NIH hibe HL89764, NSF hibe 0.546.012, 0.730.503 ve 0.725.528 tarafından sağlanan fon desteği.

DNA A. biyotinilasyon Plazmid DNA kovalent olarak üretici (Mirus Bio, Madison, WI) tarafından açıklanan, ancak ~ DNA ortalama 1-2 biotin etiketler ölçekli guanin özel biotin etiketleri ile biotinlenmiş. Plazmid DNA (pEGFP-C1, 4,9 kb, Clontech, Mountain View, CA) bu protokol etiketli oldu. <ol><li> 1μg/μL DNA çözümü DNaz-özgür ve RNaz-TE tamponu (moleküler biyoloji sınıf kaliteli) suya pDNA istenilen miktarda eritilir. </li><li> Aşağıdaki reaksiyon karışımları kullanarak etiketleme reaksiyon Davranış. BT reaktif son Etiket ekleyin. </li></ol> 100μg DNA reaksiyon için: <table border="1"><tbody><tr><td> DNaz ücretsiz ve RNaz ücretsiz su </td><td> 75 mcL </td></tr><tr><td> 10X Etiketleme Tampon A </td><td> 20 mcL </td></tr><tr><td> 1μg/μL DNA </td><td> 100 mcL </td></tr><tr><td> BT reaktif Etiket </td><td> 5 mcL </td></tr><tr><td> Toplam Hacim </td><td> 200 mcL </td></tr></tbody></table><ol start="3"><li> 37 ° C&#39;de 1 saat süreyle reaksiyon inkübe edin. </li><li> Etanol veya isopropanol yağış standart protokoller etiketli numune arındırın. </li></ol> Not: Jel filtrasyon tabanlı sütun DNA sayısallaması veya floresan karakterizasyonu etkileyebilir yüksek UV absorbans veya floresan arka plan yol açabilir. Not: biyotinilasyon düzeyi haba tabanlı testler ile belirlenebilir. Cy5 Katyonik Polimer B. Etiketleme Chitosan (% 83.5 deacetylated 390 kDa Vanson, Redmond, WA) bu çalışmada bir model katyonik polimer olarak kullanılmıştır. Kitosan polimer omurgasına ücretsiz olarak birincil aminler Cy5-NHS (Amersham Biosciences, Piscataway, NJ) ile etiketlenir. <ol><li> Cy5 boya komple konjugasyon kolaylaştırmak için, Cy5 NHS gerekli miktarı hesaplamak Cy5, molar oranda böyle: 200: birincil aminler 1. </li><li> NaOH ilavesi ile ~ 6.5 pH kitosan çözümü (25mm asetat tampon) ayarlayın. Not NHS reaksiyon bazik pH daha etkilidir, ancak burada chitosanın çözünürlük çalışma pH aralığı sınırları. </li><li> Karıştırarak iken, yavaş yavaş bir damla bırak şekilde kitosan çözüm Cy5-NHS (1 mg / ml DMSO) hesaplanan miktar ekleyin. </li><li> Gece karanlıkta, oda sıcaklığında karışımı çalkalayın. </li><li> Arındırmak için,% 1 asetat tamponu karşı karanlıkta oda sıcaklığında 2 saat süreyle 10k MWCO Slayt-a-Lyzer (Pierce) ile dialyze. </li><li> 2 saat karanlıkta, oda sıcaklığında bir başka tampon ve dialyze değiştirin. </li><li> Tampon ve 4 gecede dialyze ° C karanlıkta değiştirin. </li><li> Mağaza saflaştırılmış etiketli -20 ° C&#39;de polimer </li></ol> Not: Bu çalışmada, standart bir eğri, 670 nm Cy5-NHS ester emisyon yoğunluğu ölçümü ile inşa edilmiştir . Standart eğri Cy5 etiketli kitosan 670 nm&#39;de elde edilen emisyon ölçüm etiketleme yoğunluğu karakterize eder. Absorbans etiketleme etkinliğini belirlemek için de kullanılır olabilir ama burada değildi. QD etiketli DNA ve Cy5-Polimer C. Hazırlık QD için pDNA