Generering av skalerbare, Metallic høy Aspect Ratio Nanocomposites i en biologisk væske Medium
Summary
Her presenterer vi en protokoll for å syntetisere nye, high-sideforhold biocomposites under biologiske forhold og i flytende medier. De biocomposites skalere fra nanometer til mikrometer i diameter og lengde, henholdsvis. Kobber nanopartikler (CNPs) og kobbersulfat kombinert med cystin er de viktigste komponentene for syntesen.
Abstract
Målet med denne protokollen er å beskrive syntesen av to nye biocomposites med høy sideforhold strukturer. De biocomposites består av kobber og cystin, med enten kobber nanopartikler (CNPs) eller kobber bidrar den metalliske komponent. Syntesen utføres i flytende under biologiske betingelser (37 ° C) og selv-sammensatte kompositter formen etter 24 timer. Når dannet, disse kompositter er svært stabil i både flytende medier og i tørket form. Komposittene kalk fra nano- i mikro- varierer i lengde, og fra noen få mikron til 25 nm i diameter. Feltemisjons-scanning elektronmikroskopi med energi dispersive røntgenspektroskopi (EDX) viste at svovel var til stede i NP-avledede lineære strukturer, mens det var fraværende fra utgangs CNP materialet, noe som bekrefter cystin som kilde for svovel i slutt nanokompositter . Under syntese av disse lineære nano- og mikro kompositter, et variert utvalg av lengder på structures er dannet i-syntese-karet. Sonikering av den flytende blanding etter syntese ble påvist å hjelpe til i å kontrollere midlere størrelse av konstruksjonene ved å redusere den gjennomsnittlige lengde med øket tid for lydbehandling. Siden de dannede strukturene er meget stabile, ikke agglomererer, og er dannet i væskefase, kan også sentrifugering brukes til å hjelpe med å konsentrere seg og segregere dannet kompositter.
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
Mens evaluere potensielle toksiske effekter av nanomaterialer inkludert CNPs, ble det observert at på lang sikt, CNPs ble forvandlet fra en i utgangspunktet mer spredt partikkelfordelingen til et større, aggregert form (figur 2). I noen tilfeller er disse svært aggregerte formasjonene som ble produsert i cellekulturskål, under biologiske betingelser, dannes sterkt lineære fremspring fra den sentrale aggregat, som minner om tidligere beskrevne kobberholdige "bollene" 6. Det skal…
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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