Gli autori ringraziano il Dipartimento di Chimica e il laureato in Missouri State University per il loro sostegno di questo progetto. Riconosciamo anche la microscopia Laboratorio Electron presso il Laboratorio Nazionale di Federico per la Ricerca sul Cancro per l&#39;utilizzo del loro microscopio elettronico a trasmissione e griglie di carbonio rivestiti.

1. Sintesi di fosfuro di indio / solfuro di zinco (INP / ZnS) Quantum Dots <ol><li> Sintesi di fosfuro di indio (InP) Quantum Dot Cores <ol><li> Montare un fondo da 100 ml rotondo, 3-collo, pallone con un condensatore da 12 pollici. Aggiungere 30 ml oleilammina (OLA), 0,398 g di indio (III) cloruro (compresi 3), 0,245 g di zinco (II) cloruro (ZnCl 2) e mescolare mentre evacuare a temperatura ambiente usando un vuoto per 1 ora. La soluzione deve apparire incolore con un precipitato bianco. </li><li> Utilizzando un mantello di riscaldamento con una termocoppia e regolatore di temperatura proporzionale-integrale-derivativa (PID), aumentare la temperatura della soluzione a 120 ° C. Evacuare la soluzione sotto vuoto per 20 minuti per eliminare le impurezze a basso punto di ebollizione che possono influenzare la crescita nucleo. Nota: Mentre è possibile utilizzare un bagno di sabbia e termometro, con un mantello riscaldante e PID aumenta l&#39;uniformità e la riproducibilità dei prodotti di reazione. </li><li> Sotto gas inerte (ad esempio, N 2), riflusso della soluzione e aumentare la temperatura a 220 ° C per 15 min. Il Incl 3 e ZnCl 2 completamente dissolvono, per ottenere una soluzione di colore giallo pallido. Lasciare che la temperatura si stabilizzi per 10 minuti. </li><li> Purge una siringa monouso da 3 ml di plastica e 4 pollici, 22 G ago con azoto gassoso. Usando la siringa, fornire rapidamente 0,5 ml tris (dimetilammino) fosfina (TDMAP) alla soluzione incl 3. La temperatura della soluzione diminuisce leggermente e ritorna a 220 ° C. I cambiamenti soluzione da trasparente, giallo pallido a opaco, nero. </li><li> Dopo 9,5 min, togliere il pallone di reazione dal mantello di riscaldamento fino a quando la temperatura scende al di sotto di 200 ° C. Per proteggere l&#39;integrità dei nuclei InP, procedere direttamente al rivestimento ZnS nel passaggio 1.2.1. </li></ol></li><li> Sintesi di solfuro di zinco Conchiglie (ZnS) Quantum Dot <ol><li> Porre il pallone di reazione dal punto 1.1.5 su un mantello riscaldante e stabilizzare il temperature a 200 ° C. Aggiungere lentamente 3,58 g dodecantiolo (DDT) nel corso di 15 sec per la soluzione contenente InP QD. Lasciare la soluzione di reagire per 1 ora. Nota: spessore del guscio ZnS può essere variata aumentando o diminuendo la quantità di stearato di zinco aggiunto nel passaggio 1.2.4. Che modifica l&#39;importo di ZnCl 2 o dodecantiolo in passi 1.1.1 e 1.2.1 può avere un impatto significativo sulla qualità della QD, modificando la cinetica di reazione. <ol><li> In seguito, rimuovere il pallone di reazione dal mantello di riscaldamento e lasciar raffreddare la soluzione a circa 60 ° C. </li></ol></li><li> Una volta che la soluzione InP / ZnS raggiunge ~ 60 ° C, aggiungere 10 ml esano e trasferire