molar oranı aşan tutulur (pDNA: QD ≈ 1: 2) pDNA için QDS tam konjugasyon sağlamak. TEM görüntüleme veya eşdeğer diğer tesisleri ile her pDNA üzerine etiketli QDS sayısı tahmin edilebilir. Bizim çalışmamızda, pDNA ortalama QDS sayısı olduğu tahmin edilmektedir ~ TEM ve tek bir molekül spektroskopisi tarafından 1-3. Hazırlanması sırasında 1 Kullanım Millipore Milli-Q degrade su (0.2um süzülmüş&gt; 18.0 MW). <ol><li> 10μg pDNA istenilen N / P oranına göre chitosanın gerekli miktarda DNA çözümü olumsuz fosfat kitosan çözüm Protonlaşmış aminlerin teorik oranını hesaplayın. </li><li> Biotinlenmiş pDNA çözüm streptavidin Fonksiyonlu 605QDs (Qdot 605 ITK, Invitrogen, Carlsbad, CA) ekleyin. </li><li> 15 dakika süreyle karanlıkta, oda sıcaklığında çözüm inkübe edin. </li><li> QD-işaretli DNA son hacmi 200μL yapmak için 50 mM sodyum sülfat çözeltisi içine ekleyin. </li><li> Milli-Q su ile son hacim 200μL yapmak için, istenilen N / P oranı göre, Cy5-kitosan seyreltilir. </li></ol> Not: mümkün photobleaching önlemek için koyu reaksiyon tutun. Önemli: Bu katalogda Kuantum nokta FRET bir amaç için tasarlanmış Qdot 605 ITK ™ streptavidin konjuge (ITK serisi), kullanmak için dikkatli olun. Düzenli Qdot serisi özellikle hücresel etiketlenmesi için, non-spesifik bağlama önlemek için PEG tabakası ile konjuge. Ancak, bu ek kaplama r, donör-alıcı uzaklığı büyütür.enerji transfer verimliliği educed. Standart Fotolitografi SU-8 Master D. Fabrikasyon <ol><li> Si gofret piranha ° C &#39;de 5 dakika süreyle temizlenmelidir ve 200 pişmiş. </li><li> 25μm, belirlenen ana kalınlığı, spin kat 30 saniye boyunca 2000 rpm&#39;de Si levhası üzerindeki olumsuz fotorezist (SU-8 2025, Microchem, Newton, MA). </li><li> Yumuşak fırında 65 bir rampa ile ocak gofret ° C / saat - 95 ° C </li><li> 250mJ/cm 2 için UV ışık (365nm), mikro tasarım içeren bir maske film (CAD / Sanat Hizmetleri, Bandon, OR) yoluyla maruz . </li><li> Maruziyet sonrası, 65 bir rampa ile bir ocak gofret fırında ° C / saat - 95 ° C </li><li> SU-8 fotorezist geliştirici kullanarak gofret geliştirin. </li><li> Desenli gofret sert bir rampa, 65 ocak üzerinde pişirilir ° C / saat ile 200 ° C Oda sıcaklığına kadar gofret, daha sonra yavaş yavaş serin, en az 5 saat süreyle 200 ° C&#39;de gofret koruyun. </li></ol> Önemli: süresince kademeli Ramping SU-8 ana pişirme işlemi gereklidir, aksi takdirde SU-8 yapısı silikon gofret veya çatlak kopuk SU-8 yapısı, stres sürümü ile indüklenen olabilir . Kapak Cam ustaları ve Bağlanma PDMS E. Replica Kalıp <ol><li> SU-8 ana tartı tekne yerleştirilir. </li><li> 10 karıştırın silikon elastomer ve kür ajanı (Poli (dimethylsiloxane), PDMS, Sylgard 184, Dow Corning, Midland, MI): 1 oranında. </li><li> SU-8 ana PDMS karışımın üzerine dökün ve kabarcıklarını çıkarmak için bir vakum desikatörde tartım tekneden ayrılabilirsiniz. </li><li> 1-2 saat 65 ° C&#39;de PDMS Cure. </li><li> Si master kalıp PDMS şerit soyun. </li><li> Punch