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.).

1. Planning of Experiments
<ol>
	<li>Determine the volume of copper nanocomposites needed for synthesis. On that basis, choose a number of small volume flasks (25 cm2), or larger flasks as indicated below in preparation of materials.</li>
	<li>For this synthesis, use a 37 &#176;C incubator with 5% CO2 and at least 40% humidity. Ensure that such an incubator is available and that it will not be repeatedly disturbed over the period of synthesis (approximately 24 hr). 
	CAUTION: Repeated opening and closing of the incubator will certainly cause temperature fluctuations which may result in altered synthesis of the nanocomposite structures.</li>
</ol>
2. Preparation of Materials
<ol>
	<li>Prepare all materials fresh before the start of an experiment, by adding solid materials to solvents right before synthesis is to begin. Keeping stock solutions of cystine and copper starting materials in liquid for long times before the experiment is not recommended and may lead to variable results. Once opened from the vendor, keep starting materials dry by wrapping the top of the container with Parafilm. 
	Note: The following protocol is used as an example for reaction in a 25 cm2 cell culture flask using 7 &#181;l of cystine, 6,643 &#181;l of sterile water, and 350 &#181;l of CNPs.</li>
	<li>Prepare a 2 mg/ml solution of copper nanoparticles by weighing out at least 2 mg of CNPs. Wear disposable gloves during this step to prevent possible contact of CNPs with skin. Place the nanoparticles in an empty sterile 16 ml glass vial.
	<ol>
		<li>To the vial containing CNPs, add sterile deionized water in the appropriate volume to make a 2 mg/ml solution and vortex the solution for 20 sec to provide dispersion of the nanoparticles before synthesis starts (at least 1 ml total volume is recommended). Do not fill the vial more than half way with water as this will inhibit mixing by vortexing. CNPs will quickly settle to the bottom of the vial and will appear dark in color (grey to black).</li>
		<li>Sonicate the CNP solution for 17 min at RT to provide maximal dispersion of CNPs before start of synthesis. Periodically check to make sure that CNPs are mixing due to sonication. After a successful sonication, CNPs remain suspended in solution for at least 30 min and the solution will be dark in color.</li>
	</ol>
	</li>
	<li>Weigh out sufficient mass of cystine to make a 72.9 mg/ml solution for the synthesis. Since cystine is not directly soluble in water, place the weighed cystine in an antistatic weighing vessel.
	<ol>
		<li>To the weighing vessel containing cystine, add sufficient volume of sterile, 1 M NaOH, so that the cystine completely dissolves. For example, dissolve 7.29 mg of cystine completely in 100 &#181;l of 1 M NaOH, to make a 72.9 mg/ml solution.</li>
		<li>To maintain sterile conditions, carry out this step in a sterile flow tissue culture hood. 
		CAUTION: NaOH at 1 M concentration is caustic, so wear disposable gloves during this step to prevent contact of concentrated NaOH with skin</li>
	</ol>
	</li>
	<li>Working in a sterile tissue culture hood, add 7 &#181;l of cystine with 6,643 &#181;l of sterile water to the sterile synthesis flask first, and let incubate for 30 min in the incubator at 37 &#176;C with the flask cap vented (loose) to provide effective mixing. Resuspend the 2 mg/ml CNP solution by vortexing for 30 sec, since CNPs will have settled after the sonication step.
	<ol>
		<li>Add sufficient CNP solution to the synthesis flask (using sterile technique) to maintain the following component ratios: combine 1 parts cystine, 50 parts CNPs, and 949 parts sterile water in a 25 cm2 cell culture flask to start the synthesis. For example, for a 7 ml synthesis volume, combine 7 &#181;l of cystine stock solution, 350 &#181;l of CNPs, and 6,643 &#181;l of sterile water. Replace the cap on the flask and tighten so that it is secure.</li>
		<li>After combining all components for the synthesis, gently mix in the flask by swirling 4-5 times. Place flask in the CO2 incubator and vent the flask by loosening the cap so that there will be gas exchange in and out of the flask during synthesis.</li>
	</ol>
	</li>
	<li>Allow synthesis to run in the incubator for approximately 24 hr. During synthesis, one can observe, with microscopy and by eye, formation of highly linear composites. 
	Note: The process of formation of the structures may happen suddenly in the sense that structures are initially hard to detect, then appearance proceeds quickly to an increasing density. Formation may therefore occur before 24 hr. The process can also be observed by eye once structures become larger and their density increases. While generation of the structures can be observed over time under the microscope and by eye at later time points, continuously interrupting the synthesis conditions and temperature will lead to poor synthesis results.</li>
	<li>Terminate synthesis of biocomposites by tightly capping the synthesis flask and storing the vessel in a refrigerator (4 &#176;C). Structures, once generated, remain stable in this form for at least a year. Label the flask with synthesis conditions, including components utilized, date of the synthesis, and incubation time of the synthesis before termination.</li>
</ol>
3. Synthesis Using Copper Sulfate
<ol>
