Nazik Şekil 5&#39;te gösterilen veri toplama ve analizi ile destekli Michael Henry, Dublin City University,. Bu çalışma ile kısmen desteklenmiştir (1) George Mason Üniversitesi, (2) İtalya / ABD çerçevesinde İtalyan IstitutoSuperiore di Sanita &#39; ABD Sağlık Bakanlığı ve İnsan Hizmetleri, George Mason Üniversitesi ve Halk Sağlığı İtalyan Bakanlığı, (3) NIH, IMAT programı hibe 1R21CA137706-01 ve LAL için 1R33CA173359-01, ve (4) Ceres Nanobilimler, Inc arasında işbirliği anlaşması .

İnsan plazma ve idrar George Mason Üniversitesi Kurumsal Değerlendirme Kurulu onaylanan protokollerde aşağıdaki, bilgilendirilmiş yazılı onayı ile, sağlıklı gönüllü vericilerden toplanmıştır. Bağışçılar eşit ayrı ayrı analiz edildi 25 ve 42. Numune yaş arası Kafkas kadınlar ve erkekler arasında dağıtılmış ve havuza değil. Serum veya Plazma Örneklerinin 1. Nanoparçacık İşleme Plazma potansiyel biyo-düşük miktarda hidrojel nanopartiküller ile, çözelti içinde yakalanır. Tanecikleri, yıkandı, santrifüj ile ayrılmış plazma kuluçkaya yatırılır, ilave edilir ve çekilen proteinleri elüte edilir. Elüsyona tâbi tutulmuş proteinler alt-kütle spektrometrisi sekanslama ve tanımlanması için azot akışı altında kurutulur. <ol><li> Mikrosantrifüj tüp içinde 50 mM TrisHCI pH 7 (500 ul serum + 500 ul TrisHCI) 2: serumun 1 500 ul sulandırmak. </li><li> 500 mcL poli (Nipam / AAc) çekirdek nanop Eklemakaleler. Oda sıcaklığında 15 dakika boyunca inkübe edin. </li><li> Sabit açılı rotor ile donatılmış bir santrifüj içinde 10 dakika boyunca, 16,100 x g hızında 25 ° C örnek dönerler. Çıkarın ve süpernatant atın. </li><li> Topağa 500 ul sodyum tiyosiyanat (25 mM) eklenir. Kuvvetlice pipetleme ve birden çok kez aşağı nanopartiküller süspanse. </li><li> Sabit açılı rotor ile bir santrifüj içinde 10 dakika boyunca, 16,100 x g hızında 25 ° C örnek dönerler. Çıkarın ve süpernatant atın. </li><li> Nanopartikül pelet MiliQ suyu içinde 500 ul ilave edin. Kuvvetlice pipetleme ve birden çok kez aşağı nanopartiküller süspanse. </li><li> Sabit açılı rotor ile bir santrifüj içinde 10 dakika boyunca, 16,100 x g hızında 25 ° C örnek dönerler. Çıkarın ve süpernatant atın. </li><li> Taze elüsyon tamponu hazırlayın: temiz bir tüp içinde 300 ul amonyum hidroksite ul asetonitril 700 ekleyin. Dikkat: Amonyum hidroksit aşındırıcı olduğunu. Uygun havalandırma ile kullanın. Not: Yıkama tamponu, preparatif olmalıdırared kullanımdan hemen önce. Elüsyon tamponu tutmayın. </li><li> Nanopartikül pelet elüsyon tamponunun 300 ul ilave edin. Kuvvetlice pipetleme ve birden çok kez aşağı nanopartiküller süspanse. Oda sıcaklığında 15 dakika boyunca inkübe edin. </li><li> Sabit açılı rotor ile bir santrifüj içinde 10 dakika boyunca, 16,100 x g hızında 25 ° C örnek dönerler. Çıkarın ve temiz, etiketlenmiş bir mikrosantrifüj tüpü içinde eluatın kaydedin. </li><li> 1.10 - Tekrar 1.9 adımları. Bir mikrosantrifüj tüpe iki elüsyonların birleştirin. </li><li> 8 (Şekil 2F) ayarlanmış hava akışı ile, 42.1 ° C &#39;de bir azot evaporatör manifoldunun, azot akışı altında sıvı kısımlar kurutun. </li><li> O / N depolama için oda sıcaklığında kurutuldu eluatın saklayın, ya da uzun süreli depolama için -20 ° C, kütle spektrometresi, Western blotting, ELISA tahlilleri ya da (isteğe bağlı) öncesinde. </li></ol> İdrar Örneklerinin 2. Nanoparçacık İşleme Normal idrar az 30 mg / dl protein içerir ve1+ kandan daha az. Ancak, birçok hastalık / durumlar idrar protein ve normal kan seviyelerini değiştirebilir. Idrar örneği eklemek için nanopartiküllerin uygun hacminin belirlenmesinde yardımcı olmak için, bir idrar toplama nanopartikül önce gerçekleştirilir. İdrar biyolojik göstergeler idrar hacmi nanopartiküllerin oranını optimize gerektirebilir son derece düşük konsantrasyonlarda mevcut olabilir. Bu işlem alt Western blot analizi için idrar örnekleri nanopartikül hasat tarif etmektedir. <ol><li> Temiz, kuru bir plastik kap içinde idrar örnekleri toplamak. Bir 22 ml minimum hacim gereklidir. Analiz için hazır olana kadar mağazası idrar -80 ° C&#39;de numune. </li><li> Oda sıcaklığında ya da 4 ° de CO / N, dondurulmuş idrar çözülme. , Bir girdaplı karıştırıcı ile kısa bir süre karıştırılır. 