kanal girişleri ve çıkışları akışkan cihaz. </li><li> PDMS şerit ve etanol ile cam kapak ve hava kuru temizleyin. </li><li> Oksijen plazma ile temizlenmelidir PDMS şerit ve cam kapak (1dk için 20W) davranın. </li><li> Cam kapak ile hemen bağ PDMS şerit. </li><li> 95 ° C&#39;de gece boyunca fırında gümrüklü mikroakışkan çip bırakın. </li></ol> Önemli: Plazma tedavi ve gecede pişirme gelişmiş yapıştırma gücü için gerekli. F. Monitör mikroakışkan Aygıt DNA Nanocomplexes oluşumu <ol><li> Deney sırasında düzgün akışını sağlamak için reaktifler yüklemeden önce, mikroakışkan kanal (mikroakışkan kanal içinde herhangi bir baloncuklar olmadığından emin olmak için) su ile doldurun. </li><li> Video açıklanan boru yoluyla, iki ayrı cam şırınga QD etiketli DNA ve Cy5 etiketli kitosan çözümleri yükleyin. </li><li> Tüp mikroakışkan cihazlar iki girişleri ile bağlayın. Işlemi sırasında herhangi bir hava tanıtmak için dikkatli olun. Laminer akış koşulları altında, 20nL/min (PHD-2000 şırınga pompası, Holliston, MA) akış hızını ayarlayın. </li><li> Mikroskop altında mikro kontrol edin. </li><li> Akış stabil (~ 15 ila 20 dakika), QD-aracılı kanalın merkezi dikkat edilmelidir FRET. </li><li> Kanal boyunca farklı yerlerde floresan resimler (Soğutmalı CCD, Qimaging, BC, Kanada) ele alalım. </li><li> ImageJ ve OriginLab floresan görüntüleri analiz. </li></ol><img src="/files/ftp_upload/1432/1432fig1.jpg" alt="Şekil 1" /> Şekil 1. QD-FRET DNA Nanocomplexes self-montaj başlangıç ​​hassas bir gösterge sağlar <ol style="list-style-type:lower-alpha;"><li> Kuantum nokta-aracılı floresan rezonans enerji transferi (QD FRET) net tespiti için DNA ve gen taşıyıcı arasındaki etkileşimleri başlangıç ​​sağlayan, ekstra ya da hücre içi ortamlarda ya polyplex istikrar nicel ve çok hassas bir göstergesi sağlayabilir. FRET çifti, 605QD ve Cy5, donör ve alıcı arasındaki spektral örtüşme maksimize etmek ve potansiyel çapraz konuşma minimize dayalı seçildi. Bu çifti için, Förster mesafe 69.4Å 3 </li><li> Self-assembly QD-FRET DNA nanocomplexes. Anyonik plazmid DNA (pDNA) ve katyonik gen taşıyıcı, sırasıyla QD (enerji donör) ve Cy5 (enerji alıcı) ile işaretlenmiştir. QD-FRET nanocomplexes elektrostatik karmaşık Koaservasyon yoluyla oluşmuştur. QD-FRET aracılı Cy5 emisyon 488 nm uyarma üzerine, kompakt ve sağlam nanocomplex oluşumu göstermiştir. Kalma süresi (t R) iki dere soruşturma altında reaksiyon konumu ve ortalama akış hızı (v) buluştuğu önlemler mesafe (x) göre hesaplanabilir. Karıştırma, laminer akış doğası nedeniyle sadece hassas hesaplama t R fonksiyonu olarak toplu taşıma izin arayüzü (her görüntünün merkezi) yer alır. Zamansal çözünürlük applie değiştirerek ayarlanabilird akış oranları. (Ankastre) FRET-aracılı sinyal uygulanan akış hızlarına göre birkaç milisaniye içinde meydana gelen bu bağlama hızlı olduğunu belirten, iki dere bir araya geldi arayüzü hemen gözlendi. Ölçek Bar: 100μm. </li></ol>

