This work was supported by the National Science Foundation (NSF awards CMMI-0922946 to D.B., CMMI-1300920 to D.B. and S.O'M., and CMMI-1531789 to S.O'M., D.B., and E.A.K.) and a Busch Biomedical Research Grant to E.A.K. and S.O'M.

1. Focusing the Nanosecond Laser and Measuring Fluence
<ol>
	<li>Assemble the ablation apparatus by placing a magnetic stir bar and a porous ablation stage inside a 50 mL glass beaker. 
	NOTE: The ablation stage consists of a 3.81 cm diameter, 1.6 mm thick stainless steel platform with ten 0.65 cm diameter holes and six 0.50 cm diameter holes drilled in the pattern illustrated in Figure 1. The purpose of these holes is to allow the liquid to move across the target so ...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+Properties+of+Nanoparticles:+A+Comparative+Review+of+Chemically+Synthesized+and+Laser-Generated+Particles.">Antibacterial Properties of Nanoparticles: A Comparative Review of Chemically Synthesized and Laser-Generated Particles.</a> Adv. Sci. Eng. Med. 7 (12), 1044-1057 (2015).</li><li>Elsayed, K. A., Imam, H., Ahmed, M. A., Ramadan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+focusing+conditions+and+laser+parameters+on+the+fabrication+of+gold+nanoparticles+via+laser+ablation+in+liquid.">Effect of focusing conditions and laser parameters on the fabrication of gold nanoparticles via laser ablation in liquid.</a> Opt. &amp; Laser Tech. 45, 495-502 (2013).</li><li>Kabashin, A. 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The authors have nothing to disclose.

Reproducible antimicrobial effects of NPs require consistent production of NPs with similar sizes and concentrations. Therefore, it is critical to standardize laser parameters including fluence, wavelength, and pulse duration. While dynamic light scattering is an easy and rapid method for estimating NP size, accurate quantification of the size distribution requires direct measurement by TEM. As each laser beam has distinct characteristics in terms of mode profile and divergence, it is critical to employ an empirical proc...

Nanoparticles (NPs) are generally defined as particles that have at least one dimension that is less than 100 nm in length. Traditional chemical NP synthesis methods typically require hazardous reducing agents, such as borohydrides and hydrazines. In contrast, laser ablation of solid metal targets immersed in a liquid medium (pulsed laser-ablation in liquids &#8211; PLAL) provides an environmentally friendly route for NP synthesis that satisfies all 12 of the Principles of Green Chemistry1,2. In PLAL, a submerged metal target is irradiated by repeated laser pulses. As the laser ablates the target, a dense plume of atomic clusters and vapor is released into the liquid medium wherein NPs rapidly coalesce. NPs produced by PLAL are finely dispersed in an aqueous medium and the size, polydispersity, and composition of the NPs can be easily controlled by varying the aqueous ablation liquid as well as laser parameters1,2,3,4,5,6.
Nanoparticle characteristics can be tuned by adjusting a number of laser parameters, including: fluence, wavelength, and pulse duration (reviewed in reference7). Laser fluence is calculated as the pulse energy divided by the area of the laser spot on the target surface. The precise effects of fluence on the size and polydispersity of NPs are somewhat controversial. In general, it has been shown that for &#39;long&#39; and &#39;ultra-short&#39; pulsed laser systems there are low and high fluence regimes that produce negative and positive trends in size, respectively8,9,10,11. NP size distributions can be empirically measured using techniques such as dynamic light scattering and transmission electron microscopy (TEM), as described below.
The choice of laser wavelength can affect the physical mechanisms by which the NPs are formed. At shorter (ultraviolet) wavelengths, high energy photons are capable of breaking interatomic bonds12. This mechanism of photo-ablation is an example of a top-down NP synthesis because it results in the release of ultra-small fragments of material which tend to produce larger more polydisperse samples upon quenching in the submersion liquid12,13,14. In contrast, near-infrared ablation (&#955; = 1,064 nm) yields a bottom-up synthesis mechanism dominated by plasma ablation12. Laser absorption by the target frees electrons that collide with, and subsequently free, bound electrons. As collisions increase, the material is ionized, thus igniting a plasma. The surrounding liquid confines the plasma, enhances its stability, and further increases absorption12. As the expanding plasma is quenched by the confining liquid, NPs are condensed with various geometries4,12,15.
The choice of laser pulse duration can further impact the NP-formation process. Commonly used long-pulsed lasers, with pulse durations greater than a few picoseconds, include all milli, micro, nano and some picosecond pulsed lasers. In this pulse-width regime, the laser pulse duration is longer than the electron-phonon equilibration time, which is typically on the order of a few picoseconds4,16,17,18,19. This results in the leaking of energy into the surrounding ablation medium and the formation of NPs by thermal mechanisms such as thermionic emission, vaporization, boiling and melting1,20.
The antibacterial activity of NPs is strongly influenced by particle size21,22,23,24. In order to enhance size reduction and monodispersity, the NPs can be irradiated a second time using a laser of a wavelength near the surface plasmon resonance (SPR) of the NP. The incident laser radiation is absorbed by the NP through excitation of the SPR. Fragmentation of the NP may occur through either thermal evaporation25,26 or Coulomb explosion27,28. The photoexcitation raises the temperature of the NP above the melting point, resulting in the shedding of the outer layer of the particle. It has been shown that adding agents such as polyvinylpyrrolidone (PVP) or sodium dodecyl sulfate (SDS) to the solution can greatly enhance post-irradiation effects5. The impact of the addition of various solutes have been described in several reports1,4,6. The ease of manipulation of NP characteristics by PLAL affords a novel method to develop novel NP-based antimicrobials.