	<li>Carry out self-assembly synthesis by replacing CNPs with copper sulfate salt. Using sterile technique, dissolve at least 2 mg of copper sulfate in sufficient volume of sterile deionized water to make a 2 mg/ml solution. The copper sulfate crystals easily go into solution at this concentration, but vortex the vial if needed, and inspect by eye to ensure all crystals are dissolved.</li>
	<li>After preparation of the copper sulfate, carry out synthesis as described previously, but replacing CNPs with the copper sulfate. 
	Note: Self-assembled nanocomposites using copper sulfate as a starting material were found to be much more consistent in final shape than for structures synthesized from CNPs.</li>
	<li>Terminate the synthesis of copper sulfate biocomposites as for CNP composites (step 2.6) and store them long-term at 4 &#176;C.</li>
</ol>
4. Characterization and Handling of Biocomposites Post-synthesis
<ol>
	<li>Characterize biocomposites derived from CNPs and from copper sulfate by white light microscopy9 and by electron microscopy9.
	<ol>
		<li>For characterization and inspection of biocomposites post-synthesis by white light microscopy, use an inverted microscope as composites will settle to the bottom surface of the flask within a few minutes of laying the flask flat, and can then be brought into focus. Use the bright field setting on the microscope to maximize contrast between biocomposites and the liquid medium. Composites derived from CNPs and copper sulfate will both appear clear to opaque in color, but unreacted CNP aggregates will appear very dark in color.
		<ol>
			<li>Use a digital camera connected to the microscope to capture images of the composites. A range of lengths for the individual structures will be observed.</li>
		</ol>
		</li>
		<li>For characterization and inspection of biocomposites post-synthesis and after storage at 4 &#176;C, allow flasks to come to RT for at least 15 min as flasks will form condensation initially upon removal from refrigerator, which will obscure effective focusing while carrying out microscopy imaging. After allowing equilibration to RT, wipe the top and bottom surfaces of the flask with a clean paper towel to maximize microscopy imaging quality.</li>
		<li>When working with or imaging composites that have been stored long-term, vortex the flask for 30 sec to dissociate clumps of composites that form while in the refrigerator. After vortexing, inspect the structures with an inverted microscope to ensure that aggregates have dissociated, and repeat vortexing as necessary.</li>
		<li>Use inverted white light microscopy to assess the efficacy of the synthesis for a given experiment using CNPs. For example, document the presence or absence of unreacted CNPs in synthesis flasks used for CNP-derived biocomposites from flasks with different parameters such as time of synthesis. 
		Note: Individual CNPs are too small to observe with a light microscope, but unreacted CNP aggregates will appear as round-shape and dark objects, in contrast to the successfully synthesized CNP-composites which will have a high-aspect ratio, linear form, and will have a range of different lengths. Avoid carrying out synthesis for too long of a period of time before termination, as this will result in highly branched &#8220;urchin&#8221; type structures, which are difficult to disperse into individual structures once formed.</li>
		<li>Use inverted white light microscopy to assess the efficacy of the synthesis for a given experiment using copper sulfate. Since copper sulfate goes fully into solution using this protocol, the solution will appear less dark than the solution from synthesis using CNPs. Document the size and extent of copper sulfate composites by comparing flasks with different synthesis conditions such as time of synthesis before termination. 
		Note: Successfully synthesized composites will show a range of different lengths. Avoid carrying out synthesis for too long of a period of time before termination, as this will result in highly branched aggregates of composites, some of which will be &#8220;urchin-like&#8221; in structure, and which are difficult to disperse into individual structures once formed.</li>
	</ol>
	</li>
	<li>To concentrate biocomposites post-synthesis, centrifuge solutions of composites in a centrifuge tube. Add 6 ml of either CNP-derived structures or copper sulfate-derived structures to a 15 ml centrifuge tube. Centrifuge for 10 min at 500 x g at RT to form a pellet. For smaller volumes, add 500 &#181;l of structures in solution to 0.6 ml sized tubes. Centrifuge at 2,000 x g at RT for at least 10 min to form a pellet.
	<ol>
		<li>After centrifuging for sufficient time (at least 10 min for microfuges), save the observable pellet at the bottom of the tube where the structures are concentrated by carefully removing the supernatant liquid above the pellet. Biocomposite structures derived from copper sulfate appear blue in color and structures derived from CNPs are darker (grey to black).</li>
		<li>Add more composites to this tube and repeat the process in the same tube to concentrate structures if desired. To disperse the concentrated pellets, add the desired volume of solution to the tube, and vortex for 10-30 sec.</li>
	</ol>
	</li>
	<li>Sonicate structures once formed, to move the average population size (lengths) of the structures to lower values. Place structures in sterile deionized water and sonicate for at least 10 min. Using this process, over time, structures become fragmented and smaller in average length (see Figure 6 of the text). Document changes in composite sizes with different sonication times using an inverted white light microscope and digital camera.</li>
</ol>

