Vorremmo anche ringraziare Xiaoyu Miao e Ben Wilson per lo sviluppo della maggior parte dei metodi descritti all&#39;interno. Questo lavoro è stato finanziato dalla National Science Foundation (DBI 0454324) e l&#39;Istituto Nazionale della Salute (R21 EB005183) e da PHS NRSA T32 GM07270 da NIGMS a ECK.

1. Array casuale Au Fabrication nanoparticelle 8,10,12,14 <ol><li> L&#39;array nanoparticelle Au è formata creando prima un modello che è fatto di un denso strato di lattice in modo casuale assorbito sfere con diametro medio di 454 nm. Questo risultato è ottenuto dal primo oro evaporazione su un vetrino di vetro per uno spessore di 20 nm usando cromo come il livello di adesione. </li><li> Il monostrato sfera di polistirolo è poi auto-assemblati esponendo l&#39;oro rivestite substrato ad una miscela di 1-etil-3-(3-dimetilamminopropil) carbodiimmide cloridrato (EDC), la sospensione sfera lattice e acqua deionizzata. </li><li> Il processo di adsorbimento è consentito a durare per circa un&#39;ora e non assorbita sfere vengono lavate via con abbondante acqua. </li><li> Il monostrato formato è lasciati asciugare all&#39;aria. </li><li> Infine, un altro 20 nm di oro è evaporato in monostrato sfera di lattice per formare la serie casuale nanoparticelle d&#39;oro. </li><li> Se un SEM è disponibile, l&#39;array AuNP può essere visto sotto il SEM a guardare come figura 1 e un diagramma del processo è mostrato in figura 8. </li></ol> 2. Preparazione del campione biologico 9,11 <ol><li> Preparazione dei campioni per i nuclei del mouse ottico cattura cella è ora mostrato. </li><li> 3T3 nuclei delle cellule del mouse taggati con acridina arancio colorante sono stati ottenuti dal gruppo Tewari presso il Fred Hutchinson Cancer Research Center. </li><li> 10% di albumina di siero bovino (BSA) in tampone fosfato (PBS) si aggiunge ai nuclei delle cellule del mouse in una concentrazione di circa 1: 10 (BSA: Cell Nuclei mouse). La BSA aiuta a prevenire i nuclei di attaccarsi al substrato. </li><li> Mescolare la soluzione utilizzando sonicazione. </li><li> 5 ul della nostra soluzione si deposita sul vetrino serie alluminio grata. E &#39;preferibile eseguire questo passaggio con la grata di alluminio sul palco microscopio in modo che non c&#39;è bisogno di trasporto del campione dopo la soluzione è depositato. </li><li> Due pile di due 1 &quot;1&quot; coprioggetto vengono utilizzati per supportare un coprioggetto quinte attraverso il quale è visto il campione. </li><li> Posizionare il campione al microscopio per la visualizzazione. </li></ol> 3. Metodo di trapping <ol><li> Le pinzette ottiche sono costruite inviando un 35 mW laser elio neon attraverso un Axio Imager.D1M Zeiss dotata di un set di 17 GFP filtro che viene modificato per consentire la radiazione laser 633 nm per raggiungere il campione. </li><li> Un Zeiss LD CE Epiplan - Neofluar obiettivo 50x è usato per l&#39;immagine del nucleo delle cellule, che sono circa 5 micron di diametro. </li><li> Dopo che il campione è posto sotto l&#39;obiettivo, l&#39;attenzione al microscopio sulla matrice nanoparticelle d&#39;oro o reticolo di diffrazione. </li><li> Traduci microscopio verticalmente fino a fuoco l&#39;immagine sui nuclei che si desidera trappola. </li><li> Laser posto sulla posizione trappola particella e la particella dovrebbe mantenere la sua posizione nel punto laser anche quando il palco è tradotto. </li></ol> 4. Rappresentante dei risultati: Il caso delle nanoparticelle d&#39;oro procedure di serie dovrebbe depositare un monostrato di AuNP che può essere visto sotto un SEM ad assomigliare Figura 1. La forza di cattura creato da questi pinzette plasmoniche possono essere 10-20 volte la forza generata dalla norma pinzette ottiche. L&#39;intensità minimo richiesto dalla pinzetta plasmoniche per raggiungere confinamento delle particelle sono mostrate per particelle di diverse dimensioni in Figura 4. 