The authors would like to acknowledge Ph.D. grant of LANEF in Grenoble for support of Laurie-Amandine Gar&#231;on. This work was financially supported by the French National Research Agency (ANR-grant 06-NANO-045).

Çeşitli Çözümleri ve Protein Örneklerinin 1. Hazırlık <ol><li> , 50 mM NaH 2 PO 4, 50 mM NaCl ihtiva eden fosfat tampon çözeltisi (PBS-G) 100 mi, pH 6.8 &#39;de% 10 gliserol hazırlayın. </li><li> PH 7.4 olan 10 mM HEPES, 150 mM NaCl,% 0.005 Tween 20 içeren HEPES tampon çözeltisi 250 ml hazırlayın. </li><li> PBS-G 0.2 mM&#39;de laktoz (Şekil 1) sülfatlanmış yapı bloğunun 1 (BB1), laktoz ve yapı taşı 2 (BB2) içinde bir stok çözelti hazırlayın. </li><li> 1 uM A rakis hypogaea lektin (AHL) 500 nM, miyoglobin de ve 500 nM&#39;de lisozim: HEPES, protein çözeltisinin 1 ml hazırlayın. </li><li> Ultra-saf su içinde% 1 SDS ile 20 ml hazırlayın. </li></ol> CoCRR Array 2. Hazırlık <ol><li> % 75 oksijen,% 25 argon, 0.6 mbar, gücü 40 W: Bu koşullar altında 3 dakika boyunca plazma temizleyici ile kullanımdan önce 48 saat prizma altın yüzeyini temizleyin </li><li> Onbir saf ve karışık Solutio hazırlayın[BB1] / ile BB1 ​​ve BB2 ns ([BB1] + [BB2]), PBS-G içinde 0,1 mM toplam bb konsantrasyonda% 10 artışlar ile, 0 ile% 100 arasında oranları. </li><li> Depozit toplam 44 noktanın (Şekil 2), her oran için dört kat olarak temassız bir gözcü kullanılarak prizma yüzeyi üzerindeki bu saf ve karışık çözümler 8 nl damlacıkları. Not: PBS-G çözeltisine,% 10 gliserol, solvent buharlaşması ve çökelme sonrası bb konsantrasyon değişimini azaltmak için çok önemlidir. </li><li> Ultra saf su 1 ml içeren bir Petri kabı içinde prizma yerleştirin ve altın yüzeyde BB1 ​​ve BB2 kendini düzenlemesinden RT&#39;de göre O / N bırakın. Burada, bu karışık SAM ortalama yüzey bileşimi yerleştirme karıştırılmış çözeltisinin bileşimi (Şekil 3) 11 yansıttığı varsayılmaktadır. </li><li> Ultra-saf su ile iyice yıkayın ve daha sonra prizmanın bir argon akışı altında kurutun. </li></ol> 3. Protein Algılama SPRI tarafından <ol><li> Incubato ayarlayınR&#39;nin SPRI cihaz, protein algılama sırasında sıcaklık değişiminin neden olduğu kırılma indeksi değişiklikleri önlemek için 25 ° C sıcaklıkta yerleştirilir. </li><li> (PEEK), bir bilgisayar kontrollü bir şırınga pompası, gaz alıcı ve 6 bağlantı noktası, orta basınçlı enjeksiyon valfına bağlanan akış hücresinin (Şekil 4) 10 ul polieter eter keton olmayan bir işlevselleştirilmiş durulama prizma yerleştirin. Taze süzülmüş ve tampon HEPES çalışan degaze ile akış sistemi doldurun. </li><li> Durulama prizma çıkarın ve akış hücresi CoCRR dizi içeren prizma yerleştirin. 100 ul / dakika akış hızında, HEPES. CCD kamera yardımı ile hızlı çalışan tampon geçirilerek prizma yüzeyi üzerinde mevcut olan hava kabarcıklarını. </li><li> Dizinin iyi bir tezat görüntü ve SPRI sistemine entegre yazılım yardımı ile esaslı ilgi alanı için aynı çapa sahip bir daire çizerek, her nokta için çalışma alanı tanımlayın. </li><li> Yansıtan temsil plazmon eğrileri, İztüm noktalar için olay açısı, fonksiyonunda ivity eğrileri. </li><li> Yansıtma eğrilerinin en yüksek yamacında çalışma açısının (kinetik eğrileri kaydedilmiş olacak hangi pozisyon) seçin. Kinetik ölçümleri için seçilen çalışma açısına düzeltmek için tarama ayna döndürün. </li><li> Tüm noktalar için yansıma sinyal istikrarlı ve sürekli kadar akış sisteminde HEPES çalışmaya devam eder. </li><li> Konumu &quot;yük&quot; enjeksiyon valfi ile numune döngü (500 ul) içine şırınga ile 1 ml AHL çözüm enjekte. Ardından &quot;enjeksiyon&quot; konumuna enjeksiyon valfi koydu. Tüm protein enjeksiyonu için kullanılan akış hızı 100 ml / dak idi. </li><li> Tüm noktalar aynı anda zamana karşı yansıtma değişimlerini izleyerek kinetik ölçümler başlayın. </li><li> Proteini enjekte sonunda, 8 dakika boyunca tampon maddesi ile dizi yıkayın. Son olarak, yeniden 1 ml% 1 SDS ile enjekte edilir. </li><li> Diğer p için aynı prosedürü tekrarlayınroteinler. </li></ol> 4. Veri İşleme ve Analizi <ol><li> Akış hücreye protein çözeltisi girişi sonra, bağlayıcı molekül ortaya çıkar ve plazmon eğrilerinin bir kayma ve yansıtma bir varyasyonunu neden olur. Adı geçen görüntü edinim program sensogramlarını (Şekil 5B) vererek SPR görsel Şekil 5A verir gri skala seviyelerinin, ölçülen ışık şiddeti değerleri dönüştürür ve zamana karşı yansıtma değişimini üretir. </li><li> [BB1] karşı her proteini enjekte sonunda yansıtma (R%) çizmek için bir matematik bilgisayar yazılımını kullanın / ([BB1] + [BB2]) Her bir numune için 2D sürekli evrim profili (CEP) oluşturmak için oranı (Şekil 5C). </li><li> [BB1] / ([BB1] + [BB2]) sensörgramlar içinde oranı Ekle (Şekil 5B) her proteinin (Şekil 5D) için 3D sürekli evrim manzara (CEL) oluşturmak için. </li></ol>