l&#39;intera soluzione di circa 45 ml in una provetta da centrifuga da 50 ml in polipropilene. Centrifugare il campione (3000 xg per 10 min) per rimuovere precursori solidi non reagiti. </li><li> trasferire con cautela il surnatante in una bottiglia in polipropilene centrifuga da 250 ml, aggiungere 200 ml di acetone, e centrifugare la soluzione (3.000 xg per 10 minuti) per far precipitare QD InP / ZnS. Questo volume può essere diviso equamente in quattro 50 ml provette per centrifugazione se una centrifuga con le necessarie rotore / accessori non è disponibile. Decantare il surnatante e asciugare il pellet QD a fondo con azoto per eliminare l&#39;acetone. </li><li> Risospendere le QD in 20 ml OLA utilizzando sonicazione, il trasferimento ad un fondo da 50 ml rotondo, 3-collo, pallone contenente 0.474 g di zinco stearato, e mescolate. Evacuare la soluzione sotto vuoto per 20 minuti a RT. </li><li> Sotto azoto gassoso, aumentare la temperatura a 180 ° C e lasciare che la reazione di procedere per 3 ore. Mentre non ci sono cambiamenti visivi notevoli alla soluzione di reazione che si verificano durante questa reazione, l&#39;aggiunta di stearato di zinco aumenta ZnS spessore del guscio, aumentando così QY migliorando passivazione della superficie di QD 12.. Una volta che la reazione è completa, togliere il pallone dal mantello di riscaldamento e lasciare raffreddare la soluzione a circa 60 ° C. </li><li> Una volta che i soluti InP / ZnSsulla raggiunge ~ 60 ° C, aggiungere 20 ml esano e trasferire in una provetta da centrifuga da 50 ml in polipropilene. Centrifugare il campione (3000 xg per 10 min) per rimuovere stearato di zinco non reagito. </li><li> Trasferire accuratamente surnatante in un flacone da centrifuga da 250 ml polipropilene, aggiungere 200 ml di acetone, e centrifugare la soluzione (3000 xg per 10 min) per precipitare InP / ZnS QD. decantare con cautela il surnatante e asciugare accuratamente con azoto per eliminare l&#39;acetone. </li><li> Sciogliere il pellet InP / ZnS QD in 30 ml di esano. Vortex e Sonicare la soluzione per breve tempo per garantire la completa dispersione. </li><li> purificazione Ripetere i punti 1.2.6-1.2.8 altre due volte per assicurare la rimozione completa di leganti organici in eccesso. Le interazioni tra il polimero amfifilico ed il QD nel passo 1.2 possono essere compromessi in presenza di leganti eccesso. </li><li> Con calcoli dettagliati da Xie, et al. 16, determinare la dimensione e la concentrazione dei QD InP / ZnS sintetizzati mediante Spettroscopia UV-Vis. <br/&gt; Nota: La caratteristica spettrale importante per questa analisi è la spalla del picco di assorbimento. Il valore di lunghezza d&#39;onda e l&#39;assorbanza del massimo della spalla è utilizzato per calcolare le dimensioni e la concentrazione dei QD rispettivamente. Questa reazione produce tipicamente di circa 5 micromole di QD con un picco di emissione a 600 nm. Per sintetizzare QD di diversi colori, la durata e / o la temperatura della reazione può essere cambiato. Un tempo di reazione più lungo e / o superiore temperatura dei risultati della soluzione in un picco di emissione rossa spostata. Ad