وإزالة فقاعات الهواء من PDMS باستخدام degasifier فراغ ثم ترك الأطباق على طبق ساخن على 80 درجة مئوية لمدة 2الموارد البشرية. </li><li style=";text-align:right;direction:rtl"> إزالة احباط وقشر PDMS من رقائق. </li><li style=";text-align:right;direction:rtl"> استخدام هاريس أحادي النواة (2.5 مم) لكمة منافذ الوصول السائل في كلا طرفي القناة. قطع القناة وPDMS فارغة إلى شرائح ذات الحجم المماثل. </li><li style=";text-align:right;direction:rtl"> تحميل قناة، الفراغ PDMS قطاع وشريحة زجاجية (75 × 25 مم 2) في مؤكسد البلازما، وتقع على الأرجح في غرف الأبحاث. فضح لالبلازما الأكسجين لمدة 40 ثانية في 40 W السلطة. </li><li style=";text-align:right;direction:rtl"> الصاق قطعة قناة وشريحة زجاجية إلى طرفي نقيض في قطاع فارغة. يوضع جانبا لمدة 2 ساعة لاستكمال الربط. </li><li style=";text-align:right;direction:rtl"> وضع الجمعية على طبق ساخن عند 120 درجة مئوية. إرفاق أنابيب بلاستيكية (القطر الداخلي 1/32 &quot;، وقطره الخارجي 3/32&quot;)، كل لا يقل عن 6 بوصة طويلة، إلى خزانات لكمات باستخدام PDMS سابق البلمرة. يضعوا موصل البلاستيك فلويديك على أحد أو كلا أنابيب للسماح المرفقات حقنة، أو استخدام التجهيزات المتاحة تجاريا. </li><li style=";text-align:right;direction:rtl"> السماح للPDMS تأمين أنابيب لعلاج. إدراج 3 &quot;أطوال من 22 مقياس معزول الأسلاك النحاسية في مجموعة شرق افريقياح الخزان، بين أنبوب مدخل والقناة، وآمنة مع PDMS سابق البلمرة. يظهر المنتج النهائي في الشكل 2B. </li></ol> 3. انجذاب كهربي التجربة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع متناهية على المسرح (ويفضل XY-المنقولة) من المجهر مع كاميرا محمولة متصلة إلى جهاز (الشكل 1). </li><li style=";text-align:right;direction:rtl"> توصيل التيار الكهربائي أو الأسلاك الناتج مكبر للصوت لأقطاب متناهية ل. A بسيط العاصمة امدادات الطاقة غير كافية إذا كان المطلوب فقط إشارة DC، لكن مكبر للصوت متصلا وظيفة مولد يسمح تطبيق نابض العاصمة وإشارات AC كذلك. </li><li style=";text-align:right;direction:rtl"> إرفاق متناهية في أنبوب الإخراج إلى الحقنة. يغرق مصب أنبوب مدخل في M9 العازلة الفسيولوجية وبلطف يطمح السائل في القناة عن طريق تطبيق الضغط السلبي داخل الحقنة (إما يدويا أو باستخدام مضخة محقنة). عندما يتم ملأ كل مدخل ومخرج أنابيب مع M9، افصل جمعة حقنةROM الأنبوب. المستويين أنابيب إلى نفس الارتفاع لمنع تدفق مدفوعة hydrostatically. </li><li style=";text-align:right;direction:rtl"> تطبيق الجهد DC إلى القناة والتأكد من أن المقاومة (R = V / I) هو حوالي 0.6 MΩ (ل50 مم طويل، 0.3 ملم واسعة و~ 0.1 ملم متناهية العميقة). </li><li style=";text-align:right;direction:rtl"> إذا كنت راضيا عن سلامة القناة، اتبع الخطوات المذكورة أعلاه لتحميل الديدان من تعليق المخفف في القناة. </li><li style=";text-align:right;direction:rtl"> قطع المحقنة وhydrostatically التلاعب في تدفق من خلال تعديل ارتفاع نسبي الأنابيب &#39;. استخدام هذا الأسلوب لوضع دودة في وسط القناة ثم وضع كل من أنابيب شقة في نفس الارتفاع. </li><li style=";text-align:right;direction:rtl"> تعيين التيار الكهربائي إلى الجهد المناسب: 4-12 V / سم لL3 الحيوانات المرحلة، 4-10 V / سم لL4s، و2-4 V / سم للصغار البالغين. تفعيل إشارة كهربائية والسماح 1 دقيقة من قبل التعرض لدودة للتأقلم إلى الميدان. وينبغي أن تبدأ الدودة تتحرك نحو القطب السالب. عندما اجتاز دقيقة، استخدام الكاميرا لبدء التسجيل. </li><li style=";text-align:right;direction:rtl"> لميلان ونبضد التجارب العاصمة، ويمكن اعتماد أقصى الحقل الكهربائي استجابة من دورة أعلاه وتردد واجب يمكن أن يكون إشارة التضمين كما تريد 7، 8. </li><li style=";text-align:right;direction:rtl"> عندما يتم الانتهاء من التجربة، وإزالة جميع السائل (والديدان) من القناة، وشطفه مع DH 2 0، وترك الجهاز على طبق ساخن عند 125 درجة مئوية لتجف. </li><li style=";text-align:right;direction:rtl"> استخراج البيانات الحركي من أشرطة فيديو مسجلة يدويا باستخدام يماغيج المعاهد الوطنية للصحة ( <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ) أو العرف القائم على MATLAB تتبع البرمجيات دودة. </li></ol>