<ol>
	<li>Margulies, D., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+protein+recognition+as+an+alternative+approach+to+antibody-mimetics.">Combinatorial protein recognition as an alternative approach to antibody-mimetics.</a> Current Opinion in Chemical Biology. 14, 705-712 (2010).</li><li>Umali, A. P., Anslyn, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+approach+to+differential+sensing+using+synthetic+molecular+receptors.">A general approach to differential sensing using synthetic molecular receptors.</a> Current Opinion in Chemical Biology. 14, 685-692 (2010).</li><li>Baldini, L., Wilson, A. J., Hong, J., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern-based+detection+of+different+proteins+using+an+array+of+fluorescent+protein+surface+receptors.">Pattern-based detection of different proteins using an array of fluorescent protein surface receptors.</a> J. Am. Chem. Soc. 126, 5656-5657 (2004).</li><li>Zhou, H. C., Baldini, L., Hong, J., Wilson, A. J., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+of+proteins+based+on+an+array+of+functionalized+porphyrins.">Pattern recognition of proteins based on an array of functionalized porphyrins.</a> J. Am. Chem. Soc. 128, 2421-2425 (2006).</li><li>Wright, A. T., Griffin, M. J., Zhong, Z., McCleskey, S. C., Anslyn, E. V., McDevitt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+receptors+create+patterns+that+distinguish+various+proteins.">Differential receptors create patterns that distinguish various proteins.</a> Angew. Chem. Int. Ed. 44, 6375-6378 (2005).</li><li>Wright, A. T., Anslyn, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+receptor+arrays+and+assays+for+solution-based+molecular+recognition.">Differential receptor arrays and assays for solution-based molecular recognition.</a> Chem. Soc. Rev. 35, 14-28 (2006).</li><li>You, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+proteins+using+nanoparticle&#8211;fluorescent+polymer+&#8216;chemical+nose&#8217;+sensors.">Detection and identification of proteins using nanoparticle&#8211;fluorescent polymer &#8216;chemical nose&#8217; sensors.</a> Nature Nanotechnology. 2, 318-323 (2007).</li><li>De, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+proteins+in+human+serum+using+conjugates+of+nanoparticles+and+green+fluorescent+protein.">Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein.</a> Nature Chemistry. 1, 461-465 (2009).</li><li>Hou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+evolution+profiles+for+electronic-tongue-based+analysis.">Continuous evolution profiles for electronic-tongue-based analysis.</a> Angew. Chem. Int. Ed. 51, 10394-10398 (2012).</li><li>Campbell, C. T., Kim, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPR+microscopy+and+its+applications+to+high-throughput+analyses+of+biomolecular+binding+events+and+their+kinetics.">SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics.</a> Biomaterials. 28, 2380-2392 (2007).</li><li>Stranick, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometer-scale+phase+separation+in+mixed+composition+self-assembled+monolayers.">Nanometer-scale phase separation in mixed composition self-assembled monolayers.</a> Nanotechnology. 7, 438-442 (1996).</li></ol>

