The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).

Deney 1. Planlama <ol><li> Sentezi için gerekli olan bakır nanokompozitlerin hacmini belirleyin. Bu temelde, küçük hacimli şişeler (25 cm2), ya da malzemelerin hazırlanmasında, aşağıda gösterildiği gibi daha büyük bir şişe bir dizi seçim. </li><li> Bu sentez için,% 5 CO2 ve en az% 40 nem ile 37 ° C inkübatör kullanın. Böyle bir inkübatör mevcuttur ve tekrar tekrar sentez döneminde (yaklaşık 24 saat) boyunca rahatsız olmayacak emin olun. DİKKAT: Tekrarlanan açma ve inkübatör kapanış kesinlikle nanokompozit yapılarının değişmiş sentezinde neden olabilir sıcaklık dalgalanmalarını neden olacaktır. </li></ol> Malzemelerin 2. Hazırlık <ol><li> Sentezi başlatmak için hemen önce çözücü maddeler, katı maddeleri ekleyerek, bir deney başlamadan önce taze tüm malzemeleri hazırlayın. Uzun zamanlar b sıvı içinde sistin ve bakır başlangıç ​​maddelerinin stok çözümleri tutulmasıefore Deney tavsiye edilmez ve değişken sonuçlara yol açabilir. Bir kez Parafilm ile kabın üst sararak kuru başlangıç ​​malzemeleri, satıcıdan devam açıldı. Not: Aşağıdaki protokol sistin 7 ul steril su 6643 ul ve KNP&#39;lerle 350 ul kullanılarak 25 cm 2 hücre kültürü şişesi içinde tepkimesi için bir örnek olarak kullanılmıştır. </li><li> KNP&#39;lerle en az 2 mg tartılması bakır nanopartiküller bir 2 mg / ml çözeltisi hazırlayın. Cilt ile KNP&#39;lerle olası temasını önlemek için bu adımı sırasında tek kullanımlık eldiven giyin. Boş bir steril 16 ml cam şişede nanopartiküller yerleştirin. <ol><li> KNP&#39;lerin içeren şişeye, 2 mg / ml&#39;lik bir çözelti oluşturmak ve sentez başlamadan önce nanopartiküllerin dağılmasını temin etmek için 20 saniye için çözüm vorteks uygun hacimde steril deiyonize su ekleyin (en az 1 mL toplam hacim tavsiye edilir). Bu vorteks karıştırma inhibe edecek şekilde su ile şişe yarısından fazlası yol doldurmayın. KNP&#39;ler will hızla şişenin dibine yerleşmek ve (siyah gri) rengi koyu görünecektir. </li><li> Sentezi başlamadan önce KNP&#39;lerle maksimum dağılım temin etmek üzere, oda sıcaklığında 17 dakika boyunca CNP çözeltisi sonikasyon. Periyodik KNP&#39;ler Sonication nedeniyle karıştırma emin olmak için kontrol edin. Başarılı Sonikasyon sonrasında, KNP&#39;ler en az 30 dakika süre ile çözelti içinde askıda kalır ve çözelti, koyu renkli olur. </li></ol></li><li> Sentezi için bir 72.9 mg / ml çözelti yapmak için sistin yeterli kütleye tartılır. Sistin su içinde doğrudan çözünür olmadığı için, antistatik tartım kabında tartılır sistin yerleştirin. <ol><li> Sistin tamamen eriyene kadar tartım kabı içeren sistin için, steril, 1 M NaOH yeterli hacmi ekleyin. Örneğin, bir 72.9 mg / ml çözelti elde edilerek, 1 M NaOH içinde 100 ul tam sistin 7.29 mg çözülür. </li><li> Steril akış doku kültürü kaputu bu adımı yürütmek, steril koşullar korumak için. UYARI: NaOH1 M konsantrasyon yakıcı ile, yani deri ile konsantre NaOH temasını önlemek için bu adım sırasında atılabilir giyim eldivenleri </li></ol></li><li> Steril doku kültürü kaputu çalışan birinci steril sentezi şişeye steril su 6643 ul sistin 7 ul, ve şişe kapağı ile 37 ° C&#39;de kuluçka makinesi içinde, 30 dakika boyunca inkübe edin etkili sağlamak için (gevşek) havalandırılmış Karıştırma. KNP&#39;ler sonikasyon aşamasından sonra yerleşmiş çünkü, 30 saniye boyunca girdaplanarak 2 mg / ml CNP solüsyonu yeniden süspanse edin. <ol><li> Sentezini başlatmak için 25 cm 2 hücre kültürü şişesi steril su, 50 parça KNP&#39;ler ve 949 parça 1 parça sistin birleştirmek: Aşağıdaki bileşen oranları korumak için (steril tekniği kullanarak) sentez şişeye yeterli CNP çözüm