9,10 Il reticolo di diffrazione ottenuto l&#39;allineamento e la cattura di 20 volte maggiore efficienza rispetto alla cattura nanodots oro e potrebbe raggiungere cattura con il poco a 17 uW / um 2 (Figura 7) 11. <img alt="Figura 1" src="/files/ftp_upload/3390/3390fig1.jpg" /> Figura 1 10 (a) Micrografia SEM della auto-assemblati nanoparticelle di oro. Il diametro delle nanoparticelle d&#39;oro individuale è di circa 450 nm. (B) immagine NSOM del substrato plasmoniche dove la distribuzione delle nanoparticelle è scarsa, dimostrando la quasi-campo di radiazione. La lunghezza d&#39;onda del laser eccitazione è 633 nm. (C) vista Alto ingrandimento: la zona contrassegnata con il quadrato rosso in (b). (D) efficienza dello spettro Scattering del substrato plasmoniche, che mostra il picco a 624 nm. (E) efficienza dello spettro di assorbimento del substrato plasmoniche, che mostra il picco a 668 nm. <img alt="Figura 2" src="/files/ftp_upload/3390/3390fig2.jpg" /> Figura 2 13 (a) Au nanosfere distribuiti casualmente su un dominio 2D 1 x 1 micron 2. Ogni puntino blu rappresentano il centro del nanosfere (a = 60 nm). Distribuzioni di campo dispersione su piani di osservazione che sono parallele alla matrice casuale nanosfere sono mostrati in (b) - (e). L&#39;array nanosfere è uniformemente illuminato da un&#39;onda piana alla lunghezza d&#39;onda di 540 nm. L&#39;indice di rifrazione del mezzo circostante è 1,33. La poladirezione rizzazione dei punti d&#39;onda il piano lungo l&#39;asse X (orizzontale in (a)). L&#39;ampiezza del campo elettrico incidente è considerato uguale a 1 nel calcolo. La separazione tra il piano di osservazione e l&#39;array è definito come nanosfere h. b) h = a. c) h = 2 bis. d), h = λ. e), h = 2λ. <img alt="Figura 3" src="/files/ftp_upload/3390/3390fig3.jpg" /> Figura 3 9 Schema di configurazione personalizzata microscopio a fluorescenza tra cui un filtro di eccitazione bypassato e una dicroica sostituito beam-splitter. Questa è la configurazione utilizzata per la cattura simultanea e imaging di fluorescenza. <img alt="Figura 4" src="/files/ftp_upload/3390/3390fig4.jpg" /> Figura 4 10 L&#39;intensità del laser minima per mantenere la trappola in funzione della portata del fluido circostante utilizzando cattura plasmoniche. Tutte le intensità ottica sono misurati sul piano di campionamento nell&#39;ambito dell&#39;obiettivo microscopio. (A) - (f), mostrano i risultati di misura per perle di polistirene singolo con diametro 7.3, 6.3 (non sferica), 5,0, 3,9, 2,5 e 1,1 micron, rispettivamente. I riquadri mostrano le immagini corrispondenti di particelle microscopiche. Le barre di scala in tutte le immagini rappresentano il 5 micron di lunghezza. <img alt="Figura 5" src="/files/ftp_upload/3390/3390fig5.jpg" /> Figura 5 10 La pendenza della retta con origine in fig. 4 rispetto alle dimensioni delle particelle per la cattura plasmoniche. Le barre di errore mostrano le deviazioni standard del