The authors have no conflicts of interest to declare.

<html> Bu eT yapımı için en kritik adımlar sisteminin iyi tekrarlanabilirlik sağlamak için varız. Örneğin, BB1 ve BB2 ait on saf ve karışık solüsyonlarda gliserol% 10 unu ilave, kullanımdan önce, standart bir prosedür ile prizma altın yüzey temizleme BB prizma altın yüzey üzerinde kendi kendine montaj sırasında çözücü buharlaşmasını önlemek için, Her [BB1] için birden çok tekrar / ([BB1] + [BB2]) oranı, vs bırakılması. SPRI protein tespiti için olduğu gibi, çalışma aç?...

Hassas ve hızlı protein algılama yöntemleri tıbbi teşhis ve Proteomikte çok önemlidir. Böyle bioçipler gibi klasik protein-tespit diziler, &quot;kilit ve anahtar&quot; tanıma ilkesine dayalı ve bu aptamers, antikorlar, veya taklitçi gibi belirli reseptörleri gerektirir. Son yıllarda, insan koku duyusunun ve tadına bakma esinlenerek diferansiyel algılama alternatif 1 olarak ortaya çıkmıştır. Bu elektronik burun / dil (eN / eT) yaklaşımı hedef moleküller için son derece spesifik veya seçici olması gerekmez çapraz-reaktif reseptörleri (crrs), bir dizi Analitlerin farklı bağlanmanın dayanmaktadır böylece zahmetli aşmak için izin yüksek ölçüde seçici reseptörleri geliştirme süreci. Onun teşhisine izin veren, bir parmak izi gibi, her bir örnek için ayrı bir desen oluşturur tüm reseptörlerinin birleşik yanıttır. ELEC gelişimi için iki önemli zorluklaretkin bir protein algılama tronic burun / yay yapısal olarak benzer bir analit ve bağlanma olayı için uygun olan iletim sistemine arasında ayrım yeteneğine sahip algılama elemanlarının üretimi. Şimdiye kadar yapılan çalışmalar dizi gelişme 2 için çeşitli yaklaşımlar bildirdi. Bir çalışmada Örneğin proteinlerin bir dizi-bazlı kimlik çeşitli amino asitler veya proteinler ve ücret için farklı çevresel birimlere afinite için bir hidrofobik çekirdeğin sahip diferansiyel reseptörleri sağlamak dipeptidlerin için karboksil grupların birleştirilmesi ile, tetra-carboxyphenylporphyrin türevlerinden hazırlanabilir crrs kullanılarak geliştirilmiştir kazandıran farklı bağlama. Bu sistemi kullanarak, farklı proteinler ve protein karışımları analitlerle etkileşimin ardından 3,4 reseptörlerinin floresanlık sönmesini ölçülerek tespit edilmiştir. Başka bir çalışmada, hexasubsti bir birleştirici şekilde sentezlendi, tripeptid ve boronik asit kısımları içeren 29 crrs oluşan bir kütüphanedeğiştirilmemiş benzen iskele bir göstergesi alım kolorimetrik algılama 5,6 proteinleri algılama için geliştirilmiştir. Böyle bir tasarımla, her reseptör glikoproteinlerden proteinlerinin ayrımında destekli peptid kollardaki varyans göre protein ve boronik asitler ile farklı bağlama kapasitesini göstermiştir. Daha yakın zamanda, bir anyonik floresan polimer poli (p-phenyleneethynylene) (PPE) konjüge edilmiş farklı katyonik fonksiyonalize altın nanopartiküllerinin oluşan bir dizi tespit etmek ve tanımlamak için proteinleri 