<ol><li>Klinman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+copper-enzyme+family+of+dopamine+beta-monooxygenase+and+peptidylglycine+alpha-hydroxylating+monooxygenase%3A+resolving+the+chemical+pathway+for+substrate+hydroxylation%5BTitle%5D)+AND+%22The+Journal+of+biological+chemistry%22%5BJournal%5D)">The copper-enzyme family of dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase: resolving the chemical pathway for substrate hydroxylation.</a> The Journal of biological chemistry. 281, 3013-3016 (2006).</li>
<li>Uauy, R., Olivares, M., Gonzalez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Essentiality+of+copper+in+humans%5BTitle%5D)+AND+%22The+American+journal+of+clinical+nutrition%22%5BJournal%5D)">Essentiality of copper in humans.</a> The American journal of clinical nutrition. 67, 952S-959S (1998).</li>
<li>Karlsson, H. L., Cronholm, P., Gustafsson, J., Copper Moller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((oxide+nanoparticles+are+highly+toxic%3A+a+comparison+between+metal+oxide+nanoparticles+and+carbon+nanotubes%5BTitle%5D)+AND+%22Chemical+research+in+toxicology%22%5BJournal%5D)">oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes.</a> Chemical research in toxicology. 21, 1726-1732 (2008).</li>
<li>Parekh, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Layer-by-layer+nanoencapsulation+of+camptothecin+with+improved+activity%5BTitle%5D)+AND+%22International+journal+of+pharmaceutics%22%5BJournal%5D)">Layer-by-layer nanoencapsulation of camptothecin with improved activity.</a> International journal of pharmaceutics. 465, 218-227 (2014).</li>
<li>Harrington, M. J., Masic, A., Holten-Andersen, N., Waite, J. H., Fratzl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron-clad+fibers%3A+a+metal-based+biological+strategy+for+hard+flexible+coatings%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Iron-clad fibers: a metal-based biological strategy for hard flexible coatings.</a> Science. 328, 216-220 (2010).</li>
<li>Keyson, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CuO+urchin-nanostructures+synthesized+from+a+domestic+hydrothermal+microwave+method%5BTitle%5D)+AND+%22Materials+Research+Bulletin%22%5BJournal%5D)">CuO urchin-nanostructures synthesized from a domestic hydrothermal microwave method.</a> Materials Research Bulletin. 43, 771-775 (2008).</li>
<li>Liu, B., Zeng, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesoscale+organization+of+CuO+nanoribbons%3A+formation+of+%27dandelions%27%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Mesoscale organization of CuO nanoribbons: formation of 'dandelions'.</a> J Am Chem Soc. 126, 8124-8125 (2004).</li>
<li>Peng, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controllable+synthesis+of+self-assembled+Cu2S+nanostructures+through+a+template-free+polyol+process+for+the+degradation+of+organic+pollutant+under+visible+light%5BTitle%5D)+AND+%22Materials+Research+Bulletin%22%5BJournal%5D)">Controllable synthesis of self-assembled Cu2S nanostructures through a template-free polyol process for the degradation of organic pollutant under visible light.</a> Materials Research Bulletin. 44, 1834-1841 (2009).</li>
<li>Deodhar, S., Huckaby, J., Delahoussaye, M., DeCoster, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Aspect+Ratio+Bio-Metallic+Nanocomposites+for+Cellular+Interactions%5BTitle%5D)+AND+%22IOP+Conference+Series%3A+Materials+Science+and+Engineering%22%5BJournal%5D)">High-Aspect Ratio Bio-Metallic Nanocomposites for Cellular Interactions.</a> IOP Conference Series: Materials Science and Engineering. 64, 012014(2014).</li>