adatta lineare. La pendenza della retta (rapporto tra la soglia di intensità ottica e portata) in fig. 4 ha una relazione approssimativamente lineare con la dimensione delle particelle, come mostrato in questa figura, indicando il vantaggio di cattura plasmoniche soprattutto per le particelle più piccole. <img alt="Figura 6" src="/files/ftp_upload/3390/3390fig6.jpg" /> Figura 6 11 (a) Schema del intrappolamento ottico potenziato utilizzando nanostrutture periodici 1-D. Il fascio incidente è diffratto dalla nanostruttura periodici a campo lontano. (B) La distribuzione dell&#39;intensità della luce con due polarizzazioni ortogonali nanostruttura in campo lontano. (B) La distribuzione dell&#39;intensità della luce con due polarizzazioni ortogonali alla superficie di un reticolo di alluminio con un periodo di 417 nm ottenuti utilizzando simulazioni FDTD. La distribuzione è normalizzata sulla intensità su una superficie piatta in alluminio. (C) e (d) potenziale cattura per particelle direttamente sopra la superficie del reticolo rispetto a posizione della particella per (c) un polistirolo 350 nm tallone e (d) un polistirolo 1 micron tallone. I cerchi bianchi illustrano le dimensioni delle particelle. Riquadri mostrano il potenziale cattura di sopra di una superficie piatta in alluminio per la dimensione delle particelle stesse confronti. I valori sono normalizzati per ogni dimensione delle particelle. Per tutte le figure di simulazione FDTD il campo visivo è di 10 x 8 micron 2. <img alt="Figura 7" src="/files/ftp_upload/3390/3390fig7.jpg" /> Figura 7 11 (a) l&#39;efficienza e l&#39;intensità Trappola cattura minima misurata per perle di polistirolo di varie dimensioni, con polarizzazione del fascio perpendicolare alle linee di griglia. Inserto trappola mostra asimmetria in termini di efficienza di cattura per tradurre una perpendicolare 3,87 um polistirolo tallone e in parallelo alle regole della griglia. La linea continua (grande asimmetria) è ottenuta con luce polarizzata incidente perpendicolarmente alla grata, e la linea trattino (piccola asimmetria) è ottenuta con luce polarizzata incidente parallelo al reticolo. L&#39;unità è in (pN [mW / micron 2] -1). (B) - (d) dimostrazione cattura di una fluorescente 590 nm perline di polistirene. Il cerchio rosso indica la posizione del punto laser come la luce del laser era troppo debole per essere visto. In un primo momento la particella è intrappolata all&#39;interno del punto di maggiore potenza, come il potere si abbassa il moto browniano della particella supera la forza cattura, permettendo la particella di scappare. (E) - (g) dimostrazione cattura di un nucleo cellulare fluorescente cancro ovarico. L&#39;intensità minima necessaria per avviare cattura di 16 μW / micron 2, ottenuti utilizzando un obiettivo 20x. <img alt="Figura 8" src="/files/ftp_upload/3390/3390fig8.jpg" /> Figura 8 14 procedimento di fabbricazione del tappo a forma di nanoparticelle d&#39;oro: a) evaporazione dello strato di Cr e Au sottili sul vetro coprioggetti. b) L&#39;esposizione alla sospensione sfera di polistirolo e di adsorbimento di sfere per 1 ora. c) eliminazione delle materie non adsorbito sfere di polistirolo e l&#39;asciugatura della superficie. d) evaporazione di un altro strato di Au sulla parte superiore delle sfere modello. e) Schema del tappo a forma di array di nanoparticelle Au, dove Au copre solo la parte superiore delle sfere modello.