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وقد قدم ميكروفلويديك انجذاب كهربي تكنولوجيا الفحص لبراءات الاختراع في الولايات المتحدة الأمريكية وكندا.

الاستفادة من هذه الظاهرة السلوكية وصف لأول مرة من قبل غابل والزملاء وبناء على أعمال التلاعب dielectrophoretic من تشوانغ وزملاؤه 11،12، يوفر لنا مقايسة انجذاب كهربي المستندة ميكروفلويديك وسيلة سهلة وقوية وحساسة للتحقيق في نشاط الخلايا العصبية في الديدان باستخدام حركة ك...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CALEDON Labs</td>
			<td>1200-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>CALEDON Labs</td>
			<td>6700-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>CALEDON Labs</td>
			<td>8600-1-40</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SU-8</td>
			<td>Microchem Corp.</td>
			<td>Y131273</td>
			<td>SU-8 100</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem Corp.</td>
			<td>Y020100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92x16 mm Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>Contains elastomer base and curing agent</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Tektronix Inc.</td>
			<td>&nbsp;</td>
			<td>Model AFG3022B</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Trek Inc.</td>
			<td>&nbsp;</td>
			<td>Model 2210-CE</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4506</td>
			<td>Model 11 ELITE</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>11675916Q</td>
			<td>Model HP131725Q</td>
		</tr>
	</tbody>
</table>

microfluidic-based electrotaxis for on-demand quantitative analysis of caenorhabditis elegans' locomotion

في انواع معينة ايليجانس الخيطية هو النموذج الحي تنوعا للبحوث الطبية الحيوية بسبب صيانته من الجينات ذات الصلة بالمرض والممرات وكذلك سهولة زراعة. عدة C. وقد تم الإبلاغ عن نماذج مرض ايليجانس، بما في ذلك الاضطرابات العصبية مثل مرض باركنسون (PD)، والذي ينطوي على انحطاط الدوبامين (DA) الخلايا العصبية 1. وقد استخدمت كل من الجينات المحورة والمواد الكيميائية أعصاب للحث DA تنكس عصبي وعيوب حركة يترتب على ذلك في الديدان، والسماح لتحقيقات في أساس تنكس عصبي وشاشات للجينات الأعصاب ومركبات 2،3. شاشات في أقل حقيقيات النوى مثل C. توفير ايليجانس وسيلة فعالة واقتصادية لتحديد المركبات والجينات التي تؤثر على إشارات العصبية. وعادة ما يتم تنفيذ الشاشات التقليدية يدويا وسجل عن طريق التفتيش البصري، وبالتالي، فهي المرة الجدد<html> uming وعرضة للأخطاء البشرية. بالإضافة إلى ذلك، فإن معظم التركيز على تحليل المستوى الخلوي في حين تجاهل الحركة والتي هي معلمة أهمية خاصة بالنسبة للاضطرابات الحركة. لقد قمنا بتطوير نظام الفرز ميكروفلويديك رواية (الشكل 1) الذي يتحكم والكمي C. تنقل ايليجانس &#39;باستخدام المحفزات الحقل الكهربائي داخل microchannels. لقد أظهرنا أن تيارا الميداني المباشر (DC) يمكن أن تحفز بقوة على الطلب تحرك نحو القطب السالب (&quot;انجذاب كهربي&quot;) 4. عكس قطبية الحقل يسبب الدودة لعكس اتجاهه بسرعة كذلك. لقد أظهرنا أيضا أن العيوب في الخلايا العصبية الحسية الدوبامين وغيرها من تغيير استجابة سباحة 5. ولذلك، شذوذ في إشارة العصبية يمكن تحديدها باستخدام تنقل باعتبارها مقروءة إجراءات المغادرة. استجابة حركة يمكن أن يكون كميا بدقة باستخدام مجموعة من المعلمات مثل سرعة السباحة، هيئة تردد الانحناء والوقت الانعكاس. ق = &quot;jove_content&quot;&gt; كشفت عملنا أن الاستجابة electrotactic يختلف مع تقدم العمر. على وجه التحديد، والاستجابة الشباب إلى مجموعة أقل من الحقول الكهربائية والتحرك على نحو أسرع مقارنة اليرقات 4. أدت هذه النتائج لنا لتصميم جهاز ميكروفلويديك جديدة لفرز بسلبية الديدان حسب العمر والنمط الظاهري 6. لقد اختبرنا أيضا استجابة من الديدان إلى العاصمة نابض والمتناوب (AC) الحقول الكهربائية الحالية. الحقول DC نابض من دورات العمل المختلفة بشكل فعال انجذاب كهربي ولدت في كلا C. ايليجانس ولها ابن عم C. briggsae 7. في تجربة أخرى، متناظرة حقول AC مع ترددات تتراوح ما بين 1 إلى 3 كيلوهرتز هرتز يجمد الديدان داخل القناة 8. تنفيذ الحقل الكهربائي في بيئة ميكروفلويديك تمكن التنفيذ السريع والآلي للمقايسة انجذاب كهربي. وعود هذا النهج لتسهيل شاشات الوراثية والكيميائية عالية الإنتاجية لعواملالتي تؤثر على وظيفة الخلايا العصبية وقدرتها على البقاء.