<li>Montes-Burgos, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterisation+of+nanoparticle+size+and+state+prior+to+nanotoxicological+studies%5BTitle%5D)+AND+%22Journal+of+Nanoparticle+Research%22%5BJournal%5D)">Characterisation of nanoparticle size and state prior to nanotoxicological studies.</a> Journal of Nanoparticle Research. 12, 47-53 (2010).</li>
<li>Wiogo, H. T., Lim, M., Bulmus, V., Yun, J., Amal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stabilization+of+magnetic+iron+oxide+nanoparticles+in+biological+media+by+fetal+bovine+serum+%28FBS%29%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Stabilization of magnetic iron oxide nanoparticles in biological media by fetal bovine serum (FBS).</a> Langmuir. 27, 843-850 (2011).</li>
<li>Yunker, P. J., Still, T., Lohr, M. A., Yodh, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+the+coffee-ring+effect+by+shape-dependent+capillary+interactions%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Suppression of the coffee-ring effect by shape-dependent capillary interactions.</a> Nature. 476, 308-311 (2011).</li>
<li>Kahler, H., Lloyd Jr, B., Eden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+Microscopic+and+Other+Studies+on+a+Copper%E2%80%93Cystine+Complex%5BTitle%5D)+AND+%22The+Journal+of+Physical+Chemistry%22%5BJournal%5D)">Electron Microscopic and Other Studies on a Copper–Cystine Complex.</a> The Journal of Physical Chemistry. 56, 768-770 (1952).</li>
<li>Furia, E., Sindona, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complexation+of+L-cystine+with+metal+cations%5BTitle%5D)+AND+%22Journal+of+Chemical+%26+Engineering+Data%22%5BJournal%5D)">Complexation of L-cystine with metal cations.</a> Journal of Chemical & Engineering Data. 55, 2985-2989 (2010).</li>
<li>Hawkins, C., Perrin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polynuclear+Complex+Formation.+II.+Copper+%28II%29+with+Cystine+and+Related+Ligands%5BTitle%5D)+AND+%22Inorganic+Chemistry%22%5BJournal%5D)">Polynuclear Complex Formation. II. Copper (II) with Cystine and Related Ligands.</a> Inorganic Chemistry. 2, 843-849 (1963).</li>
<li>Hallman, P., Perrin, D., Watt, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+computed+distribution+of+copper+%28II%29+and+zinc+%28II%29+ions+among+seventeen+amino+acids+present+in+human+blood+plasma%5BTitle%5D)+AND+%22Biochem.+J%22%5BJournal%5D)">The computed distribution of copper (II) and zinc (II) ions among seventeen amino acids present in human blood plasma.</a> Biochem. J. 121, 549-555 (1971).</li>
<li>Jensen, L. S., Maurice, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+sulfur+amino+acids+on+copper+toxicity+in+chicks%5BTitle%5D)+AND+%22The+Journal+of+nutrition%22%5BJournal%5D)">Influence of sulfur amino acids on copper toxicity in chicks.</a> The Journal of nutrition. 109, 91-97 (1979).</li>
<li>Lee, Y., Choi, J. R., Lee, K. J., Stott, N. E., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+synthesis+of+copper+nanoparticles+by+chemically+controlled+reduction+for+applications+of+inkjet-printed+electronics%5BTitle%5D)+AND+%22Nanotechnology%22%5BJournal%5D)">Large-scale synthesis of copper nanoparticles by chemically controlled reduction for applications of inkjet-printed electronics.</a> Nanotechnology. 19, 415604(2008).</li>
<li>Hume, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+coiled-coil+protein+microfibers%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">Engineered coiled-coil protein microfibers.</a> Biomacromolecules. 15, 3503-3510 (2014).</li>
</ol>