50 ml&#39;lik konik tabanlı polipropilen tüp içine idrar en az 22 ml dökün. </li><li> 15 dakika boyunca 3700 x g&#39;de dışarı salınan bir rotora sahip bir santrifüj içinde idrar dönerler. Temiz bir 50 ml&#39;lik konik tabanlı polypr içine pelet bozmadan, idrar süzünopylene tüpü. Pelet atın. </li><li> Bir multi-analit &quot;idrar testi&quot; reaktif şerit kullanarak idrar tahlili gerçekleştirin. Not: Doğrudan güneş ışığı ve nem 17, 18 uzak bir sıkıca kapalı bir kapta reaktif şeritler saklayın. <ol><li> Temiz, kuru bir kağıt havlu üzerine reaktif şerit, yüz-up, yatıyordu. </li><li> Adım 2.3 hazırlanan idrar 1 ml aspire bir pipet kullanın. </li><li> 2 dakika için bir zamanlayıcı ayarlayın ama başlamayın. Reaktif şerit her test pad üzerinde idrar 2 damla - Çabuk 1 dağıtın. Hemen sayacını başlatmak. Not: idrar konteyner doğrudan reaktif şerit batırmayın. Ayıraç şerit boya / kimyasal göstergeler potansiyel idrar kabına ayıraç şeridinin dışına yapışabilen. </li><li> Reaktif şerit konteyner üzerindeki zamanda, karşılık gelen renk kodlu indica tek tek reaktif şeritlerin renk karşılaştırılarak bir ayıraç çubuğu üzerindeki çeşitli analitler için niteliksel neticelerin kayıtReaktif kabın üzerine monte edin. Tipik normal idrar Sonuçlar şu şekildedir: (normal veya negatif), kan, protein, lökosit, nitrit, glukoz, keton, bilirubin ve Ürobilinojen tayini için; İdrar pH (5,5-7,0); özgül ağırlığı (1,001-1,020). Not: Bir idrar örneğinde yüksek protein seviyelerinin nanopartiküller bağlanma alanlarında ilgilenilen protein ile rekabet edebilir. Nanopartiküller protein hasat üst düzeye çıkarmak için, nanopartikül hacim / hacim oranında idrar yüksek bolluk proteinlerinden ya da sınır rekabete ayarlanabilir ya da çok düşük konsantrasyonlarda mevcut proteinlerin toplanması için optimize edilebilir. Idrar protein değeri 1+ veya daha fazla ise, aşağıdaki adımda 2,5 nanopartiküllerinin 2x hacimlerini ekleyin. Ilgilenilen analit çok düşük bir miktarda bir protein ise, 2 ml (Şekil 3) &#39;e kadar nanopartiküllerin hacmini artırmak için gerekli olabilir. </li></ol></li><li> Aktarım, temiz 50 ml&#39;lik konik tabanlı polipropilen tüp içine adım 2.3 açıklık idrar 20 mi. Yapamazidrar tüpün alt olabilecek herhangi bir artık / pelet rahatsız. 20 ml idrar numunesi ile nanopartiküller 200 ul ilave edin. , Bir girdaplı karıştırıcı ile kısa bir süre karıştırılır. Idrar protein değeri 1+ veya daha fazla ise, 20 mi idrara nanopartiküllerinin 400 ul ekleyin: edin. </li><li> Sallanan / karıştırma olmadan, oda sıcaklığında 30 dakika için idrar / nanopartikül karışımı inkübe edin. </li><li> 10 dakika boyunca 3700 x g&#39;de dışarı salınan bir rotor ile donatılmış bir santrifüjde idrar / nanoparçacık süspansiyon dönerler. Not: pelet görünür ardından santrifüj değilse, ek bir 2 için örnekler dönmeye -, 7 dakika. </li><li> Çıkarın ve süpernatant atın. Nanopartikül pelet 500 ul, MilliQ su ekleyin. </li><li> Kuvvetlice pipetleme ve birden çok kez aşağı nanopartiküller süspanse. Temiz bir 1.5 ml mikrosantrifüj tüpüne nanopartikül çözeltisini. </li><li> 10 dakika boyunca 16.100 x g&#39;de sabit açılı rotor ile donatılmış bir santrifüj içinde nano-tanecikleri dönerler. Not: pelet görünür vr değilse7 dakika - santrifüj iyice silinir, ek bir 2 için örnekler döndürün. </li><li> Çıkarın ve süpernatant atın. </li><li> Tekrar nanopartiküller yıkamak için 200 ul 18 MilliQ su ile (iki kez) 2.8 ve 2.11 adımları. </li><li> Taze elüsyon tamponu hazırlayın: temiz bir tüp içinde 30 ul amonyum hidroksite ul asetonitril 970 ekleyin. Dikkat: Amonyum hidroksit aşındırıcı olduğunu. Uygun havalandırma ile kullanın. Not: Yıkama tamponu, kullanılmadan hemen önce hazırlanmış olmalıdır. Elüsyon tamponu tutmayın. </li><li> Nanopartikül pelet 20 ul yıkama tamponu ilave edin. Kuvvetlice pipetleme ve birden çok kez aşağı nanopartiküller süspanse. Oda sıcaklığında 15 dakika boyunca inkübe edin. </li><li> 10 dakika boyunca 16.100 x g&#39;de nanopartikül örnekleri dönerler. Çıkarın ve temiz bir 1.5 ml mikrosantrifüj tüpü süpernatant kaydedin. Nanoparçacık pelet bozmayan. Biohazard çöpe pelet atın. </li><li> Bir kimyasal davlumbaz rafa örnekleri yerleştirin. Kapaklar açın ve inc30 dakika boyunca oda sıcaklığında Ubate. (- 1, 2 saat) Alternatif olarak, kuru hale gelinceye kadar 40 ° C&#39;de azot akışı altında örnekleri yer. </li><li> Örnekler 15 ul Tris-glisin SDS numune tamponu (2x) ekleyin. Isı 100 ° C&#39;de Açık veya boru içinde kalan hacme kadar kapakları 5 dakika boyunca kuru ısı bloğu, ° C olarak en fazla 20 ul&#39;dir. Not: Bir kaynar su banyosu kullanmayın. Su banyosu nem tampon buharlaşmasını engeller. </li><li> Borular üzerinde kapağını yerleştirin. Isı bloğu tüpleri çıkarın. Numune -80 ° C&#39;de saklandı veya alt Western blot analizi için hemen kullanılabilir. </li></ol>