esempio, aumentando la temperatura di reazione a 240 ° C e mantenendo il tempo di reazione di 10 min comporterà QD con nm picco 680 emissioni massime. Analogamente, riducendo il tempo di reazione per 2 min e raddoppiando cloruro di zinco utilizzato comporterà QD con nm picco massimo di emissione 470. Questi QD InP / ZnS sono stabili per almeno un mese a 4 ° C al buio sotto gas inerte. </li></ol></li></ol> 2. acquaSolubilizzazione del InP / ZnS Quantum Dots Utilizzando un Amphiphilic Polymer <ol><li> solubilizzazione acqua <ol><li> Usando i QD InP / ZnS dal passaggio 1.2.10, diluire una parte dei QD con esano per ottenere 1 ml di 1 pM QD. <ol><li> In una provetta da centrifuga, trasferire 0,25 ml InP / ZnS QD in ogni provetta. Aggiungere 1 ml di acetone o metanolo al tubo da centrifuga e centrifugare (3000 xg per 10 min). Rimuovere con attenzione il surnatante e sciogliere ogni precipitato in 1 ml di tetraidrofurano (THF). </li><li> Trasferire i QD InP / ZnS disciolto in THF in un 100 ml pallone da fondo e diluire con 16 ml di THF. Per ridurre il numero di aggregati in soluzione, sonicare i QD per 5-10 min. </li></ol></li><li> Sciogliere 30 mg di poli (maleica andhydride- alt -1-ottadecene), 3- (dimetilammino) -1-propilammina (PMAL-d) in 10 ml di acqua grado molecolare. Bagnomaria sonicazione o blanda agitazione fino a quando la soluzione è trasparente è sufficiente per sciogliere completamente il polimero. iluso di vortice o vigorosa agitazione può produrre molte bolle, che ostacola l&#39;interazione del polimero con il QD. Aggiungere la soluzione di polimero 10 ml ai 100 ml rotonda pallone a fondo contenente InP / ZnS QDs in THF. </li><li> Evaporare THF dalla soluzione QD / polimero utilizzando un evaporatore rotante. Collocare la beuta in un bagno di ghiaccio evaporando facilitare l&#39;interazione tra il polimero e QD. A seconda della forza del vuoto, più THF viene evaporato dopo 10 minuti e la soluzione appare torbida. <ol><li> Una volta che la soluzione viene evaporata a 10 ml, togliere il matraccio dall&#39;evaporatore rotante e aggiungere 30 ml di acqua molecolare. Riportare il pallone evaporatore rotante e continuare ad evaporare a 2 ml. Questa fase di evaporazione finale può richiedere molte ore; assicurare il bagno di ghiaccio viene mantenuta. </li></ol></li><li> Rimuovere i QD InP / ZnS idrosolubili dal pallone a fondo tondo con una pipetta. Filtrare la soluzione QD usando una siringa di plastica 3 ml collegata ad un nylon sy 0,1 umRinge filtrare in una provetta da centrifuga da 5 ml. </li><li> Mettere i QD in una unità di dialisi membrana 20.000 MWCO e dializzare contro 0,05 M tampone borato pH 8,5 per rimuovere l&#39;eccesso di polimero. (Aggiungere lentamente 0,05 M di sodio tetraborato decaidrato a M di acido borico 0.05, sotto vigorosa agitazione, fino a quando il pH è 8,5 a rendere questa soluzione tampone borato.) Utilizzando un concentratore a vuoto, concentrare gli QD in tampone borato a 1 ml. </li><li> Per la conservazione, eliminare la soluzione con azoto prima di sigillare con parafilm. I QD InP / ZnS idrosolubili sono stabili per almeno 4 mesi a 4 ° C al buio. </li></ol></li></ol>