Authors have nothing to disclose.

While evaluating potential toxic effects of nanomaterials including CNPs, it was observed that over the long-term, CNPs were transformed from an initially more dispersed particulate distribution to a larger, aggregated form (Figure 2). In some cases, these highly aggregated formations which were produced in the cell culture dish, under biological conditions, formed highly linear projections from the central aggregate, reminiscent of previously described copper- containing &#8220;urchins&#8221; 6</su...

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 &#176;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 &#8220;urchin&#8221; and &#8220;nanoflower&#8221; 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 &#176;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 &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table><tbody><tr><td>Mini Vortexer</td><td>VWR (https://us.vwr.com)</td><td>58816-121</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator Model # 2425-2</td><td>VWR (https://us.vwr.com)</td><td>Contact vendor</td><td>Current model calalog # 98000-360</td></tr><tr><td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td><td>Labnet (http://labnetinternational.com/)</td><td>C2500-R</td><td>Model Prism R</td></tr><tr><td>Cell Culture Centrifuge Model Z323K</td><td>Labnet (http://labnetinternational.com/)</td><td>Contact vendor</td><td>Current model Z206A catalog # C0206-A</td></tr><tr><td>Sonicator (Ultrasonic Cleaner)</td><td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td><td>1510R-MTH</td><td></td></tr><tr><td>Balance</td><td>Sartorius (http://dataweigh.com)</td><td></td><td>Model CP225D similar model CPA225D</td></tr><tr><td>Olympus IX51 Inverted Light Microscope</td><td>Olympus (http://olympusamerica.com</td><td>Contact vendor</td><td></td></tr><tr><td>Olympus DP71 microscope digital camera</td><td>Olympus (http://olympusamerica.com</td><td>Contact vendor</td><td></td></tr><tr><td>external power supply unit - white light for Olympus microscope</td><td>Olympus (http://olympusamerica.com</td><td>TH4-100</td><td></td></tr><tr><td>10X, 20X, and 40X microscope objectives</td><td>Olympus (http://olympusamerica.com</td><td>Contact vendor</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td><td>model S-4800</td><td></td></tr><tr><td>Transmission Electron Microscope</td><td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td><td>model Libra 120</td><td></td></tr><tr><td>Table Top Work Station Unidirectional Flow Clean Bench</td><td>Envirco (http://envirco-hvac.com)</td><td>model PNG62675</td><td>Used for sterile cell culture technique</td></tr></tbody></table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Figure 1 shows a flow-chart schematic of the synthesis steps to form the linear biocomposites described in this work. CNPs or copper sulfate as starting materials are combined with sterile water to form a 2 mg/ml solution, this solution is mixed and sonicated to provide an even mixture, and this copper solution is then mixed in the following ratio for synthesis: 949 parts sterile water: 50 parts copper mixture: 1 part cystine stock solution. The actual volumes may be increased or decreased according to t...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Research

JoVE Journal

Bioengineering

9.0K Views.  Centenary College of Louisiana.  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. 

Video: Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

Engineering

Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting

We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding combined with layer transfer enables the replication of arbitrary surface patterns from a master structure onto the functional material. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes. In particular we demonstrate the fabrication of patterned transparent zinc oxide electrodes for light trapping applications in solar cells.

We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes.

We describe a nanomoulding technique which  allows low-cost nanoscale patterning of functional materials, materials stacks  and full devices. Nanomoulding ...

