Name: solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count
Uploaded: 2012-07-11T13:00:00
Description: Watch this Scientific Journal Video about Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count at JoVE.com

Dette arbejde blev finansieret af NSERC Individual Discovery programmet, NSERC / CIHR Collaborative Health Research Program (CHRP), og NSERC CREATE canadiske Astrobiology Training Program (CATP).

1. QD Solubilisering Dette er en metode egnet til CdTe. Lignende fremgangsmåder kan anvendes med andre typer af QDs såsom InP / ZnS 20 og CdSe / ZnS 21. <ol><li> Fremstilling af en opløsning af CdTe QDs i toluen ved 15 uM (optisk densitet = 2,5 i den første exciton peak). </li><li> Overførsel 200 uL af dette lager i et hætteglas. Brug ikke plastic! </li><li> Tilsættes 800 pi toluen, 1 ml 200 mM boratbuffer (pH 9) og 2 uL af 11,5 M mercaptopropionsyre (MPA). </l...

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Nanopartikler repræsenterer en lovende tilgang til at skabe nye antimikrobielle midler. Vækstkurve analyse er en måde at overvåge bakteriel celledensitet, der adskiller aktivt voksende celler fra vækst-inhiberede celler. Når kombineret med plade tæller, det giver mulighed for en grundig analyse af den antibiotiske potentialet i en konjugat. 96-brønds format tillader forholdsvis højt gennemløb variationer af koncentrationen og andre tilstande, såsom lyseksponering, sidstnævnte er afgørende for lysaktiveret m...

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Katalog nr. </td><td> Kommentarer (valgfri) </td></tr><tr><td> Boratpuffer Komponent # 1 </td><td> Fisher </td><td> Borsyre A-74 til 1 </td><td></td></tr><tr><td> Boratpuffer Komponent # 2 </td><td> Sigma-Aldrich </td><td> Natriumtetraborat B9876 </td><td></td></tr><tr><td> MPA </td><td> Sigma-Aldrich </td><td> M5801 </td><td></td></tr><tr><td> Vivaspin 500 </td><td> GE Healthcare </td><td> 28-9322 </td><td> Forskellige MWCO tilgængelig </td></tr><tr><td> Hætteglas </td><td> Fisher </td><td> 03-338C </td><td></td></tr><tr><td> EDC </td><td> Sigma-Aldrich </td><td> E6383 </td><td></td></tr><tr><td> Polymyxin B </td><td> Sigma-Aldrich </td><td> P1004 </td><td></td></tr><tr><td> Bakteriel GRowth medium (LB) Komponent # 1 </td><td> Fisher </td><td> NaCl S271 </td><td></td></tr><tr><td> Bakterievækst medium (LB) Komponent # 2 </td><td> BD </td><td> Trypton 211705 </td><td></td></tr><tr><td> Bakterievækst medium (LB) Komponent # 3 </td><td> BD </td><td> Gærekstrakt 211.929 </td><td></td></tr><tr><td> Lampe til lyseksponering </td><td> Brugerdefineret </td><td></td><td></td></tr><tr><td> Klar bund 96-brønds plader </td><td> Fisher </td><td> 07-200-567 eller 07-200-730 </td><td></td></tr><tr><td> Fluorescensspektrometer </td><td> Molecular Devices </td><td></td><td></td></tr><tr><td> Absorbans pladelæser </td><td> Molecular Devices </td><td></td><td></td></tr><tr><td> Bactoagar til faste medier </td><td> Bioshop </td><td> AGR001.1 </td><td></td></tr><tr><td> Petriskåle rundt </td><td> Fisher </td><td> 08-75-12 </td><td></td></tr><tr><td> Petriskålene rektangulære </td><td> Fisher </td><td> 08 til 757-11A </td><td></td></tr></tbody></table>

solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

Kvantepunkter (QDs) er fluorescerende halvleder nanopartikler med størrelse-afhængig emission spektre, der kan ophidset af et bredt udvalg af bølgelængder. QDs har tiltrukket stor interesse for billedbehandling, diagnostik og behandling på grund af deres klare, stabile fluorescens 1,2 3,4,5. QDs kan konjugeres til en række biologisk aktive molekyler om binding til bakterier og mammale celler 6. QDs bliver også bredt undersøgt som cytotoksiske midler til målrettet aflivning af bakterier. Fremkomsten af multipelt resistente bakteriestammer som hurtigt bliver en folkesundhedsområdet krise, især i tilfælde af Gram-negative patogener 7. På grund af den velkendte antimikrobielle virkning af visse nanomaterialer, især Ag, er hundredvis af studier af toxicitet af nanopartikler til bakterier 8. Bakterielle undersøgelser er blevet udført med andre typer af halvleder nanopartikler så godt, especially TiO 2 9,10-11, men også ZnO 12 og andre, herunder CuO 13. Sammenligninger af bakteriestammer er blevet udført i disse undersøgelser, sædvanligvis sammenligne en Gram negativ stamme med en grampositiv. Med alle disse partikler er mekanismer toksicitet skyldes oxidation: enten photogeneration af reaktive oxygenspecies (ROS) af partiklerne eller direkte frigivelse af metalioner, der kan forårsage oxidativ toksicitet. Selv med disse materialer, varierer resultaterne af forskellige undersøgelser meget. I nogle undersøgelser af Gram positive teststamme er rapporteret mere følsom end gramnegative 10, i andre er det modsatte 14. Disse undersøgelser er blevet godt revideret 15. I alle nanopartikel undersøgelser, kan partikel-sammensætning, størrelse, overflade kemi, prøve aldring / opdeling, og bølgelængde, magt, og varigheden af ​​lys eksponering alle dramatisk påvirke resultaterne. Desuden synthesis biprodukter og opløsningsmidler må anses 16 17. High-throughput screening teknikker er nødvendige for at kunne udvikle nye effektive nanomedicin midler. CdTe QDs har anti-mikrobielle virkninger alene 18 eller i kombination med antibiotika. I en tidligere undersøgelse viste vi, at kobling af antibiotika til CdTe kan forøge toksiciteten over for bakterier, men reducere toksicitet over for pattedyrceller, på grund af nedsat produktion af reaktive oxygenarter fra konjugaterne 19. Selvom det er usandsynligt, at cadmium-holdige forbindelser vil blive godkendt til anvendelse i mennesker, kan sådanne præparater anvendes til desinfektion af overflader eller sterilisering af vand. I denne protokol, giver vi en enkel tilgang til opløseliggørelse CdTe QDs med mercaptopropionsyre (MPA). De QDs er klar til brug inden for en time. Vi derefter dokumentere kobling til et antimikrobielt middel. Den anden del af protokollenviser en 96-brønds bakteriel inhibering under anvendelse af konjugerede og ukonjugerede QDs. Den optiske densitet aflæses over mange timer, tillader virkningerne af QD Ud og udsættelse for lys skal vurderes umiddelbart, samt efter en restitutionsperiode. Vi viser også en kimtal til at kvantificere bakteriernes overlevelse.

Watch this Scientific Journal Video about Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count at JoVE.com

Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

Nanopartikler såsom halvleder kvantepunkter (QDs) kan anvendes til at skabe fotoaktiverbare midler til anti-mikrobielle eller anti-cancer anvendelser. Denne teknik viser, hvordan man vand-opløse cadmiumtellurid (CdTe) QDs, konjugere dem til et antibiotikum, og udføre en bakteriel inhibering assay baseret på vækstkurver og kimtal.

Opløsning og Bio-konjugering af kvantepunkter og bakterielle Toxicity Analyser ved vækst Curve og Plate Count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. ...

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