يتم عرض شريط فيديو ممثل انجذاب كهربي البرية من نوع الديدان الخيطية الشباب الكبار وموقفها والمخرجات سرعة من برنامج تتبع دودة في التكميلية فيديو (1) والشكل (3). برنامج تحليل الحركة نفسها لا تعترف اتجاه قطبية المجال والوقت من انعكاس القطبية، بل يجب الحصول على هذ?...

Watch this Scientific Journal Video about Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion at JoVE.com

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

وهناك طريقة الصغرى الكهربائية فلويديك شبه الآلي للحث تنقل على الطلب في<em&gt; انواع معينة ايليجانس</em&gt; يوصف. ويستند هذا الأسلوب على هذه الظاهرة الفيزيولوجية العصبية من الديدان الاستجابة لمجالات كهربائية خفيفة (&quot;انجذاب كهربي&quot;) داخل قنوات ميكروفلويديك. يقدم ميكروفلويديك انجذاب كهربي باعتبارها وحساسة، تقنية السريع منخفضة التكلفة، وقابلة للكشف عن العوامل التي تؤثر على صحة الخلايا العصبية.

انجذاب كهربي المستندة ميكروفلويديك للتحليل الكمي بناء على الطلب<em&gt; انواع معينة ايليجانس</em&gt; &#39;تنقل

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis

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Bioengineering

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

9.7K Views.  McMaster University. وهناك طريقة الصغرى الكهربائية فلويديك شبه الآلي للحث تنقل على الطلب في انواع معينة ايليجانس يوصف. ويستند هذا الأسلوب على هذه الظاهرة الفيزيولوجية العصبية من الديدان الاستجابة لمجالات كهربائية خفيفة ("انجذاب كهربي") داخل قنوات ميكروفلويديك. يقدم م?...

Video: Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

تحسن التصور والتحليل الكمي لتأثيرات الدواء باستخدام خلايا Micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

A فخ هيدرودينامي ميكروفلويديك المستندة للجسيمات واحدة

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.
Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.2
C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.

دراسة كمية تحرك بحرية من حوض الكائنات الحية الدقيقة باستخدام حيود الليزر

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

الكمية الحنق (فورستر نقل الطاقة الرنين) تحليل لتحديد حركية SENP1 البروتياز

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

المخدرات التي يسببها-توعية انزيمات محلقة: تبسيط الفحص والتصغير لجزيء صغير وسيرنا تطبيقات الفحص

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

وبيوشيب الكهروكيميائية استنادا ميكروفلويديك لتسمية خالية تحليل الحمض النووي التهجين

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

الدراسات انجذاب كهربي من الرئة خلايا سرطان باستخدام القنوات المتعددة ثنائي الكهربائية الميدان ميكروفلويديك رقاقة

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

متعدد الألوان كشف الإسفار عن القطرة على microfluidics عن طريق الألياف البصرية

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

وضع بروتوكول فعال وقابل لإعادة الإنتاج لالهاء تكون العظم في نموذج الفئران مما أدى إلى عظم الفخذ المجددة الوظيفية

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

منصة موائع جزيئية "التصوير الطولي" في ايليجانس كاينورهابديتيس

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...