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	<li>Aebersold, R., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+proteomics.">Mass spectrometry-based proteomics.</a> Nature. 422 (6928), 198-207 (2003).</li><li>Gerszten, R. E., 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=Challenges+in+translating+plasma+proteomics+from+bench+to+bedside:+update+from+the+NHLBI+Clinical+Proteomics+Programs.">Challenges in translating plasma proteomics from bench to bedside: update from the NHLBI Clinical Proteomics Programs.</a> Am J Physiol Lung Cell Mol Physiol. 295 (1), L16-L22 (2008).</li><li>Merrell, K., 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=Analysis+of+low-abundance,+low-molecular-weight+serum+proteins+using+mass+spectrometry.">Analysis of low-abundance, low-molecular-weight serum proteins using mass spectrometry.</a> J Biomol Tech. 15 (4), 238-248 (2004).</li><li>Petricoin, E. F., Belluco, C., Araujo, R. P., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+peptidome:+a+higher+dimension+of+information+content+for+cancer+biomarker+discovery.">The blood peptidome: a higher dimension of information content for cancer biomarker discovery.</a> Nat Rev Cancer. 6 (12), 961-967 (2006).</li><li>Camerini, S., Polci, M. L., Liotta, L. A., Petricoin, E. F., Zhou, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+selective+isolation+and+enrichment+of+carrier+protein-bound+low-molecular+weight+proteins+and+peptides+in+the+blood.">A method for the selective isolation and enrichment of carrier protein-bound low-molecular weight proteins and peptides in the blood.</a> Proteomics Clin Appl. 1 (2), 176-184 (2007).</li><li>Geho, D., 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=Fractionation+of+serum+components+using+nanoporous+substrates.">Fractionation of serum components using nanoporous substrates.</a> Bioconjug Chem. 17 (3), 654-661 (2006).</li><li>Sennels, L., 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=Proteomic+analysis+of+human+blood+serum+using+peptide+library+beads.">Proteomic analysis of human blood serum using peptide library beads.</a> J Proteome Res. 6 (10), 4055-4062 (2007).</li><li>Zheng, X., Baker, H., Hancock, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+low+molecular+weight+serum+peptidome+using+ultrafiltration+and+a+hybrid+ion+trap-Fourier+transform+mass+spectrometer.">Analysis of the low molecular weight serum peptidome using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer.</a> J Chromatogr A. 1120 (1-2), 173-184 (2006).</li><li>Marshall, 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=Processing+of+serum+proteins+underlies+the+mass+spectral+fingerprinting+of+myocardial+infarction.">Processing of serum proteins underlies the mass spectral fingerprinting of myocardial infarction.</a> J Proteome Res. 2 (4), 361-372 (2003).</li><li>Fredolini, 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=Concentration+and+Preservation+of+Very+Low+Abundance+Biomarkers+in+Urine,+such+as+Human+Growth+Hormone+(hGH),+by+Cibacron+Blue+F3G-A+Loaded+Hydrogel+Particles.">Concentration and Preservation of Very Low Abundance Biomarkers in Urine, such as Human Growth Hormone (hGH), by Cibacron Blue F3G-A Loaded Hydrogel Particles.</a> Nano Res. 1 (6), 502-518 (2008).</li><li>Luchini, A., 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=Smart+hydrogel+particles:+biomarker+harvesting:+one-step+affinity+purification,+size+exclusion,+and+protection+against+degradation.">Smart hydrogel particles: biomarker harvesting: one-step affinity purification, size exclusion, and protection against degradation.</a> Nano Lett. 8 (1), 350-361 (2008).</li><li>Patanarut, A., 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=Synthesis+and+characterization+of+hydrogel+particles+containing+Cibacron+Blue+F3G-A.">Synthesis and characterization of hydrogel particles containing Cibacron Blue F3G-A.</a> Colloids Surf A Physicochem Eng Asp. 362 (1-3), 8-19 (2010).</li><li>Tamburro, D., 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=Multifunctional+core-shell+nanoparticles:+discovery+of+previously+invisible+biomarkers.">Multifunctional core-shell nanoparticles: discovery of previously invisible biomarkers.</a> J Am Chem Soc. 133 (47), 19178-19188 (2011).</li><li>Anderson, N. L., Anderson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+plasma+proteome:+history,+character,+and+diagnostic+prospects.">The human plasma proteome: history, character, and diagnostic prospects.</a> Mol Cell Proteomics. 1 (11), 845-867 (2002).</li><li>Fredolini, 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=Nanoparticle+technology:+amplifying+the+effective+sensitivity+of+biomarker+detection+to+create+a+urine+test+for+hGH.">Nanoparticle technology: amplifying the effective sensitivity of biomarker detection to create a urine test for hGH.</a> Drug Test Anal. 1 (9-10), 447-454 (2009).</li><li>Longo, 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=Core-shell+hydrogel+particles+harvest,+concentrate+and+preserve+labile+low+abundance+biomarkers.">Core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers.</a> PLoS One. 4 (3), e4763 (2009).</li><li>Luchini, A., 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=Nanoparticle+technology:+addressing+the+fundamental+roadblocks+to+protein+biomarker+discovery.">Nanoparticle technology: addressing the fundamental roadblocks to protein biomarker discovery.</a> Curr Mol Med. 10 (2), 133-141 (2010).</li><li>Luchini, A., 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=Application+of+Analyte+Harvesting+Nanoparticle+Technology+to+the+Measurement+of+Urinary+HGH+in+Healthy+Individuals.">Application of Analyte Harvesting Nanoparticle Technology to the Measurement of Urinary HGH in Healthy Individuals.