<ol>
	<li>Alivisatos, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semicondictor+clusters,+nanocrystals,+and+quantum+dots.">Semicondictor clusters, nanocrystals, and quantum dots.</a> Science. 271 (5251), 933-937 (1996).</li><li>Michalet, X., 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=Quantum+dots+for+live+cells,+in+vivo+imaging,+and+diagnostics.">Quantum dots for live cells, in vivo imaging, and diagnostics.</a> Science. 307 (5709), 538-544 (2005).</li><li>Jaiswal, J. K., Mattoussi, H., Mauro, J. M., Simon, 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=Long-term+multiple+color+imaging+of+live+cells+using+quantum+dot+bioconjugates.">Long-term multiple color imaging of live cells using quantum dot bioconjugates.</a> Nat. Biotechnol. 21 (1), 47-51 (2009).</li><li>Deerinck, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+fluorescent+quantum+dots+to+confocal,+multiphoton,+and+electron+microscopic+imaging.">The application of fluorescent quantum dots to confocal, multiphoton, and electron microscopic imaging.</a> Toxicol. Pathol. 36 (1), 112-116 (2008).</li><li>Smith, A. M., Duan, H., Mohs, A. M., Nie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioconjugated+quantum+dots+for+in+vivo+molecular+and+cellular+imaging.">Bioconjugated quantum dots for in vivo molecular and cellular imaging.</a> Adv. Drug Deliv. Rev. 60 (11), 1226-1240 (2008).</li><li>Jamieson, 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=Biological+applications+of+quantum+dots.">Biological applications of quantum dots.</a> Biomaterials. 28 (31), 4717-4732 (2007).</li><li>Lidke, D. S., Arndt-Jovin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+takes+a+quantum+leap.">Imaging takes a quantum leap.</a> Physiology. 19, 322-325 (2004).</li><li>Fichter, K. M., Flajolet, M., Greengard, P., Vu, T. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+G-protein-couple+receptor+endosomal+trafficking+pathways+revealed+by+single+quantum+dots.">Kinetics of G-protein-couple receptor endosomal trafficking pathways revealed by single quantum dots.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (43), 18658-18663 (2010).</li><li>Smith, A. M., Ruan, G., Rhyner, M. N., Nie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+luminescent+quantum+dots+for+in+vitro+molecular+and+cellular+imaging.">Engineering luminescent quantum dots for in vitro molecular and cellular imaging.</a> Ann. Biomed. Eng. 34 (1), 3-14 (2006).</li><li>Derfus, A. M., Chan, W. C. W., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+cytotoxicity+of+semiconductor+quantum+dots.">Probing the cytotoxicity of semiconductor quantum dots.</a> Nano Lett. 4 (1), 11-18 (2004).</li><li>Brunetti, V., 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=InP/ZnS+as+a+safer+alternative+to+CdSe/ZnS+core/shell+quantum+dots:+in+vitro+and+in+vivo+toxicity+assessment.">InP/ZnS as a safer alternative to CdSe/ZnS core/shell quantum dots: in vitro and in vivo toxicity assessment.</a> Nanoscale. 5 (1), 307-317 (2013).</li><li>Song, W., 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=Amine-derived+synthetic+approach+to+color-tunable+InP/ZnS+quantum+dots+with+high+fluorescent+qualities.">Amine-derived synthetic approach to color-tunable InP/ZnS quantum dots with high fluorescent qualities.</a> J. Nanopart. Res. 15 (1750), (1750).