7 oluşturuldu. Rekabetçi protein için farklı tanıma desen üreten bir protein analit arasındaki bağlanma ve floresan yeniden PPE / altın nano partiküler kompleksleri söndürüldü. Bu çalışmada, fonksiyonalize nanopartikül-protein etkileşimleri nanopartikül uç gruplarının fizikokimyasal özellikleri değiştirilerek ayarlanmıştır. Ayrıca bu yaklaşım, karmaşık ve protein açısından zengin bir ortamda bu protein analizi için etkili olduğu gösterilmiştirfizyolojik ilgili konsantrasyonlarda insan serumu gibi, böylece hastalık durumlarının 8 teşhisi için gerçek örnekleri profil olarak eT potansiyelini gösteren. Çok umut verici olsa da, bu sistemler bazı sınırlamaları vardır. Onlar tasarımı ve oldukça karmaşık yapılara sahip 5 ila 29 crrs sentezlenmesi gerektirir. Buna ek olarak, her bir ölçüm takip sıfırlanır koku sistemi farklı olarak, protein, numune başına bir algılama dizisi hazırlamak gerekir. Son olarak, gerçek zaman bağlanma olguları arasında son derece zordur. Bu bağlamda, bir kombinatoryal yaklaşım, farklı fiziko-kimyasal özelliklere sahip, basit ve kolayca erişilebilir moleküllerinin küçük bir numara kullanarak sürülmüştür (negatif yüklü bir nötr, pozitif yüklü, hidrofilik, hidrofobik, vb.) Yapı blokları (BB) 9 gibi. Ile karışımlar değişen BBS ve kontrollü oranlarda karıştırılması ve izin vererek bir altın yüzey tarafından monteprizma, protein bağlanması için uygun özelliklere sahip birleşik yüzeyler bir dizi oluşturuldu. Özellikle, bu sistem üzerindeki kendinden düzenlenen tek tabakalar, hızlı ve verimli bir şekilde üretilebilir olması için çeşitli birleştirici çapraz-reaktif reseptörleri (CoCRRs) sağlayacak şekilde, son derece farklı bir şekilde yüzey özellikleri, bir dizi kolay bir ayarlama sağlar. Protein algılama optik tespit sistemi kullanılarak gerçekleştirildi, plazmon rezonans görüntüleme (SPRI) yüzey. Kısaca, bir LED&#39;den gelen geniş bir ışın, polarize ışık monokromatik prizma yüzeyi üzerindeki bütün CoCRR dizi aydınlatır. Bir yüksek çözünürlüklü CCD video kamera CoCRR dizinin bütün noktalar arasında gerçek-zamanlı farkı görüntüler sağlar. Bu bağlayıcı olaylar ve kinetik süreçler 10 hakkında detaylı bilgi veren CoCRR dizinin yüzeyinde yerel tüm değişiklikleri yakalar. Bu arada, görüntüleme yazılımı yardımı ile, noktalar karşılık SPR görüntüleri otomatik olarak yansıtma Versu varyasyonları dönüştürülürSensogramlar adı verilen kinetik bağlanma eğrileri, bir dizi üreten bir zaman. Bu nedenle, bağlama SPRI etkinlikleri etiketi içermeyen, eş zamanlı, paralel ve gerçek zamanlı gözlem sağlar. Ayrıca, elde edilen CoCRR dizi protein analizi için yeniden üretilebilir ve tekrar kullanılabilir. Bu protokol, yapı blokları olarak sadece iki küçük molekülleri kullanarak elektronik dil birlikteliğinin yapımını da açıklar ve SPRI ile elde edilen kesintisiz tanıma kalıpları temel alan ortak proteinlerin analizi için uygulanmasını göstermektedir.

