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 arbeidet ble finansiert av NSERC Individuell Discovery program, NSERC / CIHR Collaborative Health Research Program (CHRP), og den NSERC CREATE kanadiske astrobiologi Training Program (CATP).

1. QD solubilization Dette er en metode som passer for CdTe. Lignende metoder kan brukes med andre typer QDs som INP / ZnS 20 og CdSe / ZnS 21. <ol><li> Forbered en løsning av CdTe QDs i toluen ved 15 mM (optisk tetthet = 2,5 ved første exciton peak). </li><li> Overføring 200 mL av denne bestanden i et hetteglass. Ikke bruk plast! </li><li> Legg 800 mL toluen, 1 mL 200 mm borate buffer (pH 9) og 2 mL av 11,5 M mercaptopropionic syre (MPA). </li><li> Cap hetteglasse...

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Nanopartikler representerer en lovende tilnærming til etablering av nye anti-mikrobielle midler. Vekstkurve analyse er en måte å overvåke bakteriell celle tetthet som skiller aktivt-voksende celler fra vekst-hemmet celler. Når dette kombineres med plate teller, gir det mulighet for en grundig analyse av antibiotika potensialet for en konjugat. Den 96-brønnen format tillater relativt high-throughput variasjoner av konsentrasjonen og andre forhold som lys eksponering, sistnevnte er avgjørende for lysaktivert agente...

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfritt) </td></tr><tr><td> Borat Buffer Component # 1 </td><td> Fisher </td><td> Borsyre A-74-1 </td><td></td></tr><tr><td> Borat Buffer Component # 2 </td><td> Sigma-Aldrich </td><td> Sodium tetraborate 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> Various MWCO tilgjengelig </td></tr><tr><td> Hetteglass </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> Bakteriell growth medium (LB) Komponent # 1 </td><td> Fisher </td><td> NaCl S271 </td><td></td></tr><tr><td> Bakterievekst medium (LB) Component # 2 </td><td> BD </td><td> Tryptone 211705 </td><td></td></tr><tr><td> Bakterievekst medium (LB) Component # 3 </td><td> BD </td><td> Gjærekstrakt 211929 </td><td></td></tr><tr><td> Lampe for lyseksponering </td><td> Custom </td><td></td><td></td></tr><tr><td> Clear-bottom 96-brønners plater </td><td> Fisher </td><td> 07-200-567 eller 07-200-730 </td><td></td></tr><tr><td> Fluorescens spektrometer </td><td> Molekylære enheter </td><td></td><td></td></tr><tr><td> Absorbans plateavleseren </td><td> Molekylære enheter </td><td></td><td></td></tr><tr><td> BactoAgar for faste medier </td><td> Bioshop </td><td> AGR001.1 </td><td></td></tr><tr><td> Petriskåler runde </td><td> Fisher </td><td> 08-75-12 </td><td></td></tr><tr><td> Petriskåler rektangulære </td><td> Fisher </td><td> 08-757-11A </td><td></td></tr></tbody></table>

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

Kvanteprikker (QDs) er fluorescerende halvledere nanopartikler med størrelse avhengig av utslipp spektre som kan bli opphisset av et bredt utvalg av bølgelengder. QDs har tiltrukket seg mye oppmerksomhet for bildebehandling, diagnostikk og terapi på grunn av sin lyse, stabile fluorescens 1,2 3,4,5. QDs kan være konjugert til en rekke biologisk aktive molekyler for å binde seg til bakterier og pattedyrceller 6. QDs blir også mye etterforskes som cytostatika for målrettet drap av bakterier. Fremveksten av formere-resistente bakteriestammer er raskt blitt en offentlig helsekrise, særlig i tilfelle av gramnegative patogener 7. På grunn av den velkjente antimikrobiell effekt av visse nanomaterialer, spesielt Ag, er det hundrevis av studier som undersøker toksisitet av nanopartikler til bakterier åtte. Bakterielle utført studier med andre typer halvledere nanopartikler også, especially TiO 2 9,10-11, men også ZnO 12 og andre, inkludert CuO 13. Noen sammenligninger av bakteriestammer har blitt utført i disse studiene, vanligvis sammenligner en Gram negativ belastning med en Gram positiv. Med alle disse partiklene, er mekanismene for toksisitet skyldes oksidasjon: enten photogeneration av reaktive oksygenforbindelser (ROS) av partikler eller direkte utslipp av metallioner som kan forårsake oksidativt toksisitet. Selv med disse materialene, resultater fra ulike studier varierer sterkt. I noen studier er Gram positive testen belastning er angivelig mer følsom enn den Gram-negative 10, i andre er det motsatt 14. Disse studiene har blitt godt anmeldt 15. I alle nanopartikkel studier, kan partikkelsammensetning, størrelse, overflatekjemi, prøve aldring / sammenbrudd, og bølgelengde, kraft og varighet av lyseksponering alle dramatisk påvirke resultatene. I tillegg synthesis biprodukter og løsemidler må vurderes 16 17. High-throughput screening teknikker for å kunne utvikle effektive nye nanomedisin agenter. CdTe QDs har anti-mikrobielle effekter alene 18 eller i kombinasjon med antibiotika. I en tidligere studie, viste vi at kobling av antibiotika til CdTe kan øke toksisiteten til bakterier, men redusere toksisitet til pattedyrceller, på grunn av redusert produksjon av reaktive oksygenforbindelser fra konjugater 19. Selv om det er usannsynlig at kadmium-holdige forbindelser vil bli godkjent for bruk i mennesker, kan slike preparater brukes til desinfeksjon av overflater eller sterilisering av vann. I denne protokollen, gir vi en grei tilnærming til solubilizing CdTe QDs med mercaptopropionic syre (MPA). De QDs er klar til bruk innen en time. Vi så demonstrere kopling til et antimikrobielt middel. Den andre delen av protokollendemonstrerer en 96-brønns bakteriell hemming analysen ved hjelp av konjugert og ukonjugert QDs. Den optiske tettheten avleses over mange timer, tillater virkningene av QD tillegg og lys eksponering skal vurderes umiddelbart og etter en restitusjonsperiode. Vi illustrerer også en kimtall for å kvantifisere overlevelse av bakterier.

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 som halvleder kvanteprikker (QDs) kan brukes til å lage photoactivatable agenter for anti-mikrobielle eller anti-kreft-applikasjoner. Denne teknikken viser hvordan vann-solubilize kadmium Telluride (CdTe) QDs, konjugat dem til et antibiotikum, og utføre en bakteriell hemming analysen basert på vekstkurver og kimtall.

Solubilization og Bio-konjugering av kvanteprikker og Bakterielle Toxicity analyser av vekst Curve og kimtall

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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