<ol>
	<li>Ho, Y. P., Chen, H. H., Leong, K. W., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+intracellular+stability+and+unpacking+of+DNA+nanocomplexes+by+quantum+dots-FRET.">Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.</a> J Control Release. 116, 83-89 (2006).</li><li>Chen, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+comparison+of+intracellular+unpacking+kinetics+of+polyplexes+by+a+model+constructed+from+quantum+dot-FRET.">Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET.</a> Mol. Ther. 16, 324-332 (2008).</li><li>Zhang, C. Y., Yeh, H. C., Kuroki, M., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Quantum-Dot-Based+DNA+Nanosensor.">Single-Quantum-Dot-Based DNA Nanosensor.</a> Nat Mat. 4, 826-831 (2005).</li></ol>

The authors have nothing to disclose.

<ul><li> Yaptığımız işin önemi: <ol><li> Bu basit bir mikroakışkan çipi içinde QD-FRET yanıtları aracılığıyla gerçek zamanlı olarak polimerik DNA nanocomplex kendini montaj kinetiği (milisaniye çözünürlük) izlemek için ilk girişimi. </li><li> Mikroakiskan mekansal, DNA nanocomplex sentezi sırasında sürecini analiz etmek için iyi kontrollü mikroçevresinin sunuyor ise QD-aracılı FRET, moleküler etkileşimlerin başlangıcı ve self-montaj süreci boyunca son derece duyarlı ve kantitatif bir gösterge sağlar. </li><li> Mikroakiskan ve sahipliği yapacaktır entegrasyonu kompleks reaksiyonlar her türlü araştırmak için yeni ve ilginç bir yaklaşım göstermektedir. </li><li> Çıkan QD-FRET polimerik DNA nanocomplexes yapı-işlev ilişkileri kurmak için kolayca uygulanabilir. 1,2 </li></ol></li></ul>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>SU-8</td>
<td>&nbsp;</td>
<td>MicroChem</td>
<td>SU-8 2025</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid DNA</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>pEGFP-C1</td>
<td>4.9 kb, MW = 3.3 x 106</td>
</tr>
<tr>
<td>LabelIT Biotin Labeling Kit</td>
<td>&nbsp;</td>
<td>Mirus Bio LLC</td>
<td>MIR 3400</td>
<td>Standard protocol yields labeling efficiency of approximately one label every 20-60 bp of double-stranded DNA, The density of labeling was adjusted in this work.</td>
</tr>
<tr>
<td>Streptavidin 605QD</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Qdot&reg; 605 ITK&trade; Streptavidin Conjugate</td>
<td>&nbsp;</td>
<tr>
<td>Cy5-NHS Ester</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>PA15101</td>
<td>&nbsp;</td>
<tr>
<td>Chitosan</td>
<td>&nbsp;</td>
<td>Vanson</td>
<td>390 kDa </td>
<td>83.5% deacetylated</td>
</tr>
<tr>
<td>Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545C</td>
<td>No. 1; Size: 40 x 22mm</td>
</tr>
<tr>
<td>Gastight Glass Syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>TLL series</td>
<td>50&mu;L to 500&mu;L depending on sample volume</td>
</tr>
<tr>
<td>Tygon Tubing</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>0.02 ID, 100ft</td>
<td>Tygon Tubes Microbore, 0.02 ID, 100ft</td>
</tr>
<tr>
<td>Connector</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>HTX-23R</td>
<td>Customized in length of 0.750"</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD-2000</td>
<td>&nbsp;</td>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Qimaging</td>
<td>Intensified Retiga Cooled</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX-51</td>
<td>100W mercury arc lamp</td>
</tr>
<tr>
<td>ImageJ</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>v1.36b</td>
<td>http://rsb.info.nih.gov/ij</td>
</tr>
<tr>
<td>Origin Pro8</td>
<td>&nbsp;</td>
<td>OriginLab</td>
<td>Student Version</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>475AF40</td>
<td>Excitation filter in both channels</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595AF60</td>
<td>Emission filter in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>670DF40</td>
<td>Emission filter in QD-FRET channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>500 DRLP</td>
<td>Long pass dichroic in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595DRLP</td>
<td>Long pass dichroic in QD-FRET channel</td>
</tr>		</tbody>
		</table>
		</div>

combining qd-fret and microfluidics to monitor dna nanocomplex self-assembly in real-time

