Michael Henry, Dublin City University, gentilmente aiutato con la raccolta e analisi dei dati illustrato in Figura 5. Questo lavoro è stato sostenuto in parte da (1) George Mason University, (2) l&#39;italiano IstitutoSuperiore di Sanita &#39;nel quadro della Italia / USA accordo di cooperazione tra il Dipartimento di Salute e Servizi Umani, George Mason University, e il Ministero della Salute pubblica italiana, (3) NIH, borse di programma IMAT 1R21CA137706-01 e 1R33CA173359-01 a LAL, e (4) Ceres Nanoscienze, Inc .

Plasma umano e nelle urine sono stati raccolti da donatori volontari sani, con il consenso informato scritto, seguendo George Mason University Institutional Review Board protocolli approvati. I donatori sono stati equamente distribuiti tra maschi caucasici e femmine di età compresa tra 25 e 42 campioni sono stati analizzati individualmente tra e non sono stati riuniti. 1 nanoparticelle Elaborazione di siero o plasma Basso potenziale di biomarcatori abbondanti nel plasma vengono catturati, in soluzione, con nanoparticelle idrogel. Le particelle vengono aggiunti al plasma, incubato, separato per centrifugazione, lavato, e le proteine ​​vengono eluite catturate. Le proteine ​​eluite vengono essiccati in corrente di azoto a valle per il sequenziamento spettrometria di massa e l&#39;identificazione. <ol><li> Diluire 500 ml di siero 1: 2 con 50 mM TrisHCl pH 7 (siero ml 500 + 500 microlitri TrisHCl) in una provetta. </li><li> Aggiungere 500 ml di poli (NIPAM / AAC) nucleo nanoparticoli. Incubare per 15 minuti a temperatura ambiente. </li><li> Centrifugare il campione a 16.100 xg, 25 ° C per 10 minuti in una centrifuga dotata di un rotore ad angolo fisso. Rimuovere e scartare il surnatante. </li><li> Aggiungere 500 microlitri tiocianato di sodio (25 mm) al pellet. Risospendere le nanoparticelle da vigorosamente pipettando su e giù più volte. </li><li> Centrifugare il campione a 16.100 xg, 25 ° C per 10 minuti in una centrifuga con rotore ad angolo fisso. Rimuovere e scartare il surnatante. </li><li> Aggiungere 500 ml di acqua MilliQ al pellet nanoparticella. Risospendere le nanoparticelle da vigorosamente pipettando su e giù più volte. </li><li> Centrifugare il campione a 16.100 xg, 25 ° C per 10 minuti in una centrifuga con rotore ad angolo fisso. Rimuovere e scartare il surnatante. </li><li> Preparare il tampone di eluizione fresco: Aggiungere 700 ml di acetonitrile idrossido di ammonio 300 microlitri in una provetta pulita. Attenzione: idrossido di ammonio è corrosivo. Usare con ventilazione adeguata. Nota: Il buffer di eluizione devono essere prepared immediatamente prima dell&#39;uso. Non conservare il tampone di eluizione. </li><li> Aggiungere 300 ml di tampone di eluizione al pellet nanoparticelle. Risospendere le nanoparticelle da vigorosamente pipettando su e giù più volte. Incubare per 15 minuti a temperatura ambiente. </li><li> Centrifugare il campione a 16.100 xg, 25 ° C per 10 minuti in una centrifuga con rotore ad angolo fisso. Rimuovere e conservare l&#39;eluato in una, provetta etichettata e pulita. </li><li> Ripetere i passaggi 1,9-1,10. Unire i due eluati in una provetta. </li><li> Essiccare gli eluati sotto flusso di azoto in un collettore evaporatore azoto a 42,1 ° C, con flusso d&#39;aria impostata a 8 (Figura 2F). </li><li> Conservare l&#39;eluato essiccata a temperatura ambiente per O / N stoccaggio, oa -20 ° C per la conservazione a lungo termine, prima della spettrometria di massa, western blotting, o test ELISA (opzionale). </li></ol> 2. nanoparticelle Trattamento dei Campioni di urina L&#39;urina normale contiene meno di 30 mg / dl e proteinemeno di 1+ sangue. Tuttavia, molte malattie / condizioni possono alterare i normali livelli di proteine ​​nelle urine e del sangue. Per aiutare a determinare il volume ottimale di nanoparticelle da aggiungere al campione di urine, un esame delle urine viene eseguito prima di nanoparticelle di raccolta. Possono esistere biomarcatori urine in concentrazioni estremamente basse, che possono richiedere l&#39;ottimizzazione del rapporto tra nanoparticelle di volume urinario. Questa procedura descrive nanoparticelle raccolta di campioni di urine per la valle analisi western blot. <ol><li> Raccogliere campioni di urina in un bicchiere di plastica pulito, asciutto. È richiesto un volume minimo di 22 ml. Urine Conservare i campioni a -80 ° C fino al momento di analizzare. </li><li> Scongelare l&#39;urina congelata a temperatura ambiente oa 4 ° CO / N. Mescolare brevemente su vortex. Versare almeno 22 ml di urina in una provetta di polipropilene fondo conico da 50 ml. </li><li> Spin urine in una centrifuga con rotore swing-out a 3.700 xg per 15 min. Decantare l&#39;urina, senza disturbare il pellet, in un ambiente pulito polypr fondo conico da 50 mltubo opylene. Eliminare il pellet. </li><li> Eseguire l&#39;analisi delle urine con un multi-analita &quot;urine con stick reattivo&quot; striscia reagente. Nota: conservare