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	<li>Jones, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electromechanics of Particles. , (1995).</li><li>Ashkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+and+manipulation+of+neutral+particles+using+lasers.">Optical trapping and manipulation of neutral particles using lasers.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 4853-4853 (1997).</li><li>Neuman, K. C., Chadd, E. H., Liou, G. F., Bergman, K., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+photodamage+to+Escherichia+coli+in+optical+traps.">Characterization of photodamage to Escherichia coli in optical traps.</a> Biophys. J. 77, 2856-2856 (1999).</li><li>Chiou, P. C., Ohta, A. T., Wu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+manipulation+of+single+cells+and+microparticles+using+optical+images.">Massively parallel manipulation of single cells and microparticles using optical images.</a> Nature. 436, 370-370 (2005).</li><li>Hsu, H. Y., Ohta, A. T., Chiou, P. Y., Jamshidi, A., Nealea, S. L., Wua, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransistor-based+optoelectronic+tweezers+for+dynamic+cell+manipulation+in+cell+culture+media.">Phototransistor-based optoelectronic tweezers for dynamic cell manipulation in cell culture media.</a> Lab Chip. 10, 165-172 (2010).</li><li>Righini, M., Ghenuche, P. S., Cherukulappurath, V., Myroshnychenko, F. J., Garcia de Abajo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quidant+Nano-optical+Trapping+of+Rayleigh+Particles+Escherichia+coli+Bacteria+with+Resonant+Optical+Antennas.">Quidant Nano-optical Trapping of Rayleigh Particles Escherichia coli Bacteria with Resonant Optical Antennas.</a> Nano Letters. 9, 3387-3391 (2009).</li><li>Righini, M., Zelenina, A. S., Girard, C., Quidant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+and+Selective+Trapping+in+a+Patterned+Plasmonic+Landscape.">Parallel and Selective Trapping in a Patterned Plasmonic Landscape.</a> Nature Physics. 3, 477-480 (2007).</li><li>Miao, X., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+dielectrophoresis+force+and+torque+induced+by+localized+surface+plasmon+resonance+of+a+cap-shaped+Au+nanoparticle+array.">Large dielectrophoresis force and torque induced by localized surface plasmon resonance of a cap-shaped Au nanoparticle array.</a> Opt. Lett. 32, 295-297 (2007).</li><li>Wilson, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulation of Nanoparticles and Biological Samples through Enhanced Optical Forces [dissertation]. , (2009).</li><li>Miao, X. Y., Wilson, B. K., Pun, S. H., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+manipulation+of+micron/submicron+sized+particles+and+biomolecules+through+plasmonics.">Optical manipulation of micron/submicron sized particles and biomolecules through plasmonics.</a> Optics Exp. 16, 13517-13525 (2008).</li><li>Wilson, B. K., Mentele, T., Bachar, S., Knouf, E., Bendoraite, A., Tewari, M., Pun, S. H., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructure-enhanced+laser+tweezers+for+efficient+trapping+and+alignment+of+particles.">Nanostructure-enhanced laser tweezers for efficient trapping and alignment of particles.</a> Optics. Exp. 18, 16005-16013 (2010).</li><li>Miao, X., Wilson, B. K., Cao, G., Pun, S. H., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+and+Rotation+of+Nanowires+Assisted+by+Surface+Plasmons.">Trapping and Rotation of Nanowires Assisted by Surface Plasmons.</a> IEEE Journal of Selected Topics in Quantum Electronics. 15, 1515-1520 (2009).</li><li>Miao, X. Y., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+and+manipulation+of+biological+particles+through+a+plasmonic+platform.">Trapping and manipulation of biological particles through a plasmonic platform.</a> IEEE Journal of Selected Topics in Quantum Electronics. 13, 1655-1662 (2007).</li><li>Miao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plasmonics for Micro/Nano Manipulation and Optofluidics [dissertation]. , (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Il significato di questi metodi di cattura è che diminuire l&#39;intensità ottica necessaria per la cattura sostenuta da qualche parte dell&#39;ordine di 10 3 μW / 2 micron a qualche parte dell&#39;ordine di 10 μW / micron 2. 10,11 I limiti su queste tecniche sono che la matrice di nanoparticelle d&#39;oro esperienze problemi di riscaldamento che deve essere superato. Per ovviare a questo problema, una struttura a cristallo fotonico 2D che è composto di un materiale dielettrico può...