</a> J Sports Med Doping Stud. 2 (6), (2012).</li><li>Eslami, A., Lujan, J., Western, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=blotting:+sample+preparation+to+detection.">blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Gallagher, S., Chakavarti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblot+analysis.">Immunoblot analysis.</a> J Vis Exp. (16), (2008).</li><li>Penna, A., Cahalan, M., , <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blotting+using+the+Invitrogen+NuPage+Novex+Bis+Tris+minigels.">Western Blotting using the Invitrogen NuPage Novex Bis Tris minigels.</a> J Vis Exp. (7), 264 (2007).</li><li>Bosch, 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=Analysis+of+urinary+human+growth+hormone+(hGH)+using+hydrogel+nanoparticles+and+isoform+differential+immunoassays+after+short+recombinant+hGH+treatment:+preliminary+results.">Analysis of urinary human growth hormone (hGH) using hydrogel nanoparticles and isoform differential immunoassays after short recombinant hGH treatment: preliminary results.</a> J Pharm Biomed Anal. 85, 194-197 (2013).</li><li>Fredolini, 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=Investigation+of+the+ovarian+and+prostate+cancer+peptidome+for+candidate+early+detection+markers+using+a+novel+nanoparticle+biomarker+capture+technology.">Investigation of the ovarian and prostate cancer peptidome for candidate early detection markers using a novel nanoparticle biomarker capture technology.</a> AAPS J. 12 (4), 504-518 (2010).</li><li>Longo, 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=A+novel+biomarker+harvesting+nanotechnology+identifies+Bak+as+a+candidate+melanoma+biomarker+in+serum.">A novel biomarker harvesting nanotechnology identifies Bak as a candidate melanoma biomarker in serum.</a> Exp Dermatol. 20 (1), 29-34 (2010).</li><li>Luchini, A., Longo, C., Espina, V., Petricoin, E. F., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+technology:+Addressing+the+fundamental+roadblocks+to+protein+biomarker+discovery.">Nanoparticle technology: Addressing the fundamental roadblocks to protein biomarker discovery.</a> J Mater Chem. 19 (29), 5071-5077 (2009).</li><li>Douglas, T. A., 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=The+use+of+hydrogel+microparticles+to+sequester+and+concentrate+bacterial+antigens+in+a+urine+test+for+Lyme+disease.">The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease.</a> Biomaterials. 32 (4), 1157-1166 (2010).</li><li>Prakash, A., 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=Interlaboratory+reproducibility+of+selective+reaction+monitoring+assays+using+multiple+upfront+analyte+enrichment+strategies.">Interlaboratory reproducibility of selective reaction monitoring assays using multiple upfront analyte enrichment strategies.</a> J Proteome Res. 11 (8), 3986-3995 (2012).</li><li>Choi, K. 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=Implication+of+lipocalin-2+and+visfatin+levels+in+patients+with+coronary+heart+disease.">Implication of lipocalin-2 and visfatin levels in patients with coronary heart disease.</a> Eur J Endocrinol. 158 (2), 203-207 (2008).</li><li>Hughes, A. D., Clunn, G. F., Refson, J., Demoliou-Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+growth+factor+(PDGF):+actions+and+mechanisms+in+vascular+smooth+muscle.">Platelet-derived growth factor (PDGF): actions and mechanisms in vascular smooth muscle.</a> Gen Pharmacol. 27 (7), 1079-1089 (1996).</li><li>Izycki, T., 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=Serum+levels+of+IGF-I+and+IGF-II+in+patients+with+lung+cancer+during+chemotherapy.">Serum levels of IGF-I and IGF-II in patients with lung cancer during chemotherapy.</a> Exp Oncol. 26 (4), 316-319 (2004).</li><li>Jalosinski, M., Karolczak, K., Mazurek, A., Glabinski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+methylprednisolone+and+mitoxantrone+on+CCL5-induced+migration+of+lymphocytes+in+multiple+sclerosis.">The effects of methylprednisolone and mitoxantrone on CCL5-induced migration of lymphocytes in multiple sclerosis.</a> Acta Neurol Scand. 118 (2), 120-125 (2008).</li><li>Kitazume, S., 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=Molecular+insights+into+beta-galactoside+alpha2,6-sialyltransferase+secretion+in+vivo.">Molecular insights into beta-galactoside alpha2,6-sialyltransferase secretion in vivo.</a> Glycobiology. 19 (5), 479-487 (2009).</li><li>Saito, T., 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=Increase+in+hepatic+NKT+cells+in+leukocyte+cell-derived+chemotaxin+2-deficient+mice+contributes+to+severe+concanavalin+A-induced+hepatitis.">Increase in hepatic NKT cells in leukocyte cell-derived chemotaxin 2-deficient mice contributes to severe concanavalin A-induced hepatitis.</a> J Immunol. 173 (1), 579-585 (2004).</li><li>Struyf, S., 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=PARC/CCL18+is+a+plasma+CC+chemokine+with+increased+levels+in+childhood+acute+lymphoblastic+leukemia.">PARC/CCL18 is a plasma CC chemokine with increased levels in childhood acute lymphoblastic leukemia.</a> Am J Pathol. 163 (5), 2065-2075 (2003).</li><li>Michael, I. P., 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=Human+tissue+kallikrein+5+is+a+member+of+a+proteolytic+cascade+pathway+involved+in+seminal+clot+liquefaction+and+potentially+in+prostate+cancer+progression.">Human tissue kallikrein 5 is a member of a proteolytic cascade pathway involved in seminal clot liquefaction and potentially in prostate cancer progression.</a> J Biol Chem. 281 (18), 12743-12750 (2006).</li></ol>