le strisce reagenti in un contenitore ermeticamente chiuso lontano dalla luce diretta del sole e dall&#39;umidità 17, 18. <ol><li> Posare la striscia reagente, a faccia in su, su un fazzoletto di carta pulito e asciutto. </li><li> Utilizzare una pipetta monouso per aspirare 1 ml di urina preparato al punto 2.3. </li><li> Impostare un timer per 2 min, ma non avviarlo. Erogare rapidamente 1 - 2 gocce di urina su ogni pad prova della striscia reagente. Avviare immediatamente il timer. Nota: Non immergere la striscia reagente direttamente nel contenitore urine. Gli indicatori colorante / chimici nella striscia reagente possono potenzialmente percolare fuori della striscia reagente nel contenitore urine. </li><li> Al tempo indicato sul flacone delle strisce di reagente, registrare i risultati qualitativi per i vari analiti sulla striscia reagente confrontando il colore delle singole strisce reagenti alla corrispondente indica codice coloretori sul contenitore del reagente. Tipici normali risultati delle urine sono: (normale o negativo) per il sangue, proteine, leucociti, nitriti, glucosio, chetoni, bilirubina e urobilinogeno; Urine pH (5,5-7,0); peso specifico (1,001-1,020). Nota: Alti livelli di proteine ​​in un campione di urina possono competere con la proteina di interesse per i siti sulle nanoparticelle vincolanti. Per massimizzare la raccolta di proteine ​​con nanoparticelle, l&#39;/ volume di urina volume di nanoparticelle può essere regolato sia per limitare la concorrenza da proteine ​​di alta abbondanza, o può essere ottimizzato per raccogliere le proteine ​​presenti in concentrazioni molto basse. Se il valore di proteine ​​nelle urine è 1+ o superiore, aggiungere 2x volumi di nanoparticelle nella fase 2.5. Se l&#39;analita di interesse è molto basso abbondante proteina, può essere necessario aumentare il volume delle nanoparticelle fino a 2 ml (Figura 3). </li></ol></li><li> Trasferire 20 ml di urina chiarito dal punto 2.3 in una provetta da 50 ml pulito polipropilene fondo conico. Nondisturbare eventuali detriti / pellet che possono essere nel fondo della provetta urina. Aggiungere 200 ml di nanoparticelle per il campione di urina 20 ml. Mescolare brevemente su vortex. Nota: Se il valore di proteine ​​nelle urine è 1+ o superiore, aggiungere 400 ml di nanoparticelle a 20 ml di urina. </li><li> Incubare la miscela urine / nanoparticelle per 30 minuti a temperatura ambiente, senza che a dondolo / miscelazione. </li><li> Centrifugare la sospensione urina / nanoparticelle in una centrifuga dotata di un rotore oscillante a 3.700 xg per 10 min. Nota: se un pellet non è visibile Dopo la centrifugazione, girare i campioni per un ulteriore 2 - 7 min. </li><li> Rimuovere e scartare il surnatante. Aggiungere acqua 500 microlitri MilliQ al pellet nanoparticelle. </li><li> Risospendere le nanoparticelle da vigorosamente pipettando su e giù più volte. Trasferire la soluzione di nanoparticelle di un ambiente pulito 1,5 ml provetta. </li><li> Spin le nanoparticelle in una centrifuga dotata di un rotore ad angolo fisso a 16.100 xg per 10 min. Nota: se un pellet non è fol visibileguendo centrifugazione, girare i campioni per un ulteriore 2-7 min. </li><li> Rimuovere e scartare il surnatante. </li><li> Ripetere i punti 2.8 e 2.11 (due volte) con 200 ml 18 MilliQ acqua per lavare le nanoparticelle. </li><li> Preparare il tampone di eluizione fresco: Aggiungere 970 ml di acetonitrile idrossido di ammonio 30 ml in una provetta pulita. Attenzione: idrossido di ammonio è corrosivo. Usare con ventilazione adeguata. Nota: Il buffer di eluizione deve essere preparata immediatamente prima dell&#39;uso. Non conservare il tampone di eluizione. </li><li> Aggiungere 20 microlitri tampone di eluizione al pellet nanoparticelle. Risospendere le nanoparticelle da vigorosamente pipettando su e giù più volte. Incubare per 15 minuti a temperatura ambiente. </li><li> Spin i campioni di nanoparticelle a 16.100 xg per 10 min. Rimuovere e conservare il surnatante in una provetta pulita da microcentrifuga da 1,5 ml. Non disturbare il pellet nanoparticelle. Eliminare il pellet nel rischio biologico spazzatura. </li><li> Mettere i campioni in un rack in una cappa. Aprire i tappi e incUbate a temperatura ambiente per 30 min. In alternativa, posizionare i campioni sotto flusso di azoto a 40 ° C fino a secco (1 - 2 ore). </li><li> Aggiungere 15 microlitri tampone Tris-glicina SDS (2x) per i campioni. Riscaldare a 100 ° C in un blocco a calore secco per 5 min con i tappi aperti o fino a quando il volume lasciato nel tubo non è più di 20 microlitri. Nota: non utilizzare un bagno di acqua bollente. L&#39;umidità dal bagno di acqua impedirà l&#39;evaporazione del buffer. </li><li> Posizionare il tappo sui tubi. Rimuovere i tubi dal blocco di calore. Il campione può essere conservato a -80 ° C o utilizzato immediatamente a valle per l&#39;analisi western blot. </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.