</li><li>Dabbousi, B. O., 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=(CdSe)ZnS+core-shell+quantum+dots:+Synthesis+and+characterization+of+a+size+series+of+highly+luminescent+nanocrystallites.">(CdSe)ZnS core-shell quantum dots: Synthesis and characterization of a size series of highly luminescent nanocrystallites.</a> J. Phys. Chem. B. 101 (46), 9463-9475 (1997).</li><li>Micic, O. I., Curtis, C. J., Jones, K. M., Sprague, J. R., Nozik, A. J. <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+InP+quantum+dots.">Synthesis and characterization of InP quantum dots.</a> J. Phys. Chem. 98 (19), 4966-4969 (1994).</li><li>Qi, L., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dot-amphipol+nanocomplex+for+intracellular+delivery+and+realtime+imaging+of+siRNA.">Quantum dot-amphipol nanocomplex for intracellular delivery and realtime imaging of siRNA.</a> ACS Nano. 2 (7), 1403-1410 (2008).</li><li>Xie, R., Zheng, L., Peng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleation+kinetics+vs+chemical+kinetics+in+the+initial+formation+of+semiconductor+nanocrystals.">Nucleation kinetics vs chemical kinetics in the initial formation of semiconductor nanocrystals.</a> J. Am. Chem. Soc. 131 (42), 15457-15466 (2009).</li><li>Williams, A. T. R., Winfield, S. A., Miller, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+fluorescence+quantum+yields+using+a+computer-controlled+luminescence+spectrometer.">Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer.</a> Analyst. 108, 1067-1071 (1983).</li><li>Schindelin, 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li><li>Schnieder, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+image+to+ImageJ:+25+years+of+image+analysis.">NIH image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Jin, Y., Kannan, S., Wu, M., Zhao, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+luminescent+silica+nanoparticles+to+living+cells.">Toxicity of luminescent silica nanoparticles to living cells.</a> Chem. Res. Toxicol. 20 (8), 1126-1133 (2007).</li><li>Corazzari, I., Gilardino, A., Dalmazzo, S., Fubini, B., Lovisolo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+CdSe/ZnS+quantum+dots+in+the+lysosomal+acidic+compartment+of+cultured+neurons+and+its+impact+on+viability:+potential+role+of+ion+release.">Localization of CdSe/ZnS quantum dots in the lysosomal acidic compartment of cultured neurons and its impact on viability: potential role of ion release.</a> Toxicol. In Vitro. 27 (2), 752-759 (2013).</li><li>Pons, T., Uyeda, H. T., Medintz, I., Mattoussi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+dimensions,+electrophoretic+mobility,+and+stability+of+hydrophilic+quantum+dots.">Hydrodynamic dimensions, electrophoretic mobility, and stability of hydrophilic quantum dots.</a> J. Phys. Chem. B. 110 (41), 20308-20316 (2006).</li><li>Durisic, N., Wiseman, P., Grutter, P., Heyes, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+mechanism+underlies+the+dark+fraction+formation+and+fluorescence+blinking+of+quantum+dots.">A common mechanism underlies the dark fraction formation and fluorescence blinking of quantum dots.</a> ACS Nano. 3 (5), 1167-1175 (2009).</li><li>Vermehren-Schmaedick, 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=Heterogeneous+intracellular+trafficking+dynamics+of+brain-derived+neurotropic+factor+complexes+in+the+neuronal+soma+revealed+by+single+quantum+dot+tracking.">Heterogeneous intracellular trafficking dynamics of brain-derived neurotropic factor complexes in the neuronal soma revealed by single quantum dot tracking.</a> PLoS ONE. 9 (4), e95113 (2014).</li></ol>