Benjamin Espina is an employee of Ceres Nanosciences, Inc. that produces reagents used in this Article. Lance Liotta, Alessandra Luchini and Virginia Espina hold patents (US patent 7,935,518 and/or 8,497,137) on the nanoparticle technology used in this Article. As university employees they are entitled to receive royalty from these patents per university policy. Lance Liotta and Alessandra Luchini are shareholders in Ceres Nanosciences and serve on the Scientific Advisory Board.

Klinik Uygunluk Bir serum ya da plazma numunesi, bir bütün olarak organizmanın durumu ile ilgili bilgiler için zengin bir kaynak temin edebilmektedir proteinler ve peptidler, dolaşımdaki düşük bol miktarda içerir düşünülmektedir. Serum proteomik söze rağmen, klinik yarar 10, 11, 16, 25 biyomarkır keşif ve çeviri thwarting üç temel ve ciddi fizyolojik engeller vardır. 1. Önemli teşhis biyomarkerler kanda çok dü...

Insan genomu dizileme tamamlanmasına rağmen, önemli ilerlemeler erken evre hastalığın öngörü belirlenmesi biyomarkerlerindeki yapılmamıştır, ya da tedavi sonucu veya prognoz 1 ile ilişkilidir. Bu ilerleme olmaması için bir nedeni olarak birçok potansiyel önemli belirteçler konvansiyonel kütle spektrometresi ve diğer biyobelirteç keşif platformlarda saptama sınırının altında bir konsantrasyonda mevcut olduğunu. Kütle spektrometrisi (MS) ve çoklu reaksiyon takip işlemi (MRM), tipik olarak, bir algılama hassasiyeti daha büyük olan 50 ng / ml olarak ise 50 pg / ml ve 10 ng / ml arasında değişen bir klinik laboratuar düşme ile ölçülen bağışıklık analitlerin çoğunluğu . Bu, özellikle bir hastalığın erken evrede pek biyomarkırlar, geleneksel MS ve MRM 2 ile tespit edilemez anlamına gelir. Ayrıca bu gibi kompleks biyolojik sıvılarda albümin ve immunoglobülin gibi yüksek bolluk proteinlerin varlığı genellikle milyar kat exc ile maskeesini düşük miktardaki, düşük molekül ağırlıklı proteinler ve peptitler 3, 4. bu nedenle birçok örnek hazırlık adımları kütle spektrometrisi sekanslama ve kimlik önce gerekmektedir. Bu gibi bir hazırlık aşaması ticari olarak temin edilebilen tükenme sütunlar 5-8, yüksek miktardaki proteinlerin tükenmesi kullanmaktadır. Genellikle kovalent olmayan bir şekilde kaldırılmakta olan taşıyıcı proteinler ile ilişkili olduğundan, ne yazık ki, bu adım aday biyobelirteç verim azalmaya yol açar. Örnekler toplandığı bir zamanlar başka bir meydan aday biyomarkerların ex-vivo stabilitesi ile temsil edilmektedir. Proteinler, endojen veya eksojen proteazlar 9 ile degradasyona tabi tutulur. Bozulması 10-13 protein korurken hidrojel nanopartiküller, tahlilin aralığı içinde bir düzeye farazi biyolojik işaretleyici konsantrasyonunu yükselterek bu kritik zorlukları aşmak olabilir. Bu th dikkat etmek önemlidirLMW, kanda proteinlerin küçük sağlam proteinler ile birlikte, bu büyük proteinlerin parçalarının bir karışımı bulunmaktadır. 60 kDa&#39;dan daha büyük bir Doku türevli proteinler pasif vasküler bazal zar boyunca kan akışına girmesine kadar büyük, ancak peptidler veya protein parçacıkları 14 kanın temsil edilebilir. Amacımız Hastalığın erken teşhisi, tedavisi için hasta tabakalaşma ve tedaviye yanıtın izlenmesinde için aday olabilir roman dolaşan biyomarkerları ölçmektir. Bizim nanopartiküller anda küçük proteinler ve peptidler yakalama ve başlangıç ​​hacmine göre 100 kat kadar onları konsantre olurken seçici, yüksek bolluk immünoglobinlerin ve albumin dışlamak için oluşturulur. Bizim grup başarıyla proteinler ve peptidler için yüksek afinite moleküler yemler hareket edebilen küçük organik