Rilevanza clinica Un campione di siero o plasma è pensato per contenere bassa abbondanza di proteine ​​e peptidi in grado di fornire una ricca fonte di informazioni per quanto riguarda lo stato dell&#39;organismo nel suo complesso circolante. Nonostante la promessa di proteomica del siero, ci sono tre barriere fisiologiche fondamentali e gravi contrastare scoperta di biomarcatori e la traduzione di beneficio clinico 10, 11, 16, 25. <p class="jove_content"...

Nonostante il completamento del sequenziamento del genoma umano, non sono stati compiuti progressi significativi in biomarcatori predittivi di identificazione di malattia in stadio precoce, o che la correlazione con il risultato terapeutico, o la prognosi 1. Uno dei motivi di questa mancanza di progresso è che esistono molti biomarcatori potenzialmente significative ad una concentrazione al di sotto del limite di rilevazione di spettrometria di massa convenzionale e altre piattaforme di scoperta biomarker. La spettrometria di massa (MS) e multipla Reaction Monitoring (MRM) hanno una sensibilità di rilevamento tipicamente superiore a 50 ng / ml, mentre la maggior parte degli analiti misurati da test immunologici caduta laboratorio clinico nell&#39;intervallo tra 50 pg / ml e 10 ng / ml . Questo significa che molti biomarcatori, in particolare nella fase iniziale di una malattia non possono essere rilevati da MS convenzionale e MRM 2. Inoltre la presenza di proteine ​​ad alta abbondanza come albumina e immunoglobuline nei fluidi biologici complessi maschera spesso da miliardi di volte excess bassa abbondanza, proteine ​​a basso peso molecolare e peptidi 3, 4. Per questo motivo sono necessari prima di sequenziamento spettrometria di massa e di identificazione diversi passaggi di preparazione del campione. Un tale passo preparatorio impiega l&#39;esaurimento di proteine ​​di alta abbondanza con colonne di esaurimento disponibili in commercio 5-8. Purtroppo questo passaggio porta alla riduzione del rendimento di biomarker candidati perché sono spesso non covalentemente legato al proteine ​​carrier che vengono rimossi. Un&#39;altra sfida è rappresentata dalla stabilità dei biomarcatori candidati ex-vivo una volta che i campioni vengono raccolti. Le proteine ​​sono soggetti a degradazione da proteasi endogene o esogene 9. Nanoparticelle idrogel possono superare queste sfide critiche amplificando la concentrazione biomarcatore putativo a un livello entro l&#39;intervallo del dosaggio, proteggendo la proteina dal degrado 10-13. E &#39;importante notare tha LMW proteine ​​nel sangue sono una miscela di piccole proteine ​​intatte così come frammenti di proteine ​​di grandi dimensioni. Proteine ​​derivate dal tessuto superiore a 60 kDa sono troppo grandi per entrare passivamente il flusso sanguigno attraverso la membrana basale vascolare, ma possono essere rappresentati nel sangue come peptidi o frammenti di proteine ​​14. Il nostro obiettivo è quello di misurare nuovi biomarcatori circolanti che possono essere candidati per la diagnosi precoce della malattia, stratificazione dei pazienti per la terapia e monitorare la risposta alla terapia. Le nostre nanoparticelle sono creati per escludere selettivamente immunoglobuline alta abbondanza e albumina, e contemporaneamente catturare proteine ​​e peptidi più piccoli e la loro concentrazione fino a 100 volte a seconda del volume di partenza. Il nostro gruppo ha identificato una serie di piccoli coloranti organici che possono agire con successo esche molecolari ad alta affinità per le proteine ​​e peptidi. Binding protein-dye è pensato per essere a causa di una combinazione di interazione idrofobica ed elettrostaticas. Gli anelli aromatici