Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

The overall goal of this procedure is to examine cupric oxide nanoparticle treatment as a contaminant removal process in production, bleed water, and to evaluate it cytotoxicity using cultured human cells. This is accomplished by first treating production bleed water with cupric oxide nanoparticles. The concentration changes of individual elements are evaluated before and after treatment.

Next, concentrated cell culture media is diluted with either untreated production bleed water or cupric oxide. Nanoparticle treated production bleed water in addition to standard cell culture supplements to prepare the test media untreated production bleed, water media and cupric oxide, nanoparticle treated production bleed. Water media is then applied to human kidney and liver cells and culture, and the viability is measured over seven days.

Ultimately, the M-T-T-S-A demonstrates the removal of arsenic, selenium, vanadium, and uranium from production. Bleed water by cupric oxide nanoparticles is associated with increased viability of human kidney and liver cells. This is the first study to investigate the removal of specific contaminants using cooper oxide nanoparticles while assessing changes in cytotoxicity associated with the removal in a cell culture format.

The main advantage of this technique over existing methods is that the environmental sample as it exists in nature, is tested alongside the nanoparticles in a inexpensive hydro putt format. Cupric oxide nanoparticles tested in this study can be applied to other groundwater systems such as domestic groundwater associated with small communities. The regeneration of cupric oxide nanoparticles creates less waste and minimizes waste to disposal problems.

To begin this procedure, prepare cupric oxide nanoparticles and collect the production bleed water or PBW as described in the text protocol to treat PBW with cupric oxide nanoparticles at 50 milligrams of the prepared nanoparticles to a 50 milliliter conical tube, followed by 50 milliliters of PBW seal the tube and react for 30 minutes on a benchtop orbital shaker at 250 RPM centrifuge, the sample tubes at 250 times G for 30 minutes alter the centrifuge speed and time depending on the nanoparticle to ensure the nanoparticles become compact in the centrifuge tube Following centrifugation, filter the supernatant using a 0.45 micron syringe. Filter samples are then sent for elemental analysis To prepare cell culture media. First, use RO water from the laboratory.

These include 100%75%50%and 25%untreated PBW or CIC oxide nanoparticle treated PBW Next at 25 milliliters of concentrated 10 X-E-M-E-M to 190 milliliters of the ro. As well as each of the treated and untreated PBW concentrations. Adjust the pH of each solution to 7.4 with sodium hydroxide or hydrochloric acid.

Then supplement each concentration of untreated and treated PBW plus media, as well as the RO control media with standard components that are listed in the text protocol. After each concentration of untreated, untreated PBW plus media as well as RO control water plus media is made, the osmolality is adjusted. A drop of each solution is applied to a solute free paper disc and the disc is placed into a vapor pressure OSM ter.

The osmolality is then adjusted to between 290 and 310 milli osmoles per kilogram by adding RO water. Finally, filter each solution using a zero point 22 micron vacuum filter unit and store at four degrees Celsius. Prepare a culture of human embryonic kidney cells and human hepatocellular carcinoma cells two to three days before they are to be plated.

In 96. Well plates per manufacturers and instructions after removing the cells from their culture dishes, using trypsin centrifuge at 1000 times G for five minutes and decant the trypsin. Add five milliliters of phosphate buffered saline or PBS and mix the cells to obtain a single cell solution.

Then apply 20 microliters of the single cell solution to a hemo cytometer to obtain a cell count per milliliter of solution after centrifuging. Again, as before and decanting, the PBS used to rinse the cells at the appropriate amount of one X-E-M-E-M to adjust the concentration of cells to 500 cells per 100 microliters per well. Next, fill the perimeter wells of the plate with 200 microliters of PBS to control for evaporation.

Seat the cells at a density of 500 cells per well, adding 100 microliters to each well except for the perimeter wells. Incubate the cells for 24 hours at 37 degrees Celsius, allowing them to recover before performing baseline. MTT readings of cell density.

Perform baseline MTT readings of cell density by removing the seeding media from the first column and adding 100 microliters of MTT to the wells for one hour. After one hour. Remove the MTT and add 100 microliters of dimethyl sulfoxide to dissolve the MTT Informason produced by viable cells.

Then read the optical density of the first column at an absorption wavelength of 570 nanometers. To obtain a baseline reading, adding one solution per plate. Replace the seeding media with 100 microliters of one X-E-M-E-M RO control media as well as each of the treated and untreated PPW plus media concentrations.

Incubate the cells in their test concentrations or control solutions for a total of seven days each day. Following the baseline MTT reading, remove the control and test solutions from the next column of their respective plate and repeat the MTT protocol. Repeat the protocol every day for seven days average.

The OD results were each row and report against time to generate a seven day growth curve to assess the effect of copper chelation on cell viability and cupric oxide. Nanoparticle treated PBW plus media follow the same procedure except at 100 micromolar d penicillin to control and test solutions before adding the solutions to their respective plates. As a final step, perform data analysis using scientific graphing software, cupric oxide nanoparticle treatment removed arsenic, selenium, uranium and vanadium from PBW speciation.

Modeling results support the analytical results protecting that in PBW 99%of arsenic, 94%of selenium and 99%of vanadium are present as negative species capable of absorbing to cupric oxide nanoparticles. However, only 35.5%of dissolved uranium species are negatively charged, which would limit absorption of uranium to cupric oxide. Nanoparticles viability was impaired in a concentration dependent manner in cells grown in untreated PBW plus media, whereas cupric oxide nanoparticle treatment improved cellular viability in both cell lines.

Heck and hep cellular growth on day five in control media show confluent well attached healthy looking cells. Conversely, heck and cells grown in untreated. PBW plus media are less confluent showed, detachment and appear rounded or unhealthy.

Moreover, heck, and he cells grown in capic oxide. Nanoparticle treated PBW plus media showed improved co fluency, attachment and health compared to cells grown in untreated PBW plus media Following this procedure. Other toxicity testing methods such as flow cytometry could be used to answer additional questions about mechanistic changes, cytotoxicity of mixtures before and after treatment.

After watching this video, you should have a good understanding of to test the effectiveness cupric and nanoparticles in removal of water, contaminants, and its effects on cell culture. However, it is important to remember to monitor copper concentrations in treated waters.