<table border="1"><tr><td> Nome del materiale </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commento </td></tr><tr><td> Axio Imager Microscopio </td><td> D1M </td><td> Zeiss </td><td> D1M </td><td> Zeiss Axio Imager.D1M </td></tr><tr><td> Obiettivo microscopio </td><td> 50x/0.55 </td><td> Zeiss </td><td></td><td> LD CE Epiplan - Neofluar 50x/0.55 HD DIC </td></tr><tr><td> Microscopio Zeiss Camera </td><td> AxioCam MRC </td><td> Zeiss </td><td></td><td></td></tr><tr><td> Elio neon Laser </td><td> 35 mW </td><td> Ricerca Electro-Optics </td><td></td><td></td></tr><tr><td> Attenuatore variabile </td><td> ND variazione continua </td><td> Thorlabs </td><td> NDC-100C-4M </td><td> Per regolare l&#39;intensità microscopio </td></tr><tr><td> Zeiss filtro Set </td><td> Filtro Set # 17 </td><td> Zeiss </td><td> 488017-9901-000 </td><td> Filtro Set # 17 </td></tr><tr><td> Microscopio diapositive </td><td> 0,5 millimetri di spessore </td><td> VWR </td><td></td><td></td></tr><tr><td> 3T3 topo nuclei cellulari </td><td></td><td colspan="2"> Fred Hutchinson Cancer Research Center </td><td> Conservare freddo come possibile </td></tr><tr><td> Acridina arancio colorante </td><td></td><td colspan="2"> Fred Hutchinson Cancer Research Center </td><td></td></tr><tr><td> Albumina di siero bovino </td><td> 1-10 razione in PBS </td><td colspan="2"> Fred Hutchinson Cancer Research Center </td><td></td></tr><tr><td> 454 nm polistirolo sfere in lattice </td><td></td><td> Polysciences, Inc. </td><td></td><td></td></tr><tr><td> carbodiimmide cloridrato (EDC) </td><td> 1-etil-3-(3-dimetilamminopropil) </td><td> G-Biosciences </td><td> BC25-1 </td><td></td></tr><tr><td> oro (per la deposizione) </td><td></td><td></td><td></td><td></td></tr><tr><td> Riflettente reticolo di diffrazione governato </td><td></td><td> Edmund Optics </td><td></td><td></td></tr><tr><td> Soluzione tampone fosfato (PBS) </td><td> Fosfato-Buffered Saline Dulbecco (D-PBS) (1X) </td><td> Invitrogen </td><td> 14190-144 </td><td></td></tr></table>