sulla colorante interleave con proteine ​​tramite tasche idrofobiche sulla superficie della proteina 11. Le esche, a seconda della loro chimica, mostrano una particolare affinità per le classi selezionate di analiti. Le esche sono in concorrenza con le proteine ​​di trasporto, come l&#39;albumina, per le proteine ​​o peptidi. Il peso proteine ​​/ peptidi a basso peso molecolare diventano intrappolati nella particella. Proteine ​​di elevato peso molecolare come albumina e immunoglobuline viene impedito di entrare particella a causa della capacità setacciatura dovuta al poro restrittiva del idrogel 11 (Figura 1). Nanoparticelle idrogel sono sintetizzati per precipitazione polimerizzazione iniziata da persolfato di ammonio 11. N-isopropilacrilammide (NIPAM), co-monomeri di acido acrilico (AAC) e allilamina (AA) e cross-linker N, N&#39;-Methylenebisacrylamide (BIS) sono autorizzati a reagire a 70 ° C per 6 ore in condizioni diluite 11, 13. L&#39;affinità di legame ad alta percentuale proteica di poli (N isopropilacrilammide-co-acrilico-acido) (poli (NIPAM-co-AAC) nanoparticlesis raggiunto da covalentemente incorporando coloranti-aminoacidi contenenti (es., Coloranti sulfonatedanthraquinonetriazine) nelle nanoparticelle attraverso una reazione ammidazione eseguita in solventi acquosi o organici a seconda delle caratteristiche idrofiliche / idrofobiche dei coloranti 11, 13. sostituzione nucleofila dei gruppi amminici della nanoparticella con l&#39;atomo di cloruro di un colorante anthraquinonetriazine è utilizzata per creare-colorante contenente poli (NIPAM -CO-allilamina) (AA) nanoparticelle 11, 12. Un processo di polimerizzazione in due fasi viene utilizzato per creare nanoparticelle idrogel che contengono un guscio esterno di acido vinylsulfonic (VSA) 11, 13. Nanoparticelle idrogel possono essere applicati a vari fluidi biologici, compreso il sangue intero, plasma, siero, liquido cerebrospinale, il sudore e l&#39;urina. In un passo, in soluzione, il nanoparticles eseguire una rapida (entro pochi minuti) il sequestro e la concentrazione di basso peso molecolare analiti 10, 11, 13, 15-18. Le proteine ​​sono successivamente eluiti dalle nanoparticelle e rilevati mediante Western Blotting 19-21, spettrometria di 10, 11, 13, 15, 18, ​​22 di massa, 23, saggi immunologici / ELISA 10, 11, 15, 18, ​​o fase inversa proteina microarray 16, 24 saggi. Le nanoparticelle funzionalizzate con esche chimiche, e presentando un nucleo o nucleo shell architettura, la cattura e concentrano le proteine ​​in base alle proprietà fisico-chimiche esca / shell. Diversi coloranti incorporati nelle nanoparticelle saranno quindi catturare diversi sottoinsiemi di proteine ​​con diversa efficacia basata sull&#39;affinità colorante, il pH della soluzione, e la presenza / assenza di competere proteine ​​ad alto abbondante 13. Inoltre, la quantità di nanoparticelle in rapporto al volume della soluzione influenzerà la resa di proteine ​​dalle nanoparticelle. Questi aspetti diidrogel nanoparticelle raccolta sono dimostrata utilizzando tre diverse esche nanoparticelle per le proteine ​​raccolta di campioni di plasma che contengono elevate quantità di proteine, e da campioni di urina che in genere non contengono grandi quantità di proteine. In questo protocollo dimostriamo la raccolta e concentrazione fattore di necrosi tumorale alfa (TNF-alfa) da campioni di plasma mediante poli (NIPAM-co-AAC), Poli (NIPAM / colorante) e nanoparticelle core-shell (Poly (NIPAM-co-VSA)) . Poli (NIPAM / colorante) nanoparticelle sono mostrati a concentrarsi Mycobacterium antigene specie che è stato aggiunto a campioni di urina umana, per imitare Mycobacterium tuberculosis individui infetti.