<table border="0" cellpadding="0" cellspacing="0" width="1511">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:225px;">Catalog Number</td>
			<td style="width:799px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPRi apparatus&#160;</td>
			<td>Horiba Scientific-GenOptics</td>
			<td style="width:225px;"></td>
			<td>SPRi apparatus is placed in a temperature regulated incubator at 25&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Memmert</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump&#160;</td>
			<td>Cavro scientific instruments</td>
			<td style="width:225px;">Cavro XLP 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Micro Elite Degasser&#160;</td>
			<td style="width:203px;">Alltech</td>
			<td style="width:225px;">AT590507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-port medium pressure injection valve</td>
			<td>Upchurch Scientific</td>
			<td style="width:225px;"></td>
			<td>The volume of injection loop used is 500 &#181;l.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Femto plasma cleaner (version 7)</td>
			<td>Diener Electronic</td>
			<td style="width:225px;"></td>
			<td>On-line degassing system with 2 channel.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spotter</td>
			<td style="width:203px;">Siliflow</td>
			<td style="width:225px;"></td>
			<td>&#160;It is a non-contact piezoelectric spotter.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SPRi-Biochip</td>
			<td>Horiba Scientific-GenOptics</td>
			<td align="right" style="width:225px;">36000067</td>
			<td>The prism is made of a high refractive index glass prism coated with a thin gold film (45 nm) and specially developed for imaging purposes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">erythrina cristagalli lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L5390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">arachis hypogaea lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L0881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">myoglobin</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">M1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lysozyme</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#945;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#947;&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sulfated lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">glycerol</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">G5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SDS</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">tween 20</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td align="right" style="width:225px;">274348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">HEPES</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S0751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium chloride</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S3014</td>
			<td></td>
		</tr>
	</tbody>
</table>

electronic tongue generating continuous recognition patterns for protein analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

<html> Ortak protein analizi için elektronik dil yeteneğini prob, üç proteinler kullanıldı: AHL, miyoglobin ve lizozomunu. Şekil 6&#39;da gösterildiği gibi, her bir protein için, ayrı bir 2D sürekli bir gelişim profil CEP, ve arkadaşları tarafından elde edilmiştir. Buna ek olarak, her bir protein için, zamana bağlı bir sürekli model tanıma gerçek zamanlı adsorpsiyon ve desorpsiyon kinetiğini takip edebilir SPRI, sayesinde, 3D sürekli evrim yatay (CEL...

Watch this Scientific Journal Video about Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis at JoVE.com

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

Protein Analiz Sürekli Tanıma Desenler oluşturma Elektronik Dil

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

electronic-tongue-generating-continuous-recognition-patterns-for

Research

JoVE Journal

Bioengineering

7.8K Görüntüleme.  Institut Nanosciences et Cryogénie, CEA-Grenoble. A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination. 

Video: Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Asimetrik Reseptör Desenler Hücre Rolling Yörüngeler incelenmesi

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Biyomühendislik

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Biofabrication ile Bio-Elektronik Arayüz Bridging

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Kantitatif, Deri Yara Yönetimi için bir konformal Cilt benzeri elektronik Sistemi İmalat ve Karakterizasyonu

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Planar on Tek Molekül Floresans Mikroskopi bilayers Desteklenen

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

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

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

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

Tek Karbon Nanotüp Bio-mimetik Polimerler ile Mühendislik Moleküler Tanıma

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

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Parçacıkların Mekansal Takip için Çoklanmış Elektronik Algılama ile mikroakışkan Platformu