ekleyin. Örneğin, bir 7 mi sentezi hacmi için, sistin stok çözeltisi 7 ul, KNP&#39;lerle 350 ul steril su 6643 ul birleştirir. Balona kapağını takın ve böylece secur olduğunu sıkıne. </li><li> Sentez için tüm bileşenleri birleştirdikten sonra, yavaşça 4-5 kez dönen tarafından şişede karıştırın. CO2 inkübatör şişeyi koyun ve ve sentez sırasında balonun dışına gaz değişimi olacak şekilde kapağı gevşeterek şişeyi havalandırma. </li></ol></li><li> Sentez yaklaşık 24 saat inkübatör çalışmasına izin ver. Sentez sırasında, bir, mikroskopi ve gözle yüksek doğrusal kompozit oluşumunu gözlemleyebilirsiniz. Not: yapı oluşumu süreci yapıları tespit etmek için ilk olarak sabit olduğu anlamında anda meydana gelebilir, o zaman görünümü artan bir yoğunluğa hızlı bir şekilde ilerler. Formasyon bu nedenle 24 saat önce oluşabilir. yapılar büyük ve yoğunluğu artar haline kez süreç aynı zamanda gözle görülebilir. Yapıların üretimi daha sonraki zaman noktalarında mikroskop altında ve göz zaman içinde görülmekte iken, sürekli biçimde sentez koşullarının kesintiye ve sıcaklık zayıf yol açacaktırSentez sonucu. </li><li> Sıkı sentezi şişesi kapak kapama ve bir buzdolabına (4 ° C), teknenin depolayarak biocomposites sentezini iptal eder. Bir kez üretilen yapıları, en az bir yıl boyunca bu şekilde sabit kalır. Kullanılan bileşenlerin sentez tarihi ve sona ermeden sentez inkübasyon süresi de dahil olmak üzere sentez koşulları ile balon etiketleyin. </li></ol> Bakır Sülfat Kullanılması 3. Sentezi <ol><li> Bakır sülfat tuzu ile KNP&#39;lerin değiştirerek kendini montaj sentezini yürütmek. Steril teknik kullanılarak, 2 mg / ml&#39;lik bir solüsyon yapmak için, steril deiyonize su, yeterli hacimde bakır sülfat, en az 2 mg çözülür. bakır sülfat kristalleri kolayca bu konsantrasyonda çözelti içine gitmek, ama gerekirse flakon vorteks ve tüm kristalleri çözülür sağlamak için göz tarafından kontrol edin. </li><li> Daha önce tarif edilen, ancak bakır sülfat ile KNP&#39;lerin değiştirilmesi gibi bakır sülfat hazırlanmasından sonra, sentez yapmak. Not: Bir başlangıç ​​malzemesi olarak, bakır sülfat kullanarak kendi kendine bir araya nanokompozitler KNP&#39;lerle sentezlenen yapılar için daha kesin bir şekil çok daha tutarlı olduğu bulunmuştur. </li><li> CNP kompozitler gibi bakır sülfat biocomposites sentezini sonlandırma (2,6 aşama) ve 4 ° C de, uzun dönemli saklayın. </li></ol> 4. Karakterizasyonu ve Biocomposites Post-sentez taşınması <ol><li> KNP&#39;lerle gelen ve beyaz ışık mikroskobu 9 bakır sülfat gelen ve elektron mikroskobu 9 türetilen biocomposites karakterize. <ol><li> Kompozit bir şişeye, düz döşeme birkaç dakika içinde şişenin alt yüzeyine yerleşmek gibi karakterizasyonu ve biocomposites beyaz bir ışık mikroskobu ile sentez sonrası incelenmesi için, bir ters mikroskop kullanarak ve daha sonra odak noktası haline getirilebilir. Biocomposites ve sıvı ortamın arasındaki kontrastı en üst düzeye çıkarmak için mikroskop parlak alan ayarını kullanın. KNP&#39;lerle ve polis türetilen kompozitlersülfat başına hem renkli opak açıkça görünür, ancak girmemiş CNP agrega rengi çok koyu görünecektir. <ol><li> Kompozit görüntü yakalamak için mikroskop bağlı bir dijital kamera kullanın. Ayrı ayrı yapılar için uzunluklarının bir aralığı gözlenecektir. </li></ol></li><li> Karakterizasyonu ve biocomposites sentez sonrası denetimi ve 4 ° C&#39;de depolamadan sonra, şişeler mikroskopi görüntüleme yaparken odaklama etkili belirsiz olacaktır buzdolabından çıkarıldıktan üzerine ilk yoğunlaşmayı oluşturmak gibi şişeler, en az 15 dakika boyunca oda sıcaklığına gelmesine izin. Oda sıcaklığına dengede