The overall goal of this procedure is to produce nanoparticle colloids with control over their size and composition for the purpose of assessing their antibacterial properties. This method can help answer key questions in the development of antimicrobial nanoparticles, including the impact of particular nanoparticle characters, such as size, shape, and composition on toxicity. The main advantage of this technique is the production of nanoparticles from various material systems with ease, which is done in the absence of potentially hazardous chemical byproducts.

Demonstrating this experiment will be Matthew Ratti, and undergraduate, and Julianne Griepenburg, a faculty member at Rutgers Camden. Assemble the ablation apparatus by placing magnetic stir bar and a porous ablation stage inside a 50 milliliter glass beaker. Place the beaker on a magnetic stir plate, and set the stir plate upon an XY translation stage to enable movement of the target during ablation.

Set the Nd:YAG laser to operate at the fundamental wavelength of 1, 064 nanometers with a pulse duration of five nanoseconds and a pulse repetition rate of 10 hertz. Measure the energy per pulse with a laser power and energy meter. Focus the beam beneath the target on the ablation stage using a 250 millimeter focal length converging lens.

For the synthesis of silver nanoparticles, first weigh a flat silver target using a microbalance to obtain the pre-ablation mass. Then, adhere the silver target to the porous stage using double-sided carbon tape. Add 40 milliliters of ablation liquid to the beaker.

The liquid height above the target is 11 millimeters. Determine the spot size by ablating the silver target with several laser pulses. To measure the spot size, view the ablated target together with a micrometer slide on a light microscope equipped with a CCD camera.

To synthesize the silver nanoparticles, move the computer-controlled, motorized XY stage in an elliptical pattern at a speed of 0.42 centimeters per second under constant stirring and ablate the target for 20 to 40 minutes. Collect the nanoparticle solution from the beaker by decanting. Confirm the presence of nanoparticles by measuring their UV visible light spectra.

Measure the hydrodynamic diameter of the nanoparticles using dynamic light scattering by diluting the nanoparticle solution 1 to 40 in the ablation solution. A total volume of one milliliter of the diluted solution is required when using a one centimeter plastic cuvette. Utilizing a measurement angle of 180 degrees, measure the light scattering at a wavelength of 633 nanometers to determine the nanoparticle diameter according to the Stokes-Einstein equation.

Confirm the nanoparticle size and shape using transmission electron microscopy by first diluting the nanoparticle solution 1 to 40 in double-distilled water to remove any excess additives that may interfere with imaging. Then, drop two microliters of the solution onto a copper TEM grid pre-coated with Lacey thin carbon film. Dry the grid overnight at room temperature under vacuum in a desiccator before imaging the nanoparticles as referenced in the text protocol.

To calculate the nanoparticle concentration, dislodge any loosely attached nanoparticles from the ablated metal target by placing the target in a sonicating water bath containing distilled water for one minute. Dry the target under a stream of compressed air for one minute. Then, measure the mass of the target on a microbalance.

Dilute the nanoparticles to a maximum concentration of 100 micrograms per milliliter in the same ablation solution. Transfer 15 to 17 milliliters of the diluted nanoparticles to a quartz cuvette containing a stir bar. Use an Nd-YAG laser system to produce 25 picosecond, 532 nanometer laser pulses.

Also, use a 75-millimeter focal length lens to focus the laser on the center of the solution. Irradiate the solution for 30 minutes to multiple hours depending on the desired size. To measure the antibacterial properties of the nanoparticles, first grow E.coli cultures overnight and then dilute these to an optical density of 0.01 in fresh LB.If the nanoparticles were synthesized in ablation media containing additives, add the respective chemical to the LB, such that the concentration remains constant upon adding the nanoparticles.

Next, add the nanoparticles to the diluted cultures at concentrations ranging from zero to 50 micrograms per milliliter. Grow the cultures with shaking at 37 degrees Celsius for an additional two hours. After the two-hour incubation, serially dilute the culture samples one to ten in fresh LB.And spot 10 microliter drops of each dilution onto LB augur plates.

Once the droplets have been absorbed, incubate the plates overnight at 37 degrees Celsius. The next morning, count the colony-forming units. Here, nanoparticles produced by pulse laser ablation in liquids are characterized by their absorbent spectra.

The ultraviolet visible light spectrum of silver nanoparticles shows a characteristic peak at the surface plasmon resonance wavelength of 400 nanometers. To further characterize the nanoparticles, their size distribution is measured by transmission electron microscopy. Treating bacteria, such as E.coli, with nanoparticles reduces viability as determined by measuring colony-forming units.

Cells were treated with kanamycin as a positive control. Note that the cells not receiving silver nanoparticles were grown in the presence of six millimolar SDS to ensure that the surfactants did not independently result in toxicity. Once mastered, nanoparticles can be produced and characterized in four to five hours.

Testing the antibacterial toxicity of the nanoparticles requires an additional 24 hours. While attempting this procedure, it's important to remember that the consistency between batches requires careful attention to details, such as pulse energy and the lens positioning. DOn't forget, this work can be extremely hazardous and precautions, such as appropriate eye protection, should always be taken while performing this procedure.

Thanks for watching, and good luck with your experiments.