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Tekstil Organik Elektronik Cihazlar Basit ve Ölçeklenebilir Fabrikasyon Yöntemi

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

En iyi duruma getirilmiş kurulum ve manyetik etki alanı görüntüleme ile Situ histeresis ölçüm için iletişim kuralı

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in this field have driven technological progress in a back-and-forth process that ultimately determined the impressive achievements of the past 50 years. However, the most employed devices used for cellular interfacing (namely, the microelectrode arrays and microelectronic devices based on transistors) still present several limitations such as high cost, the rigidity of the materials, and the presence of an external reference electrode. To partially overcome these issues, there have been developments in a new scientific field called organic bioelectronics, resulting in advantages such as lower cost, more convenient materials, and innovative fabrication techniques.
Several interesting new organic devices have been proposed during the past decade to conveniently interface with cell cultures.&#160;This paper presents the protocol for the fabrication of devices for cellular interfacing based on the organic charge-modulated field-effect transistor (OCMFET). These devices, called micro OCMFET arrays (MOAs), combine the advantages of organic electronics and the peculiar features of the OCMFET to prepare transparent, flexible, and reference-less tools with which it is possible to monitor both the electrical and the metabolic activities of cardiomyocytes and neurons in vitro, thus allowing a multiparametric evaluation of electrogenic cell models.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

In Vitro Mikro Organik Yük modüle Edilmiş Alan Etkili Transistör Dizileri ile ÇokParametrik Hücresel Analiz

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in ...

SARPE'den Genleşme Modellerinin Sonlu Elemanlar Modelleri Kullanılarak Analizi

Finite Element Analysis Model for Assessing Expansion Patterns from Surgically Assisted Rapid Palatal Expansion

Surgically assisted rapid palatal expansion (SARPE) was introduced to release bony resistance to facilitate skeletal expansion in skeletally mature patients. However, asymmetric expansion between the left and right sides has been reported in 7.52% of all SARPE patients, of which 12.90% had to undergo a second surgery for correction. The etiologies leading to asymmetric expansion remain unclear. Finite element analysis has been used to evaluate the stress associated with SARPE in the maxillofacial structures. However, as a collision of the bone at the LeFort I osteotomy sites occurs only after a certain amount of expansion, most of the existing models do not truly represent the force distribution, given that the expansion amount of these existing models rarely exceeds 1 mm. Therefore, there is a need to create a novel finite element model of SARPE that could perform a clinically required amount of expander activation for further analysis of the expansion patterns of the hemimaxillae in all three dimensions. A three-dimensional (3D) skull model from cone beam computed tomography (CBCT) was imported into Mimics and converted into mathematical entities to segment the maxillary complex, maxillary first premolars, and maxillary first molars. These structures were transferred into Geomagic for surface smoothing and cancellous bone and periodontal ligament creation. The right half of the maxillary complex was then retained and mirrored to create a perfectly symmetrical model in SolidWorks. A Haas expander was constructed and banded to the maxillary first premolars and first molars. Finite element analysis of various combinations of buccal osteotomies at different angles with 1 mm clearance was performed in Ansys. A convergence test was conducted until the desired amount of expansion on both sides (at least 6 mm in total) was achieved. This study lays the foundation for evaluating how buccal osteotomy angulation influences the expansion patterns of SARPE.

Yazar Gündemi: Gelişmiş Ortognatik Cerrahi Teknikler için Yeni Bir Sonlu Elemanlar Analiz Modelinin Geliştirilmesi 

A set of novel finite element models of surgically assisted rapid palatal expansion (SARPE) that could perform a clinically required amount of expander activation with various angles of buccal osteotomy was created for further analysis of the expansion patterns of the hemimaxillae in all three dimensions.

Cerrahi Destekli Hızlı Damak Genişlemesinden Genişleme Modellerini Değerlendirmek için Sonlu Elemanlar Analiz Modeli

Surgically assisted rapid palatal expansion (SARPE) was introduced to release bony resistance to facilitate skeletal expansion in skeletally mature ...