<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

Scoperta di biomarcatori romanzo gioca un ruolo cruciale nel fornire il rilevamento delle malattie più sensibile e specifica. Purtroppo molti biomarcatori bassa abbondanza che esistono nei fluidi biologici non possono essere facilmente individuati con la spettrometria di massa o immunologici perché sono presenti in concentrazioni molto basse, sono labili, e sono spesso mascherati da proteine ​​ad alta abbondanza, come l&#39;albumina e immunoglobulina. Bait contenente poli (N-isopropilacrilammide) (NIPAM) nanoparticelle a base sono in grado di superare queste barriere fisiologiche. In un passo sono in grado di catturare, concentrare e preservare biomarcatori da fluidi corporei. Analiti basso peso molecolare entrano nel nucleo della nanoparticella e vengono catturati da diversi coloranti chimici organici, che fungono da alta affinità esche proteiche. Le nanoparticelle sono in grado di concentrare le proteine ​​di interesse di parecchi ordini di grandezza. Questo fattore di concentrazione è sufficiente aumentare il livello di proteina tale che le proteine ​​sono all&#39;internolimite di rilevazione di spettrometri di massa attuali, western blotting e saggi immunologici. Le nanoparticelle possono essere incubate con una pletora di fluidi biologici e sono in grado di arricchire notevolmente la concentrazione di proteine ​​a basso peso molecolare e peptidi, escludendo albumina e altre proteine ​​ad alto peso molecolare. I nostri dati mostrano che una piega amplificazione 10.000 nella concentrazione di un particolare analita può essere raggiunto, consentendo spettrometria di massa e test immunologici per rilevare biomarker precedentemente non rilevabili.

Hydrogel nanoparticelle Dimensioni e uniformità Poli (NIPAM-AAC) le particelle sono state prodotte con altissima resa e riproducibilità tra e all&#39;interno di lotti. Le particelle hanno una buona stabilità colloidale a RT per il tempo necessario per l&#39;acquisizione, la memorizzazione e l&#39;eluizione delle proteine ​​(almeno 48 ore), e nanoparticelle precipitazione non è stato osservato (Figura 1) 11. La stabilità colloidale può es...

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.

Hydrogel nanoparticelle raccolta di plasma o nelle urine per il rilevamento di proteine ​​a basso Abbondanza

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

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JoVE Journal

Bioengineering

13.8K Views.  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.

Plasma Surface Patterning litografia per la creazione di reti di cella

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

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Bioingegneria

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.

La coltivazione di cellule progenitrici neurali umane in un 3-dimensionale di auto-assemblaggio 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 ...

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.

Progettazione e Costruzione di Artificiale matrice extracellulare (AECM) Proteine ​​da<em&gt; Escherichia coli</em&gt; Per la pelle Ingegneria dei Tessuti

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.

Fabrication Rod a base di personalizzabili morbide robotizzati pneumatici Gripper dispositivi per la manipolazione dei tessuti delicati

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.

Targeted membrana plasmatica di consegna di un idrofobo Cargo incapsulato in un cristallo nanoparticelle Carrier Liquid

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.

Patterning bioattivi proteine o peptidi su idrogel utilizzando fotochimica per applicazioni biologiche

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.

Apparato per la raccolta del tessuto microcolonne

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.

Fabbricando un rene corteccia extracellulare Matrix-derivato idrogel

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.

Interfacce idrogel fotodegradabili per lo screening, la selezione e l'isolamento dei batteri

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.

Sintesi di idrogel adesivo forte, gelatina O-nitrosobenzaldeide

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

Hydrogel nanoparticelle raccolta di plasma o nelle urine per il rilevamento di proteine ​​a basso Abbondanza

Hydrogel nanoparticelle raccolta di plasma o nelle urine per il rilevamento di proteine a basso Abbondanza