bırakıldıktan sonra, mikroskopi görüntü kalitesi üst düzeye çıkarmak için temiz bir kağıt havlu ile balonun üst ve alt yüzeylerini silin. </li><li> Buzdolabında ise oluşturan kompozit kümeleri ayırmak için 30 saniye boyunca uzun süreli saklanan ya da görüntüleme kompozitler, girdap şişesi çalışırken. Vorteks sonra ters mikrofon ile yapıları incelemekroscope agrega ayrışmış emin olun ve gerekirse vorteksleme tekrarlamak için. </li><li> KNP&#39;lerin kullanarak belirli bir deney için sentez etkinliğini değerlendirmek için ters beyaz ışık mikroskobu kullanarak. Örneğin, bu tür sentez süresi olarak farklı parametrelerle şişelerinden CNP türetilmiş biocomposites için kullanılan sentez şişelerinde reaksiyona girmemiş KNP&#39;lerle varlığını ya da yokluğunu belge. Bağımsız KNP&#39;ler bir ışık mikroskobu ile gözlenmek için çok küçük, ancak reaksiyona girmemiş CNP yüksek en boy oranı, doğrusal bir biçime sahip olacaktır başarıyla sentezlenmiştir CNP-kompozitler aksine, yuvarlak şekil ve koyu renkli bir nesne olarak görülür agregatlar ve Not: Farklı uzunluklarda bir dizi olacaktır. Bu bir kez meydana getiren bireysel yapılar halinde dağılması zor olan yüksek ölçüde dallanmış &quot;kestanesi&quot; tipi yapılarda neden olacağı için, sona önce bir süre çok uzun için sentez gerçekleştirilmesi kaçının. </li><li> Etkinliğini değerlendirmek için ters beyaz ışık mikroskopisi kullanınBakır sülfat kullanılarak, belirli bir deney için sentez. Bakır sülfat, bu protokolü kullanarak çözelti içine tamamen gider yana, çözüm KNP&#39;lerin kullanarak sentezden çözümün daha az karanlık görünür. Böyle bir fesih öncesi sentez zaman gibi farklı sentez koşulları şişeler karşılaştırarak bakır sülfat kompozit boyutunu ve kapsamını belgeleyin. Not: Başarıyla sentezlenen kompozit farklı uzunluklarda bir dizi gösterecektir. Bu &quot;kestanesi gibi&quot; bir yapıda olacaktır bazıları kompozitler, yüksek ölçüde dallanmış agrega neden olacağı için, sona önce bir süre çok uzun için sentez gerçekleştirilmesi önlemek ve hangi bir kez meydana getiren bireysel yapılar halinde dağılması zor olan . </li></ol></li><li> Biocomposites sonrası sentezi, bir santrifüj tüpü içinde kompozitlerin santrifüj çözümler yoğunlaştırmak. 15 ml santrifüj tüpüne CNP-türevi yapılar ya da bakır sülfat türevi yapılar, ya 6 ml ilave edilir. 10 dakika boyunca santrifüjoda sıcaklığında, 500 x g&#39;de topak elde etmek için. Daha küçük miktarlar için, 0.6 ml boyutlu tüpler çözüm yapıların 500 ul ekleyin. En az 10 dakika süreyle oda sıcaklığında 2000 x g&#39;de santrifüjleyin bir topak oluşturmak için. <ol><li> Yeterli bir süre boyunca (mikrofüjler için en az 10 dakika) santrifüje tabi tutulmasının ardından yapıları dikkatli bir şekilde pelet üzerinde yüzer sıvının çıkarılması ile konsantre edilir tüpün altındaki gözlemlenebilir pelet kaydedin. Bakır sülfat elde edilen Biyokompozit yapıları renk ve KNP&#39;lerle türetilen yapılarda mavi koyu (siyah gri) vardır görünür. </li><li> Bu tüp daha kompozitler ekleyin ve istenirse yapıları konsantre aynı tüpte işlemi tekrarlayın. Konsantre granül dağıtmak için, 10-30 saniye süre ile, istenen tüp çözeltisinin hacmi ve vorteks ekleyin. </li></ol></li><li> Sonikasyon yapılar bir zamanlar değerlerini düşürmek için yapıların ortalama nüfus büyüklüğü (uzunlukları) taşımak için, kurdu. En le steril deiyonize su ve sonikasyon yerleştirin yapılarıast 10 dk. Zamanla, bu yöntemi kullanarak, yapılar parçalanmış, ortalama uzunluk (metin bakınız Şekil 6) daha küçük hale gelir. Ters beyaz ışık mikroskobu ve dijital kamera kullanarak farklı sonication kez kompozit boyutlarda Belge değişir. </li></ol>

