The overall goal of this procedure is to generate linear, high aspect ratio, micro and nano composites using copper containing starting materials and cysteine. This is accomplished by first combining sonicated copper nanoparticles or copper sulfate with cystine and water in a sterile vented culture flask. The second step of the synthesis is to place the combined components in the flask in a 5%CO2 incubator at 37 degrees Celsius for at least six hours.
Next, the flask is inspected by eye and by white light microscopy To determine the extent of linear structure formation, the final step is to terminate the synthesis through placing the synthesis flask in a refrigerator maintained at four degrees Celsius. Ultimately, digital microscopy is used to characterize the synthesized structures. The main advantage of this technique over existing methods like electrode position is that they describe synthesis can be easily scaled up in liquid form and the process occurs at physiological conditions.
The implications of this technique extend toward therapy or diagnosis of diseases such as cancer because synthesized structures can be degraded by cells potentially providing a means to deliver drugs. I first had the idea for this method when we were carrying out experiments in investigating the potential toxicity of different nanomaterials. Visual demonstration of this method is critical as the final synthesis steps can be difficult to learn.
Incorrect procedures may result in aggregated structures or no structures at all. To perform the self-assembly synthesis using copper nanoparticles or CMPs, prepare a solution of copper nanoparticles by weighing out at least two milligrams of CMPs. Wear disposable gloves during this step to prevent possible contact of the CMPs with skin.
Place the nanoparticles in an empty sterile 16 milliliter glass vial to the vial containing CMPs. Add sterile deionized water in the appropriate volume to make a two milligram per milliliter solution and vortex the solution for 20 seconds to provide dispersion of the nanoparticles before synthesis starts. Do not fill the vial more than halfway with water as this will inhibit.
Mixing by vortexing CMPs will quickly settle to the bottom of the vial and will appear dark in color. Sonicate the CNP solution for 17 minutes at room temperature to provide maximal dispersion of CMPs before the start of synthesis. Periodically check to make sure that CMPs are mixing due to son after a successful son CMPs remain suspended in solution for at least 30 minutes, and the solution will be dark in color.
Weigh out 7.29 milligrams of cystine for the synthesis. Since cystine is not directly soluble in water, place the wade cystine in an antistatic weighing vessel to the weighing vessel containing cysteine. Add a sufficient volume of sterile one molar sodium hydroxide so that the cystine completely dissolves to make a 72.9 milligram per milliliter solution.
Dissolve the cysteine completely in 100 microliters of one molar sodium hydroxide. Then combine seven microliters of the dissolved cysteine with 6, 643 microliters of sterile water in a sterile synthesis flask. Place the flask with combined solution in the incubator for 30 minutes at 37 degrees Celsius with the flask cap vented to provide effective mixing.
This will be the cysteine working solution. Resuspend the two milligram per milliliter CNP solution by Vortexing for 30 seconds since CMPs will have settled after the sonication step for a seven milliliter total synthesis volume. Transfer the cysteine working solution to a 25 square centimeter cell culture flask and add 350 microliters of resuspended CMPs.
Replace the cap on the flask and tighten it so that it is secure. After combining all components for the synthesis, gently mix in the flask by swirling four to five times. Place the flask in the carbon dioxide incubator and vent the flask by loosening the cap so that there will be gas exchange in and out of the flask.
During synthesis, allow the synthesis to run in the incubator for approximately 24 hours. During the synthesis, one can observe with microscopy and by eye the formation of highly linear composites. This step is critical to ensure that some structures are forming and that the self-assembly process does not go on too long.
Terminate synthesis of bio composites once linear structures are observed in the flask by tightly capping the synthesis flask, label the flask with synthesis conditions, including utilized date of the synthesis and incubation, time of the synthesis before termination. Then store the vessel in a refrigerator once generated structures remain stable in this form for at least a year. Alternatively, to perform the self-assembly synthesis using copper sulfate, replace CMPs with copper sulfate salt using sterile technique, dissolve at least two milligrams of copper sulfate in a sufficient volume of sterile deionized water to make a two milligram per milliliter 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 after preparation of the copper sulfate. Carry out synthesis as just demonstrated, but replace CMPs with the copper sulfate. Characterize bio composites derived from CMPs and from copper sulfate by white light microscopy and by electron microscopy for characterization and inspection of bio composites.
Parasynthesis by white light microscopy use an inverted microscope. 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 bio composites and the liquid medium composites derived from CMPs and copper sulfate will both appear clear to opaque in color, but unre reacted CNP aggregates will appear very dark in color.
Use inverted white light microscopy to assess the efficacy of the synthesis for a given experiment. For example, document the presence or absence of unreactive CMPs in synthesis. Flasks used for CNP derived bio composites from flasks with different parameters such as time of incubation or duration of sonication.
Individual CMPs are too small to observe with a light microscope, but unreactive CNP aggregates will appear as round shape and dark objects. This is in contrast to the successfully synthesized CNP composites, which will have a high aspect ratio, linear form, and a range of different lengths. Avoid carrying out synthesis for too long a period of time before termination, as this will result in highly branched urchin type structures which are difficult to disperse into individual structures once formed.
Also 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 CMPs.
Document the size and extent of copper sulfate composites by comparing flasks with different synthesis conditions such as time of synthesis before termination. To concentrate bio composites parasynthesis first, add six milliliters of either CNP derived structures or copper sulfate derived structures to a 15 milliliter centrifuge tube. Centrifuge for 10 minutes at 500 times G and room temperature to form a pellet for smaller volumes.
Add 500 microliters of structures in solution to 0.6 milliliters, size tubes, centrifuge at 2000 times G and room temperature for at least 10 minutes to form a pellet. Place the structures in sterile deionized water and sonicate for at least 10 minutes to resuspend. Using this process over time, structures become fragmented and smaller in average length.
Document changes in composite sizes with different sonication times using an inverted white light microscope and digital camera shown our representative results from the discovery of bio composites in cell culture media over 82 hours initial even dispersions of CMPs aggregate into microstructures. Over time, particles are cleared and larger aggregates form. Finally, larger aggregates with linear structures appear forming urchin type structures.
Transformation of CMPs to linear bio composites is shown. CMPs were combined with cysteine and water from initial even dispersions of CMPs. Aggregates form microstructures after three hours, intermediate structures form by six hours.
High aspect ratio structures form electron microscopy. Characterization of synthesized bio composites is shown transmission electron microscopy shows CNP starting material with the forming linear composites starting CMPs are round as shown by scanning electron microscopy or SEM. Nano and micro features of composites formed from CMPs and cysteine are shown by SEM.
Features of composites formed from copper sulfate and cysteine are also depicted by SEM Energy dispersive. X-ray spectroscopy elemental analysis of starting materials and synthesized linear components is shown. Starting CMPs show a prominent copper peak with no sulfur.
Bio composites from CMPs and cystine show emergence of sulfur and carbon peaks due to cystine starting copper sulfate. Material shows peaks for copper and sulfur. Bio composites from copper sulfate and cystine show emergence of peaks for carbon due to cystine Once mastered, this technique can be done in 12 to 24 hours if it is performed properly.
While attempting this procedure, it is important to remember to monitor the synthesis progress both by eye and by the microscope, so that synthesized structures do not become too aggregated. Following this procedure, other methods like fluorescent labeling of the structures can be performed in order to answer additional questions like the synthesized structures bind to cells. After watching this video, you should have a good understanding of how to prepare reagents for synthesis, combine them correctly, monitor synthesis progression, and concentrate and modify structures after synthesis.