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Nanosecond Nd:YAG laser</td>
			<td style="width:184px;">Ekspla</td>
			<td style="width:119px;">NL303</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Motorized xy scanning stage</td>
			<td style="width:184px;">Standa</td>
			<td style="width:119px;">8MTF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">UV-VIS spectrophotometer</td>
			<td style="width:184px;">Agilent</td>
			<td style="width:119px;">Cary 60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dynamic light scattering unit</td>
			<td style="width:184px;">Malvern</td>
			<td style="width:119px;">Zetasizer ZS 90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Microbalance</td>
			<td style="width:184px;">Maktek</td>
			<td style="width:119px;">TM 400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Transmission electron microscope</td>
			<td style="width:184px;">Zeiss</td>
			<td style="width:119px;">EM 902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Silver foil target</td>
			<td style="width:184px;">Alfa Aesar</td>
			<td align="right" style="width:119px;">12127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">250 mm focal length lens</td>
			<td style="width:184px;">Edmund Optics</td>
			<td style="width:119px;">69-624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Copper TEM grids</td>
			<td style="width:184px;">Pacific Grid-Tech</td>
			<td style="width:119px;">Cu-400LD</td>
			<td>Lacey/thin film coated grid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">E. coli MG1655</td>
			<td style="width:184px;">ATCC</td>
			<td align="right" style="width:119px;">47076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bacto-tryptone</td>
			<td style="width:184px;">BD Biosciences</td>
			<td align="right" style="width:119px;">211705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Yeast extract</td>
			<td style="width:184px;">BD Biosciences</td>
			<td align="right" style="width:119px;">212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sodium chloride</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td style="width:119px;">BP3581</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bacto-agar</td>
			<td style="width:184px;">BD Biosciences</td>
			<td align="right" style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sodium dodecyl sulfate</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td>BP166-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Polyvinylpyrrolidone</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td style="width:119px;">BP431-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Stainless steel disc (for ablation stage)</td>
			<td style="width:184px;">Metal Remnants, Inc.</td>
			<td style="width:119px;">N/A</td>
			<td>1.5 inch diameter, 16 gauge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Beaker</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td style="width:119px;">02-540G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Magnetic stir bar</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td style="width:119px;">14-513-57</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Magnetic stir plate</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td style="width:119px;">11-100-49SH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Laser energy and power meter</td>
			<td style="width:184px;">Coherent</td>
			<td style="width:119px;">1098579</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Carbon tape</td>
			<td style="width:184px;">Shinto Chemitron Co. Ltd.</td>
			<td style="width:119px;">STR Tape</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sonicating water bath</td>
			<td style="width:184px;">Branson</td>
			<td align="right" style="width:119px;">1510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Air compressor</td>
			<td style="width:184px;">GMC</td>
			<td style="width:119px;">Syclone 3010</td>
			<td>For drying ablation target</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">75 mm focal length lens</td>
			<td style="width:184px;">Edmund Optics</td>
			<td style="width:119px;">34-096</td>
			<td>Focusing lens for post-irradiation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Quartz cuvette</td>
			<td style="width:184px;">Precision Cells Inc</td>
			<td style="width:119px;">21UV40</td>
			<td>50 mm light path (for post-irradiation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Kanamycin</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:269px;">Light microscope</td>
			<td style="width:184px;">Nikon</td>
			<td style="width:119px;">50i</td>
			<td style="width:279px;">This microscope is used to focus the laser on the ablation stage. This particular model is no longer available, but any light microscope with a 4X objective will work.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CCD camera</td>
			<td style="width:184px;">AmScope</td>
			<td style="width:119px;">MT5000-CCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Micrometer slide</td>
			<td style="width:184px;">Ted Pella</td>
			<td style="width:119px;">2280-70</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of metal nanoparticles by pulsed laser-ablation in liquids: a tool for studying the antibacterial properties of nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

Using silver targets, the laser parameters described above, and 60 mM SDS in the ablation liquid, silver NPs are generated with the characteristic UV-VIS absorbance at the SPR (Figure 2A). TEM and DLS measurements reveal a mean NP diameter of approximately 25 nm before post-irradiation (Figure 2B). Ablation of the silver target for 30 min typically yields an NP concentration of 200 &#181;g/mL. In assessing the antimicrobial toxic...

Watch this Scientific Journal Video about Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles at JoVE.com

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

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.

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Bioengineering

14.7K Views.  Rutgers University-Camden. 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, whic...