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Authors have nothing to disclose.

KNP&#39;lerle dahil Nanomalzemelerin potansiyel toksik etkileri değerlendirirken, uzun vadede, KNP&#39;ler büyük, birleştirilmiş forma başlangıçta daha dağınık partikül dağılımı (Şekil 2) dönüştürülmüş olduğu gözlendi. Bazı durumlarda, biyolojik şartlar altında, hücre kültürü çanağı üretildi, bu son derece birleştirilmiş formasyonlar &quot;kestanesi&quot; içeren daha önce tarif edilen bakır andıran merkez agrega oldukça lineer çıkıntılar oluşturulmuştur ...

Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 &#176;C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called &#8220;urchin&#8221; and &#8220;nanoflower&#8221; type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 &#176;C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described. 
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells. 
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mini Vortexer</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Model # 2425-2</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>Contact vendor</td>
			<td>Current model calalog # 98000-360</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>C2500-R</td>
			<td>Model Prism R</td>
		</tr>
		<tr>
			<td>Cell Culture Centrifuge Model Z323K</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>Contact vendor</td>
			<td>Current model Z206A catalog # C0206-A</td>
		</tr>
		<tr>
			<td>Sonicator (Ultrasonic Cleaner)</td>
			<td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius (http://dataweigh.com)</td>
			<td></td>
			<td>Model CP225D similar model CPA225D</td>
		</tr>
		<tr>
			<td>Olympus IX51 Inverted Light Microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus DP71 microscope digital camera</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>external power supply unit- white light for Olympus microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>TH4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>10x, 20, and 40x microscope objectives</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td>
			<td>model S-4800</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td>
			<td>model Libra 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Work Station Unidirectional Flow Clean Bench</td>
			<td>Envirco (http://envirco-hvac.com)</td>
			<td>model PNG62675</td>
			<td>Used for sterile cell culture technique</td>
		</tr>
	</tbody>
</table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