Gli autori non hanno nulla da rivelare.

dettagli Questo protocollo sintesi altamente fluorescenti QD InP / ZnS che possono essere utilizzati in molti sistemi biologici. I prodotti QD sintetizzati qui esposti un singolo picco di emissione di fluorescenza centrato a 600 nm con una FWHM di 73 nm (figura 1), che è paragonabile ad altre sintesi precedentemente descritti 12. Tempo di reazione e temperatura di reazione sono passi estremamente importanti a causa del loro effetto profondo sulla qualità sintesi QD e ripetibilità. Dopo sol...

A correction was made to: <a href="http://jove.unicartagenaproxy.elogim.com/video/53684/synthesis-cd-free-inpzns-quantum-dots-suitable-for-biomedical" target="_blank">Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications</a>. There was an error with an author's given name. The author's name was corrected to:
Katye M. Fichter
from:
Kathryn M. Fichter

A correction was made to: <a href="http://jove.unicartagenaproxy.elogim.com/video/53684/synthesis-cd-free-inpzns-quantum-dots-suitable-for-biomedical" target="_blank">Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications</a>.

Erratum: Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Punti quantici (QDs) sono semiconduttori nanocristalli che presentano proprietà fluorescenti quando irradiato con luce 1. A causa delle loro piccole dimensioni (2-5 nm), che è simile a molte biomolecole più grandi, e facilità di biofunzionalizzazione, QD sono uno strumento estremamente interessante per applicazioni biomediche. Hanno trovato l&#39;uso in etichettatura biologica, imaging singola molecola live-cell, la somministrazione di farmaci, imaging in vivo, il rilevamento degli agenti patogeni, e il monitoraggio delle cellule, tra molti altri usi 2-8. QD basata su CD sono stati più comunemente usato in applicazioni biomediche a causa della loro intensa fluorescenza e una ridotta larghezza picco di emissione 9. Tuttavia, le preoccupazioni sono state sollevate a causa della potenziale tossicità del Cd 2+ ioni 10 che possono essere immesse attraverso la degradazione della nanoparticella. Recentemente, QD InP basati sono stati esplorati come alternativa alla QD Cd basati perché mantengono molte caratteristiche fluorescenzadi QD Cd-based e può essere più biocompatibile 11. QD basata su CD sono stati trovati per essere significativamente più tossico QD InP a base di saggi in vitro a concentrazioni basse quanto 10:00, dopo solo 48 ore 11. Il colore emissione di fluorescenza di QD è formato-sintonizzabile 1. Cioè, come la dimensione degli aumenti QD, l&#39;emissione di fluorescenza è spostata verso il rosso. Le dimensioni e la dimensione dispersity dei prodotti QD può essere modificata variando la temperatura, la durata di reazione, o condizioni di concentrazione precursori durante la reazione 12. Mentre il picco di emissione di InP QD è in genere più ampio e meno intensa rispetto QD basata su CD, InP QD può essere fatta in una grande varietà di colori progettate per evitare la sovrapposizione spettrale, e sono sufficientemente intenso per la maggior parte delle applicazioni biomediche 12. La sintesi descritto in questo protocollo produce QD con un picco di emissione rosso centrato a 600 nm. Diversi passaggi sono prese afSintesi ter delle anime QD per mantenere l&#39;integrità ottica dei QD e la compatibilità per applicazioni biologiche. La superficie del nucleo QD deve essere protetto da difetti di ossidazione o di superficie che possono causare tempra; di conseguenza, un guscio di ZnS è rivestito sopra il nucleo di produrre InP / ZnS (core / shell) QD 13. Questo rivestimento è stato dimostrato di proteggere la fotoluminescenza del prodotto QD. La presenza di ioni zinco durante la sintesi InP QD ha dimostrato di limitare difetti superficiali, nonché distribuzione dimensionale diminuzione 12. Anche con la presenza di Zn 2+ nel mezzo di reazione, sintesi di InZnP sono altamente improbabili 12. Dopo il rivestimento, con conseguente QD InP / ZnS sono rivestiti in ligandi idrofobici come l&#39;ossido di trioctylphosphine (TOPO) o oleilammina 12,14. Un polimero amfifilico può interagire con leganti idrofobi sulla superficie QD e molecole di acqua bulk per conferire solubilità in acqua 15. polimeri anfifilici con carbogruppi chimici xylate possono essere utilizzate come &quot;maniglie chimici&quot; di funzionalizzare ulteriormente i QD. Dettagli Questo protocollo la sintesi e funzionalizzazione di idrosolubili InP / ZnS QD con molto intensa emissione di fluorescenza e dimensioni relativamente piccole-dispersity. Questi QD sono potenzialmente meno tossici comunemente usati QD CdSe / ZnS. Qui, la sintesi di InP / ZnS QD offre una pratica alternativa per QD Cd-based per applicazioni biomediche.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Oleylamine</td>
			<td style="width:92px;">Acros</td>
			<td style="width:119px;">129540010</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Zinc (II) chloride</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:119px;">030-003-00-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Indium (III) chloride</td>
