可扩展的，金属的高宽比纳米复合材料在生物液体中产生

Summary

在这里，我们提出了一个协议，以合成下生物条件和在液体介质新颖的，高宽比生物复合材料。的生物复合材料从纳米扩展到微米直径和长度。铜纳米粒子（CNPS）和硫酸铜结合胱氨酸是关键部件的合成。

Abstract

该协议的目的是描述两种新型生物复合材料具有高纵横比结构的合成。该生物复合材料包括铜和胱氨酸，无论是与铜纳米颗粒（CNPS）或硫酸铜贡献的金属部件。合成是在生物条件下（37℃）和24小时后的自组装的复合材料的形式下是液体。一旦形成，这些复合材料是在两种液体介质和干燥形式高度稳定。该复合物由纳米规模向微型范围在长度和从几微米到直径25纳米。能量色散X射线光谱仪（EDX）的场发射扫描电子显微镜表明，硫存在于NP衍生线性结构，而这是从起始CNP材料不存在，从而证实胱氨酸作为硫在最终纳米复合材料的源。在综合这些线性纳米和微观复合材料，STR的长度多种多样uctures形成在合成容器。合成后的液体混合物的超声处理被证明有助于通过用超声处理的时间增加减少的平均长度控制的结构的平均尺寸。因为所形成的结构是高度稳定的，不结块，并形成有在液相，离心也可用于帮助浓缩和分离形成的复合材料。

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

1.规划实验确定所需的合成铜纳米复合材料的体积。在此基础上，选择一些体积小烧瓶（25 平方厘米），或在准备材料如下所示大瓶。 对于此合成中，使用一个37℃培养箱，用5％的CO 2和至少40％的湿度。确保这种培养箱可用，并且，它不会被重复干扰比合成的周期（大约24小时）。 注意：反复开闭的保育箱的肯定会引起温度的波动，这可能会导致该纳米复合材?…

Representative Results

图1示出的合成步骤，以形成在这项工作中描述的线性生物复合材料的流程图示意图。 CNPS或硫酸铜作为起始原料相结合，与无菌水，以形成2毫克/毫升溶液，该溶液混合并超声处理以提供更混合物，使该铜溶液然后在下面的比例混合用于合成：949份无菌水：50份混合铜：1份胱氨酸原液。实际体积可以根据这些比率来放大或缩小最后合成收率而增加或减少。孵育至少2小时所指示的，线?…

Discussion

而评估纳米材料包括CNPS的潜在毒性作用，有人指出，在长期，CNPS被从初始较为分散的颗粒分布到较大 ​​的聚集形式（ 图2）转化。在某些情况下，这些中生产在细胞培养皿，生物条件下高度聚集的地层，形成了从中央骨料含有“海胆”高度线性突起，令人联想起先前所述铜的^6。应当指出的是，这里所示的条件下，CNPS的浓度加入到细胞中是次最大，因此不杀死所有的细胞?…

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