boyaların bir dizi tanımlanır. Protein boya bağlama bağlı olarak, hidrofobik ve elektrostatik etkileşimin bir kombinasyonu olduğu düşünülmektedirs. Boya üzerindeki aromatik halkalar protein yüzey 11 ile hidrofobik cepleri proteinleri ile dönüşümlü olarak çalışır. Yemler, kimyalarına bağlı olarak seçilmiş analitlerin sınıfları için özel bir çekim gücü gösterirler. Yemler proteinler veya peptidlere, örneğin albumin gibi, taşıyıcı proteinler ile rekabet eder. Düşük molekül ağırlıklı proteinler / peptidler parçacık içinde sıkışır. , Albümin ve immunoglobülin gibi yüksek molekül ağırlıklı proteinler nedeniyle hidrojel 11 (Şekil 1) yer alan sınırlayıcı gözenek çünkü eleme yeteneğinin parçacık girmesini engellenir. Hidrojel nanopartiküller amonyum persülfat 11 tarafından başlatılan çöktürme polimerizasyonu ile sentezlenir. N-izopropil (Nipam), akrilik asit (AAC) ve alilamin (AA) ve çapraz bağlayıcı, N, N&#39;-metilenbisakrilamid (BIS) eş-monomerler arasında 1 seyreltik koşullar içinde, 6 saat boyunca 70 ° C&#39;de reaksiyona girmesine imkan verilir1, 13. Poli yüksek protein bağlanma afinitesi (N-izopropil akrilamid-ko-akrilik asit) kovalent (amino içeren boya maddelerinin katılmasıyla, yani., Sulfonatedanthraquinonetriazine boyalar) nanopartiküllerin içine yoluyla elde (poli (Nipam-ko-AAC) nanoparticlesis Boyaların hidrofobik / hidrofilik özelliklere bağlı olarak, sulu ya da organik çözücü maddelerin içinde, bir amidasyon reaksiyonu 11, bir anthraquinonetriazine boyanın klorür atomu ile nanopartikül amin grubuna ait 13. nükleofilik ikamesi, boya ihtiva eden poli (Nipam oluşturmak için kullanılmaktadır CO-allilamin) (AA) 11 12 nanoparçacıklar. iki aşamalı polimerizasyon işlemi vinilsülfonik asit (VSA), 11, 13, bir dış kabuk içeren hidrojel nano-tanecikleri oluşturmak için kullanılır. Hidrojel nanopartiküller, tam kan, plazma, serum, serebrospinal sıvı, ter ve idrar dahil olmak üzere çeşitli biyolojik sıvılar, uygulanabilir. Bir adım olarak, çözelti içinde, nanoparticles (birkaç dakika içinde) hızlı bir haciz ve düşük moleküler ağırlığa sahip konsantrasyonu, 10, 11, 13, 15-18 analitlerini gerçekleştirin. Proteinler daha sonra kullanılarak nanopartiküller yıkanır ve tespit edilir batı spektrometrisi 10, 11, 13, 15, 18, ​​22 kütle, 19-21 lekeleme, 23, bağışıklık / ELISA 10, 11, 15, 18, ​​ya da ters faz, protein mikro-dizi 16 24 deneyleri. Nanopartiküller, kimyasal yem ile fonksiyonalize ve bir çekirdek ya da çekirdek-kabuk mimari, yakalama sunulması ve yem / kabuk fizikokimyasal özelliklerine göre proteinlerin konsantrasyonu. Nanopartiküller dahil farklı boyaların bu nedenle boya afinitesi göre verimlilik, çözeltinin pH değeri ve yüksek miktardaki proteinlerin 13 rakip varlığını / yokluğunu değişen proteinlerin, farklı alt çekecektir. Bundan başka, çözeltinin hacmi ile ilgili olarak nanopartiküllerin miktarı nanopartiküller gelen protein verimi etkileyecektir. Bu yönleriHidrojel nanoparçacık hasat Yüksek miktarda protein içeren plazma numuneleri hasat proteinleri için üç farklı nanopartikül yemler kullanılarak gösterilebilir, ve tipik olarak yüksek miktarda protein içermeyen idrar örnekleri vardır. Bu protokolde, hasat ve poli (Nipam-ko-AAC), Poli (Nipam / boya) ve çekirdek-kabuk nano-tanecikleri kullanılarak plazma numuneleri, tümör nekroz faktörü alfa (TNFa) konsantre göstermektedir (Poli (Nipam-ko-VSA)) . Poli (Nipam / boya) nanopartiküller Mycobacterium tuberculosis ile enfekte bireyler taklit insan idrar örneklerinde eklendi Mycobacterium türleri antijeni, konsantre gösterilmiştir.