Bu protokolün amacı yüksek en-boy oranı yapıları ile iki yeni biocomposites sentezini tarif etmektir. biocomposites bakır nanopartiküller (KNP&#39;ler) ya da metalik bileşen katkı bakır sülfat ile ya, bakır ve sistin oluşmaktadır. Sentez biyolojik koşullar (37 ° C) ve 24 saat sonra kendi kendini monte kompozit malzemelerin oluşturulması altında sıvı içinde gerçekleştirilir. Oluşturulduktan sonra, bu kompozitler hem sıvı ortam içinde ve kuru halde son derece stabildir. Kompozit uzunluğu aralığı Mikro-nano ölçekleme ve birkaç mikron çapı 25 nm. Enerji dispersif X-ışını spektroskopi (EDX) ile alan emisyonu tarama elektron mikroskobu böylece nihai nanokompozitlerin kükürt kaynağı olarak sistin teyit başlangıç ​​CNP malzemeden yokken kükürt, NP-türevi doğrusal yapılar içinde mevcut olduğunu gösterdi . Bu lineer nano ve mikro-kompozitlerin sentezi str uzunlukları ve çeşitli aralığındauctures sentez teknesine oluşturulmaktadır. Sentezden sonra, sıvı karışımın sonikasyonu sonication artan zamanla ortalama uzunluğu azaltarak yapıların ortalama boyuta kontrol edilmesine yardımcı olduğu gösterilmiştir. Oluşan yapılar yüksek toplanma olmayan, kararlı, sıvı faz içinde oluşturulmaktadır olduğu için, santrifüj, aynı zamanda konsantre edilmesi ve oluşturulan bileşikler ayrılmasında yardımcı olmak için kullanılabilir.

Şekil 1, bu çalışmada tarif lineer biocomposites oluşturmak için sentez adımlarına ait bir akım şeması şemasını göstermektedir. Başlangıç ​​materyalleri olarak KNP&#39;ler ya da bakır sülfat, 2 mg / ml&#39;lik bir çözelti oluşturmak üzere, steril su ile birlikte, bu çözüm, karıştırılır ve eşit bir karışım sağlamak için sonike ve bu çözelti daha sonra, bakır sentezi için, aşağıdaki oranda karıştırılır edilir: 949 parça, steril su: 50 parça bakır ka...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Burada biyolojik şartlar altında ve sıvı ortam içinde, yeni, yüksek en-boy oranı biocomposites sentezlemek için bir protokol mevcut. biocomposites sırasıyla çapı ve uzunluğu mikrometre nanometre kadar ölçeklendirilebilir. Sistin ile birlikte bakır nanopartiküller (KNP&#39;ler) ve bakır sülfat sentezi için önemli bileşenleridir.

Biyolojik Sıvı Ortamda Ölçeklenebilir, Metalik Yüksek Aspect Ratio Nanokompositlerin Üretimi

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Research

JoVE Journal

Bioengineering

9.0K Görüntüleme.  Centenary College of Louisiana.  Burada biyolojik şartlar altında ve sıvı ortam içinde, yeni, yüksek en-boy oranı biocomposites sentezlemek için bir protokol mevcut. biocomposites sırasıyla çapı ve uzunluğu mikrometre nanometre kadar ölçeklendirilebilir. Sistin ile birlikte bakır nanopartiküller (KNP'ler) ve bakır sülfat sentezi için önemli bileşenleridir. 

Video: Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Hücresel Lipid Ekstraksiyon Hedefli Kararlı İzotop Seyreltme Sıvı Kromatografi-Kütle Spektrometresi Analizi

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Biyomühendislik

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Için Mekanik-adaptif Polimer Nanokompozitler bir çevre kontrollü Mikrogerilim Test<em&gt; Ex vivo</em&gt; Karakterizasyonu

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Yerinde Sıvıda Biyolojik Montajlar TEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Biyomedikal Mühendisliği için keratin bazlı nanofiber sentezi

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Kontrollü İlaç Release için Manyetik Alan-Duyarlı Hibrid Jelatin mikroj Alternatif

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

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

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

Monodomain Sıvı Kristal Elastomerler ve Likit Kristal Elastomer Nanokompozitlerinin Hazırlanması

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

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Robot sıvı işleme derleme modüler DNA cihazların otomatik

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

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

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

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

3D Mekansal Hücre Kültürleri için hücre yapı iskeleleri olarak biyo-uyumlu sıvı kristal Elastomer Köpüklerin sentezi

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

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Kanser Hücre Kültürlerinden Toplanan Ortamda Sıvı Kromatografi-Kütle Spektrometresi ile Analiz Edilen Seçilmiş Kynureninlerin Eşzamanlı Nicelemesi

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

İnsan Pluripotent Kök Hücrelerinden Olgun Serebellar Organoidlerin Ölçeklenebilir Üretimi ve İmmünboyama ile Karakterizasyonu

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...