Name: hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins
Uploaded: 2014-08-07T20:45:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

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>

<ol>
	<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><tbody><tr><td>Hydrogel nanoparticles</td><td>Ceres Nanoscience</td><td>CS003</td><td>NanoTrap ESP particles</td></tr><tr><td>18 M&amp;Omega;-cm water</td><td></td><td></td><td>Type 1 reagent grade water</td></tr><tr><td>Tris HCl, 50 mM pH 7.0</td><td>VWR</td><td>IC816116</td><td>50 mM, pH 7</td></tr><tr><td>Acetonitrile</td><td>BDH</td><td>BDH1103-4LP</td><td>Available from VWR</td></tr><tr><td>Ammonium Hydroxide NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>BDH</td><td>BDH3014</td><td>Available from VWR, assayed at 28-30% NH&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium thiocyanate 25 mM</td><td>Acros Organics</td><td>419675000</td><td>For serum/plasma samples</td></tr><tr><td>Multi-analyte Urine Reagent Strips</td><td>Siemens</td><td>2161</td><td>For urine samples</td></tr><tr><td>Tris-Glycine SDS Sample Buffer (2X)</td><td>Life Technologies</td><td>LC2676</td><td>Use at RT to prevent SDS from precipitating</td></tr><tr><td>Dry bath incubator (100 &lt;sup&gt;o&lt;/sup&gt;C) with heating block</td><td>Barnstead</td><td>11-715-125DQ</td><td>Do not substitute a boiling water bath</td></tr><tr><td>Nitrogen evaporator manifold</td><td>Organomation Associates</td><td>Microvap118</td><td>For serum/plasma samples</td></tr><tr><td>Centrifuge, swing-out rotor</td><td>Sorvall</td><td>Legend series</td><td>50 ml tube capacity, rcf 3,700 x g</td></tr><tr><td>Centrifuge, fixed angle rotor</td><td>Eppendorf</td><td>5424</td><td>1.7 ml microcentrifuge capacity, rcf 16,000 x g</td></tr><tr><td>50 ml conical centrifuge&amp;nbsp;tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td>With screw caps for urine samples</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td>50-949-755</td><td></td></tr><tr><td>Timer</td><td>Fisher Scientific</td><td>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

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Biyomarker Discovery için Mezogözenekli Silika İnce Filmler üzerine düşük molekül ağırlıklı Protein Zenginleştirme

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Biyomühendislik

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Protein sindirimi, Ultrafiltrasyon ve boyutu dışlama Kromatografi insan kan plazması ve Serum Exosomes yalıtım en iyi duruma getirmek için

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Biyokimya

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Bir Modifiye Yağış Yöntemi İdrar eksozomlar yalıtmak için

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

Tıp

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Protein-Hedefleme Küçük Molekül İlaç Tarama için hızlı ve Nicel florimetrik Yöntemi

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Tüm Blood adlı ultrasensitive Protein Algılama için Tam Otomatik Santrifüj mikroakışkan Cihazı

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

Nanomalzemeler Tarafından Elde Edilen Protein ve Metabolit Koronanın Karakterizasyonu için Kılcal Elektroforez Kütle Spektrometresi Yaklaşımları

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Kimya

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

Düşük Bolluktaki Proteinlerin Hedeflenen Kütle Spektrometrisine Dayalı Protein Analizinde Mikro Örnekleme

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Protein Saflaştırma Kullanım için Otomatik Hidrofobik Etkileşim Kromatografisi Sütun Seçimi

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Biyoloji

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 ...

Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.

This protocol describes the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. It is based on the separation of EVs by polymer precipitation, followed by ACSA-1 based immunocapture of ADEVs. Analysis of ADEVs may offer clues to study changes in inflammatory pathways of living patients, non-invasively by liquid biopsy.

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a ...