Genomik gelişmeler, hücresel ve moleküler düzeyde patogenezi hedef terapötikler gelişme yakıt devam ediyor. Genelde hücre içinde fonksiyonel, nükleik asit temelli tedaviler etkili bir hücre içi dağıtım sistemi gerektirir. Bir yaygın olarak kabul edilen bir yaklaşım bozulması karşı koruyarak DNA hücresel alımını kolaylaştıran, elektrostatik kendini montaj yoluyla nanocomplexes oluşturmak için bir gen taşıyıcı ile karmaşık DNA. , Erken disosiasyon ya da çok istikrarlı bağlama hücresel alımı ve terapötik etkinliği için zararlı olabilir çünkü meydan okuma, etkin gen taşıyıcıların rasyonel tasarım yatıyor. Toplu karıştırma tarafından sentezlenen Nanocomplexes nanocomplexes büyüklüğü ve istikrar içinde heterojen atfedilen açma hücre içi ve ticareti davranış, çeşitli gösterdi. Böyle bir heterojenite, kendini montaj kinetiği doğru değerlendirme engelleyen ve onların fiziksel özellikleri transfeksiyon verimliliği veya bioactivities ilişkilendirerek zorluk ekler. Biz sahipliği yapacaktır (yani QD-FRET) ve Mikroakiskan laminer akış altında nanocomplex kendini montaj gerçek zamanlı kinetiği karakterize bir roman yakınsama sunuyoruz. Mikroakiskan yüksek temporal çözünürlük (~ milisaniye) ile mekansal sürecini analiz etmek için iyi kontrollü mikroçevresinin sunar ise, QD-FRET sentez süreci boyunca moleküler etkileşimler ve kantitatif ölçümü başlamasından son derece hassas bir göstergesi sağlar. Polimerik nanocomplexes modeli sistem için, iki ayrı self-montaj işlemi aşamalarında bu analitik platformu tarafından ele geçirildi. Microscale elde edilen öz-montaj işlemi kinetik yönü birçok mikro ve nano ölçekli uygulamalar için ilgili microreactor tabanlı reaksiyonlar için özellikle değerli olacaktır. Ayrıca, nanocomplexes microfludic cihazların uygun tasarımı sayesinde özelleştirilmiş olabilir, ve elde edilen polimerik DNA nanocomplexes yapı-işlev ilişkileri kurmak için kolayca uygulanabilir QD-FRET.

Watch this Scientific Journal Video about Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time at JoVE.com

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

Biz gelecekteki nükleik asit temelli tedaviler için daha iyi kontrol ve sentez üniforma ve özelleştirilebilir polyplexes sağlaması bekleniyor polielektrolit polyplexes oluşumunu araştırmak için yeni ve güçlü bir entegrasyon sahipliği yapacaktır (QD-FRET) ve Mikroakiskan sunuyoruz.

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

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

combining-qd-fret-microfluidics-to-monitor-dna-nanocomplex-self

Research

JoVE Journal

Biology

11.0K Görüntüleme.  Johns Hopkins University. Biz gelecekteki nükleik asit temelli tedaviler için daha iyi kontrol ve sentez üniforma ve özelleştirilebilir polyplexes sağlaması bekleniyor polielektrolit polyplexes oluşumunu araştırmak için yeni ve güçlü bir entegrasyon sahipliği yapacaktır (QD-FRET) ve Mikroakiskan sunuyoruz.

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

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

Mikro Ölçekli Besin Katmanlar Deniz Mikro-organizmalar kemotaktik Tepki

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Biyoloji

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

Mikroakiskan kullanarak Bakteriyel Kemotaksis Çalışmaları - Röportaj

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

Elektrofizyoloji için Mikroakiskan uygulama

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Otomatik Proteomik İşleme Dijital Mikroakiskan

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Genetik İfade Profilleri Belirlenmesi C. elegans Mikroarray ve Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Protein-DNA Etkileşiminin Çalışmaları Tek molekül Manipülasyon ve Görüntüleme birleştiren

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-tabanlı Real-time PCR microRNA Kantitatif Ölçüm Yaklaşımları

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Kristalin Selüloz Birikimin Yüksek Çözünürlüklü Niceleme<em&gt; Arabidopsis</em&gt; Kökler Doku spesifik Hücre Duvarı Değişiklikler Monitör

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

pHluorin etiketli Reseptör kullanarak Subselüler Yerelleşme ve Ticareti Monitör

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...