utilization of plasmonic and photonic crystal nanostructures for enhanced micro- and nanoparticle manipulation

Un metodo per manipolare la posizione e l&#39;orientamento delle particelle inferiori al micron non distruttivo sarebbe uno strumento incredibilmente utile per la ricerca biologica di base. Forse la forza più utilizzato per raggiungere la manipolazione fisica non invasiva di piccole particelle è stato dielettroforesi (DEP). 1 Tuttavia, DEP da sola non ha la versatilità e la precisione che si desidera quando manipolare le cellule in quanto è tradizionalmente fatto con elettrodi fissi. Pinzette ottiche, che utilizzano un tridimensionale gradiente di campo elettromagnetico a esercitano delle forze sulle piccole particelle, ottenere questa versatilità e precisione desiderata. 2 Tuttavia, un grave inconveniente di questo approccio è l&#39;intensità della radiazione di alta richiesti per raggiungere la forza necessaria per intrappolare una particella che possono danneggiare i campioni biologici 3 Una soluzione che permette di cattura e di smistamento con minore intensità ottici sono optoelettronici pinzette (OET) ma OET hanno limitazioni con la manipolazione multa di piccole particelle;.. essere DEP tecnologia basata mette anche vincolo sulla proprietà della soluzione 4 , 5 Questo articolo video descrivono due metodi che diminuire l&#39;intensità della radiazione necessaria per manipolazione ottica di cellule viventi e anche descrivere un metodo per il controllo di orientamento. Il primo metodo è pinzette plasmoniche che utilizzano un casuale nanoparticelle d&#39;oro (AuNP) array come substrato per il campione, come mostrato nella Figura 1. L&#39;array AuNP converte i fotoni incidenti in plasmoni di superficie localizzata (LSP) che consistono in risonanza momenti di dipolo che irradia e generare un campo di radiazione fantasia con un gradiente di grandi dimensioni nella soluzione delle cellule. Il lavoro iniziale sulla cattura plasmonica di superficie maggiore di Righini et al modeling e la nostra stessa hanno mostrato i campi generati dal substrato plasmoniche ridurre l&#39;intensità iniziale necessario, migliorando il gradiente di campo che intrappola le particelle. 6,7,8 L&#39;approccio plasmoniche permette di multa controllo l&#39;orientamento delle particelle ellissoidali e cellule con una bassa intensità ottico a causa di una conversione più efficiente l&#39;energia ottica in energia meccanica e un dipolo dipendente dal campo di radiazione. Questi campi sono mostrati in figura 2 e la cattura a bassa intensità sono dettagliati nelle figure 4 e 5. I principali problemi con le pinzette plasmoniche sono che il LSP&#39;s generano una considerevole quantità di calore e la cattura è solo bidimensionale. Questo calore genera flussi convettivi e thermophoresis che può essere abbastanza potente da espellere particelle inferiori al micron dalla trappola. 9,10 Il secondo approccio che descriveremo è l&#39;utilizzo di nanostrutture dielettrico periodici per diffondere la luce incidente in modo molto efficiente nelle modalità di diffrazione, come mostrato in figura 6 11. Idealmente, si potrebbe rendere questa struttura di un materiale dielettrico per evitare i problemi di riscaldamento stessa esperienza con la plasmoniche le pinzette, ma nel nostro approccio di alluminio rivestita reticolo di diffrazione viene utilizzato come unidimensionale nanostruttura dielettrica periodica. Anche se non è un semiconduttore, non ha subito un riscaldamento significativo ed efficace intrappolato piccole particelle con intensità di cattura basso, come mostrato in figura 7. L&#39;allineamento delle particelle con il substrato grata convalida concettualmente l&#39;idea che un 2-D cristalli fotonici potrebbe consentire la rotazione precisa di non sferiche micron di dimensioni. 10 L&#39;efficienza di queste trappole ottiche sono aumentati a causa della maggiore campi prodotti dalle nanostrutture descritto nella questo documento.

Watch this Scientific Journal Video about Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation at JoVE.com

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

Pinzette plasmoniche e nanostrutture a cristalli fotonici sono mostrati utili per produrre miglioramenti nel controllo efficienza e l&#39;orientamento ottico cattura micro-e nano-particelle.

L&#39;utilizzo di nanostrutture di cristallo plasmoniche e fotonici per Enhanced manipolazione micro e nanoparticelle

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

utilization-plasmonic-photonic-crystal-nanostructures-for-enhanced

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Bioengineering

12.2K Views.  University of Washington. Pinzette plasmoniche e nanostrutture a cristalli fotonici sono mostrati utili per produrre miglioramenti nel controllo efficienza e l'orientamento ottico cattura micro-e nano-particelle.

Video: Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Stampa planare e tridimensionale di inchiostri conduttivi

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Ricerca

Bioingegneria

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Sintesi, assemblaggio e caratterizzazione di film monostrato Protetta delle nanoparticelle d&#39;oro per Elettrochimica proteine ​​monostrato

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Valutazione della consegna del gene polimerici Nanoparticelle per l&#39;analisi delle nanoparticelle Monitoraggio e High-throughput Citometria a Flusso

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-Particle Image Velocimetry per Velocity Misure profilo dei flussi Micro sangue

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

Nanofibre ECM proteine ​​e Nanostrutture Engineered Uso Assembly-Surface avviato

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

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

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

Preparazione di Monodomain Liquid Crystal Elastomeri e cristallo elastomero Nanocompositi Liquid

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

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Tecniche per l&#39;evoluzione della robusta lievito Pentoso-fermentazione per bioconversione della lignocellulosa a etanolo

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Scambio Buffer Microfluidic per senza interferenze micro / nanoparticelle cellulare Ingegneria

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

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. ...

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

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

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

Sintesi di biocompatibili a cristalli liquidi in elastomero Schiume come impalcature cellulari per 3D spaziali colture cellulari

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