			<td style="width:92px;">Chem-Impex&#160;</td>
			<td style="width:119px;">24560</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:232px;">Tris(dimethylamino)phosphine</td>
			<td style="width:92px;">Encompass</td>
			<td style="width:119px;">50-901-10500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">1-dodecanethiol</td>
			<td style="width:92px;">Acros</td>
			<td style="width:119px;">117625000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Hexanes</td>
			<td style="width:92px;">Fisher Sci</td>
			<td style="width:119px;">H292-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Acetone</td>
			<td style="width:92px;">TransChemical</td>
			<td style="width:119px;">UN 1090</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Zinc Stearate</td>
			<td style="width:92px;">Aldrich Chem</td>
			<td style="width:119px;">307564-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tetrahydrofuran</td>
			<td style="width:92px;">Acros</td>
			<td style="width:119px;">34845-0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Molecular Water</td>
			<td style="width:92px;">Fisher Sci</td>
			<td style="width:119px;">BP2470-1</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Poly(maleic anhyrdride-alt-1-tetradecene), 3-(dimethylamino)-1-propylamine derivative</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:119px;">90771-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Boric acid</td>
			<td style="width:92px;">Fisher Sci</td>
			<td style="width:119px;">BP168-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:92px;">Fisher Sci</td>
			<td style="width:119px;">BP175-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Rhodamine B</td>
			<td style="width:92px;">Aldrich Chem</td>
			<td style="width:119px;">R95-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Nitrogen gas</td>
			<td style="width:92px;">Airgas</td>
			<td style="width:119px;">UN1066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Trypan blue</td>
			<td style="width:92px;">Thermo Sci</td>
			<td style="width:119px;">SV30084.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">3 mL plastic Luer-lock syringe</td>
			<td style="width:92px;">BD</td>
			<td style="width:119px;">309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Luer-lock Needle</td>
			<td style="width:92px;">Air-Tite</td>
			<td style="width:119px;">8300014471</td>
			<td>4 inch, 22 gauge</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">50 mL polypropyene centrifuge tube</td>
			<td style="width:92px;">Falcon</td>
			<td style="width:119px;">352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">250 mL centrifuge bottle</td>
			<td style="width:92px;">Thermo Sci</td>
			<td>05-562-23</td>
			<td>Nalgene PPCO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">5 mL centrifuge tubes</td>
			<td style="width:92px;">Argos-Tech</td>
			<td style="width:119px;">T2076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">1.5 mL microcentrifuge tubes</td>
			<td style="width:92px;">Bio Plas</td>
			<td style="width:119px;">4150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">0.1 &#956;m Syringe filter</td>
			<td style="width:92px;">Whatman</td>
			<td style="width:119px;">6786-1301</td>
			<td>Puradisc 13 mm nylon filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Slide-A-Lyzer MINI Dialysis Unit</td>
			<td style="width:92px;">Thermo Sci</td>
			<td style="width:119px;">69590</td>
			<td>20,000 MWCO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Rotary Evaporator</td>
			<td style="width:92px;">Heidolph</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Centrifuge 5072</td>
			<td style="width:92px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Swinging Bucket with 50 mL tube adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Lambda 650 UV/VIS Spectrometer</td>
			<td style="width:92px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td style="width:283px;">UV-Vis Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">LS 55 Fluorescence Spectrometer</td>
			<td style="width:92px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Fluorometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Axio Observer.A1</td>
			<td style="width:92px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>epifluorescence microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">AxioCam MRm</td>
			<td style="width:92px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>CCD Camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tecnai TF20 Microscope</td>
			<td style="width:92px;">FEI</td>
			<td style="width:119px;"></td>
			<td>Transmisison Electron Miscroscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">TEM Eagle CCD</td>
			<td style="width:92px;">FEI</td>
			<td style="width:119px;"></td>
			<td>TEM CCD Camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">NanoBrook Omni DLS</td>
			<td style="width:92px;">Brookhaven</td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Dynamic Light Scattering Instrument</td>
		</tr>
	</tbody>
</table>