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">hydrogel nanoparticles</td>
			<td style="width:187px;">Ceres Nanoscience</td>
			<td style="width:119px;">CS003</td>
			<td style="width:336px;">NanoTrap ESP particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl, 50mM pH7.0</td>
			<td style="width:187px;">VWR</td>
			<td style="width:119px;">IC816116</td>
			<td>50mM, pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH1103-4LP</td>
			<td>available from VWR</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium Hydroxide NH4OH</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH3014</td>
			<td>available from VWR, assayed at 28-30% NH3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium thiocyanate 25mM</td>
			<td style="width:187px;">Acros Organics</td>
			<td style="width:119px;">419675000</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-analyte Urine Reagent Strips</td>
			<td style="width:187px;">Siemens</td>
			<td style="width:119px;">2161</td>
			<td style="width:336px;">for urine samples</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tris-Glycine SDS Sample Buffer (2X)</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">LC2676</td>
			<td style="width:336px;">use at room temperature to prevent SDS from precipitating</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dry bath incubator (100 oC) with heating block</td>
			<td style="width:187px;">Barnstead</td>
			<td>11-715-125DQ</td>
			<td>do not substitute a boiling water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen evaporator manifold</td>
			<td style="width:187px;">Organomation Associates</td>
			<td>Microvap118</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, swing-out rotor</td>
			<td style="width:187px;">Sorvall</td>
			<td style="width:119px;">Legend series</td>
			<td>50ml tube capacity, rcf 3700 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, fixed angle rotor</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:119px;">5424</td>
			<td>1.7ml microcentrifuge capacity, rcf 16,000 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50ml conical centrifuge&#160;tubes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td>with screw caps for urine samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1.5ml microcentrifuge tubes</td>
			<td style="width:187px;">Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Disposable plastic transfer pipettes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7M</td>
			<td>at least 1ml capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td>50-949-755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Timer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">S04782</td>
			<td>seconds/minutes</td>
		</tr>
	</tbody>
</table>

hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins

Roman biyomarkör keşif daha duyarlı ve özgül hastalık tespiti sağlayan çok önemli bir rol oynar. Biyolojik sıvılarda mevcut Ne yazık ki çok düşük miktardaki biyolojik göstergeler kolayca kütle spektrometrisi ya da çok düşük bir konsantrasyonda mevcut olan değişken ve çoğu zaman, albümin veya immünoglobülin gibi proteinler, yüksek miktardaki maskelendiğinden, çünkü bağışıklık ile tespit edilemez. Bait içeren poli (N-izopropil akrilamid) (Nipam) göre nanopartiküller bu fizyolojik engelleri aşmak mümkün. Bir adım olarak onlar yakalama konsantre ve vücut sıvılarından biyomarkerları korumak mümkündür. Düşük molekül ağırlıklı analitler nanopartikül çekirdeğini girip daha yüksek bir afiniteye proteini yemler etki gösteren farklı organik kimyasal boyalar tarafından yakalanır. Nanopartiküller büyüklükte birkaç emir tarafından ilgi proteinlerin konsantre edebiliyoruz. Bu konsantrasyon faktörü proteinleri içindedir böylece protein seviyesini yükseltmek için yeterlidirGeçerli Massenspektrometer, batı lekeleme ve immunoassay&#39;lerin saptama sınırı. Nanopartiküller biyolojik sıvıların bir bolluk ile inkübe edilebilir ve albümin ve diğer yüksek molekül ağırlıklı proteinler hariç ederken, büyük ölçüde düşük molekül ağırlıklı proteinler ve peptidlerin konsantrasyonu arttırmakta edebiliyoruz. Bizim veriler, belirli bir analit konsantrasyonunun 10,000 kez amplifikasyon önceden belirlenemeyen biyolojik işaretleri tespit etmek için kütle spektrometrisi ve immün sağlayarak elde edilebileceğini göstermektedir.

Hidrojel Nanoparçacık Boyutu ve tekdüzeliği Poli (Nipam-AAC) partikülleri ile kümeler içinde son derece yüksek verim ve üretkenlik ile üretilmiştir. Parçacıklar yakalama, depolama ve proteinlerin elüsyonu (en az 48 saat) için gerekli olan süre boyunca, oda sıcaklığında çok iyi bir stabiliteye sahip kolloidal ve nano partiküler çökeltme (Şekil 1) 11 gözlenmemiştir. Koloidal stabilite nanopartiküller ile hızlı bir şeki...

Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Düşük Bolluk Proteinler saptamak için plazma veya idrar hidrojel Nanoparçacık Hasat

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

hydrogel-nanoparticle-harvesting-plasma-or-urine-for-detecting-low

Research

JoVE Journal

Bioengineering

13.8K Görüntüleme.  George Mason University. Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Video: Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Plazma Litografi Yüzey Desenlendirme Hücre Ağları Oluşturma

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Biyomühendislik

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3 boyutlu montaj Self-Peptid Hidrojel İnsan Sinir Progenitör Hücreler Yetiştiriciliği

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Tasarım ve Yapay Ekstrasellüler Matrix İnşaatı (aECM) Proteinler gelen<em&gt; Escherichia coli</em&gt; Cilt Doku Mühendisliği

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

Narin Doku Manipülasyon için Özelleştirilebilir Yumuşak Robotik Pnömatik Tutucu Cihazlar Rod tabanlı Fabrikasyon

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

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

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

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

Sıvı Kristal Nanopartikül Carrier bir Hidrofobik Kargo Encapsulated, hedeflenen Plazma Zarı Teslimat

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

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Biyoaktif proteinler veya peptidler hidrojel Photochemistry biyolojik uygulamalarda kullanarak üzerinde desenlendirme

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

Doku Microcolumns ürün toplama aparatı

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Bir böbrek korteks ekstraselüler matrisi elde edilen hidrojel imalatı

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Bakteri Taraması, Seçimi ve İzolasyonu için Fotobozunur Hidrojel Arayüzleri

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Güçlü yapışkan hidrojel, jelatin o-nitrosobenzaldehit sentezi

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...