Generatie van Scalable, Metaalkleur High-breedteverhouding Nanocomposieten in een Biological Liquid Medium
Summary
Hier presenteren we een protocol om nieuwe, high-aspect ratio biocomposieten synthetiseren onder biologische omstandigheden en in vloeibare media. De biocomposieten schaal van nanometers tot micrometers in diameter en lengte respectievelijk. Koperen nanodeeltjes (CNPs) en kopersulfaat in combinatie met cystine zijn de belangrijkste componenten voor de synthese.
Abstract
Het doel van dit protocol is de synthese van twee nieuwe biocomposieten met hoge aspectverhouding structuren beschrijven. De biocomposieten bestaan uit koper en cystine, met ofwel koper nanodeeltjes (CNPs) of kopersulfaat dragen de metallische component. Synthese wordt uitgevoerd in vloeistof onder biologische omstandigheden (37 ° C) en de zelf-geassembleerde samengestelde vorm na 24 uur uitgevoerd. Eenmaal gevormd, deze composieten zijn zeer stabiel in zowel vloeibare media in een gedroogde vorm. De composieten schaal van nano- tot range micro- lengte en van enkele microns tot 25 nm in diameter. Veldemissie scanning elektronen microscopie met energie dispersieve röntgenspectroscopie (EDX) toonden aan dat zwavel aanwezig was in de NP-afgeleide lineaire structuur, hoewel het afwezig het uitgangsmateriaal CNP materiaal, waardoor cystine als bron van zwavel in de definitieve bevestiging nanocomposieten . Tijdens de synthese van deze lineaire nano- en micro-composieten, een breed scala aan lengtes van structures wordt gevormd in de synthese vat. Sonicatie van het vloeistofmengsel na synthese werd aangetoond te helpen bij het beheersen gemiddelde grootte van de structuur van de vermindering van de gemiddelde lengte met verhoogde moment van sonicatie. Aangezien de gevormde structuren zijn zeer stabiel, niet agglomereren, en worden gevormd in de vloeibare fase, kunnen centrifugeren worden gebruikt om te helpen bij het concentreren en scheiden gevormde composieten.
Introduction
Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 °C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called “urchin” and “nanoflower” type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 °C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described.
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells.
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described “coffee ring effect” 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.
Protocol
Representative Results
Discussion
Terwijl evalueren van potentiële toxische effecten van nanomaterialen zoals CNPs werd waargenomen dat op lange termijn, CNPs omgevormd vanuit een aanvankelijk meer verspreid deeltjesvormige distributie naar een grotere, geaggregeerde vorm (Figuur 2). In sommige gevallen zijn deze zeer geaggregeerde formaties die zijn geproduceerd in de cel kweekschaal, onder biologische omstandigheden, vormden zeer lineaire projecties van de centrale aggregaat denken aan eerder beschreven koperhoudende "egels"…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).
Materials
| Mini Vortexer | VWR (https://us.vwr.com) | 58816-121 | |
| CO2 Incubator Model # 2425-2 | VWR (https://us.vwr.com) | Contact vendor | Current model calalog # 98000-360 |
| Eppendorf Centrifuge (Refrigerated Microcentrifuge) | Labnet (http://labnetinternational.com/) | C2500-R | Model Prism R |
| Cell Culture Centrifuge Model Z323K | Labnet (http://labnetinternational.com/) | Contact vendor | Current model Z206A catalog # C0206-A |
| Sonicator (Ultrasonic Cleaner) | Branson Ultrasonics Corporation (http://www.bransonic.com/) | 1510R-MTH | |
| Balance | Sartorius (http://dataweigh.com) | Model CP225D similar model CPA225D | |
| Olympus IX51 Inverted Light Microscope | Olympus (http://olympusamerica.com | Contact vendor | |
| Olympus DP71 microscope digital camera | Olympus (http://olympusamerica.com | Contact vendor | |
| external power supply unit- white light for Olympus microscope | Olympus (http://olympusamerica.com | TH4-100 | |
| 10x, 20, and 40x microscope objectives | Olympus (http://olympusamerica.com | Contact vendor | |
| Scanning Electron Microscope | Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html) | model S-4800 | |
| Transmission Electron Microscope | Zeiss (http://zeiss.com/microscopy/en_de/products.html) | model Libra 120 | |
| Table Top Work Station Unidirectional Flow Clean Bench | Envirco (http://envirco-hvac.com) | model PNG62675 | Used for sterile cell culture technique |
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