生物学的液体培地中でスケーラブルな、メタリック高アスペクト比のナノコンポジットの生成
Summary
ここでは、生物学的条件下で液体培地中で小説、高アスペクト比のバイオ複合材料を合成するためのプロトコルを提示します。バイオ複合材料は、それぞれ、直径および長さにマイクロメートルのナノメートルスケール。シスチンと組み合わせた銅ナノ粒子（CNPS）および硫酸銅は、合成のための重要なコンポーネントです。
Abstract
このプロトコルの目的は、高アスペクト比の構造を有する2つの新規なバイオ複合材料の合成を記載することです。バイオ複合は、銅ナノ粒子（CNPS）または金属成分に寄与する硫酸銅のいずれかで、銅およびシスチンで構成されています。合成は、生物学的条件（37℃）で液体で行い、自己組織化複合体は、24時間後に形成されます。一旦形成されると、これらの複合材料は、両方の液体培地中で、乾燥した形で非常に安定しています。複合材料の長さは、数ミクロンから、直径25 nmの範囲をマイクロするナノスケールから。エネルギー分散型X線分光法（EDX）を用いて電界放出走査電子顕微鏡は、それが、最終的なナノ複合材料中の硫黄の供給源としてシスチンを確認し、出発CNP材料から不在であった硫黄は、NP由来の線状構造中に存在することを実証しました。これらの線状ナノ·マイクロ複合材料の合成、STRの長さの多様な範囲の間ucturesは、合成容器内で形成されています。合成後の液体混合物の超音波処理は、超音波処理時間の増加とともに平均の長さを減少させることにより、構造物の平均の大きさを制御するのを助けることが示されました。形成された構造は、非常に安定であり、凝集しない、液相中で形成されているので、遠心分離はまた、形成された複合物を濃縮し、分離を補助するために使用することができます。
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
CNPSを含むナノ材料の潜在的な毒性効果を評価するが、それは長期的に、CNPSが大きく、凝集形態に最初により分散微粒子分布（ 図2）から変換されたことが観察されました。いくつかの場合において、生物学的条件下で、細胞培養皿に製造されたこれらの高度に凝集形成は、「ウニ」を含む前述の銅を連想させる中央集合から非常に線状突起が形成された6。これは、ここ…
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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