synthesis of cd-free inp/zns quantum dots suitable for biomedical applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

I nuclei InP non rivestiti non dimostrano sostanziale fluorescenza visibile ad occhio. Tuttavia, InP / ZnS (core / shell) punti quantici sembrano fluorescenza brillantemente ad occhio sotto irraggiamento UV. La fluorescenza di InP / ZnS QD è stato caratterizzato mediante spettroscopia di fluorescenza. Lo spettro di fluorescenza della QDs in esani (figura 1) eccitato a 533 nm mostra un picco principale centrato a 600 nm con una larghezza a metà massimo (FWHM) di 73 nm. ...

Watch this Scientific Journal Video about Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications at JoVE.com

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

Sintesi di Cd-libera InP / ZnS Quantum Dots adatto per applicazioni biomediche

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

synthesis-cd-free-inpzns-quantum-dots-suitable-for-biomedical

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

13.5K Views.  Missouri State University. In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

Video: Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Preparazione del idrofobi Frameworks Metal-Organic tramite Plasma avanzata Chemical Vapor Deposition del perfluoroalcani per la rimozione di ammoniaca

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Ricerca

Chimica

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Sintesi di Immunotargeted magneto-plasmonica nanocluster

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Sintesi di indoxyl-glicosidi per il rilevamento di glicosidasi Attività

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

Fabbricazione e collaudo di termometri fotonici

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Sintesi di nanocristalli di Upconversion drogato con lantanidi Core-shell per applicazioni cellulari

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal structure and probing the hydrogen-bond (H-bond) dynamics of the water molecules adsorbed on solid surfaces are fundamental issues of water science, which remains a great challenge owing to the light mass and small size of hydrogen. Scanning tunneling microscopy (STM) is a promising tool for attacking these problems, thanks to its capabilities of sub-&#197;ngstr&#246;m spatial resolution, single-bond vibrational sensitivity, and atomic/molecular manipulation. The designed experimental system consists of a Cl-terminated tip and a sample fabricated by dosing water molecules in situ onto the Au(111)-supported NaCl(001) surfaces. The insulating NaCl films electronically decouple the water from the metal substrates, so the intrinsic frontier orbitals of water molecules are preserved. The Cl-tip facilitates the manipulation of the single water molecules, as well as gating the orbitals of water to the proximity of Fermi level (EF) via tip-water coupling. This paper outlines the detailed methods of submolecular resolution imaging, molecular/atomic manipulation, and single-bond vibrational spectroscopy of interfacial water. These studies open up a new route for investigating the H-bonded systems at the atomic scale.

Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

La struttura e la dinamica di acqua interfaccia con Tunneling di microscopia e spettroscopia di sondaggio

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Sintesi di nanofili Au associato a substrato tramite un meccanismo di crescita superficiale attiva

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Escherichia coli-base della sintesi proteica Cell-Free: protocolli per la tecnologia della piattaforma robusta, flessibile e accessibile

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Synthesis of In37P20(O2CR)51 Clusters and Their Conversion to InP Quantum Dots

This text presents a method for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots. The In37P20(O2CR)51 clusters have been observed as intermediates in the synthesis of InP quantum dots from molecular precursors (In(O2CR)3, HO2CR, and P(SiMe3)3) and may be isolated as a pure reagent for subsequent study and use as a single-source precursor. These clusters readily convert to crystalline and relatively monodisperse samples of quasi-spherical InP quantum dots when subjected to thermolysis conditions in the absence of additional precursors above 200 &#176;C. The optical properties, morphology, and structure of both the clusters and quantum dots are confirmed using UV-Vis spectroscopy, photoluminescence spectroscopy, transmission electron microscopy, and powder X-ray diffraction. The molecular symmetry of the clusters is additionally confirmed by solution-phase 31P NMR spectroscopy. This protocol demonstrates the preparation and isolation of atomically-precise InP clusters, and their reliable and scalable conversion to InP QDs.

Synthesis of In37P20O2CR51 Clusters and Their Conversion to InP Quantum Dots

A protocol for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots is presented.

Sintesi di in37P20(O2CR)51 cluster e la loro conversione in INP Quantum Dots

This text presents a method for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots. The In37P20(O2CR)51 ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Aspetto tecnico della sintesi automatizzata e valutazione cinetica in tempo reale di [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...