Manufacturing of Three-dimensionally Microstructured Nanocomposites through Microfluidic Infiltration

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite in&#64257;ltration of 3D interconnected microfluidic networks. The manufacturing of the reinforced beams begins with the fabrication of microfluidic networks, which involves layer-by-layer deposition of fugitive ink filaments using a dispensing robot, filling the empty space between filaments using a low viscosity resin, curing the resin and finally removing the ink.&#160;Self-supported 3D structures with other geometries and many layers (e.g.&#160;a few hundreds layers) could be built using this method. The resulting tubular micro&#64258;uidic networks are then infiltrated with thermosetting nanocomposite suspensions containing nanofillers (e.g.&#160;single-walled carbon nanotubes), and subsequently cured. The infiltration is done by applying a pressure gradient between two ends of the empty network (either by applying a vacuum or vacuum-assisted microinjection). Prior to the infiltration, the nanocomposite suspensions are prepared by dispersing nanofillers into polymer matrices using ultrasonication and three-roll mixing methods. The nanocomposites (i.e.&#160;materials infiltrated) are then solidified under UV exposure/heat cure, resulting in a 3D-reinforced composite structure. The technique presented here enables the design of functional nanocomposite macroscopic products for microengineering applications such as actuators and sensors.

Three-dimensional (3D) microstructured composite beams are fabricated through the directed and localized infiltration of nanocomposites into 3D porous microfluidic networks. The flexibility of this manufacturing method enables the utilization of different thermosetting materials and nanofillers in order to achieve a variety of functional 3D reinforced nanocomposite macroscopic products.

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite ...

Chemistry

Generation of Zerovalent Metal Core Nanoparticles Using n-(2-aminoethyl)-3-aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of n-(2-aminoethyl)-3-aminosilanetriol is presented. This compound can be used to effectively reduce and complex metal salts into metal core nanoparticles coated with the compound. By controlling the concentrations of salt and silane one is able to control reaction rates, particle size, and nanoparticle coating. The effects of these changes were characterized through transmission electron microscopy (TEM), UV-Vis spectrometry (UV-Vis), Nuclear Magnetic Resonance spectroscopy (NMR) and Fourier Transform Infrared spectroscopy (FTIR). A unique aspect to this reaction is that usually silanes hydrolyze and cross-link in water; however, in this system the silane is water-soluble and stable. It is known that silicon and amino moieties can form complexes with metal salts. The silicon is known to extend its coordination sphere to form penta- or hexa-coordinated species. Furthermore, the silanol group can undergo hydrolysis to form a Si-O-Si silica network, thereby transforming the metal nanoparticles into a functionalized nanocomposites.

Generation of Zerovalent Metal Core Nanoparticles Using n-2-aminoethyl-3-aminosilanetriol

A novel method for metal core nanoparticle synthesis using a water stable silanol is described.

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the two metal oxides&#39; corresponding nanoparticles&#8212;the former being as small as 22 nm and the latter 15 nm. We fed two strains of bacteria, Shewanella algae and Pandoraea sp., with various concentrations of tungstate or molybdate ions. The concentrations of tungstate and molybdate were adjusted to make microcapsules of different length-to-diameter ratios. We found that the higher the concentration the smaller the nanoparticles were. The nanoparticles came in with three length-to-diameter ratios: 10:1, 3:1 and 1:1, which were achieved by feeding the bacteria respectively with a low concentration, a medium concentration, and a high concentration. The images of the hollow microcapsules were taken via the scanning electron microsphere (SEM). Their crystal structures were verified by X-ray diffraction (XRD)&#8212;the crystal structure of molybdate microcapsules is Na2MoO4 and that of tungstate microcapsules is Na2WO4 with Na2W2O7. These syntheses all were accomplished under a near ambient condition.

This work presents a protocol for manufacture sodium tungstate and sodium molybdate microcapsules via bacteria and their corresponding nanoparticles.

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the ...

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and high-density data storage have demonstrated the potential benefit of their use in industrial applications. These applications require precise control over nanoparticle size, spacing, and sometimes shape. These requirements have resulted in the use of time and cost intensive processing steps to produce nanoparticles, thus making the transition to industrial application unrealistic. This protocol will resolve this issue by providing a scalable and affordable method for the large-area production of nanoparticle films with improved nanoparticle control compared to the current techniques. In this article, the process will be demonstrated with gold, but other metals can also be used.

This protocol details a novel nano-manufacturing technique that can be used to make controllable and customizable nanoparticle films over large areas based on the self-assembly of dewetting of capped metal films.

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

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