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 that particles do not accumulate immediately above the target. Insufficient mixing leads to deleterious interactions between the laser and the already-formed particles. Additionally, three #29 (8-32) tapped holes are located near the perimeter of the platform to accept set screws which serve as legs to raise the platform and provide space for a magnetic stir bar (Figure 1A).
	<ol>
		<li>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 (Figure 1A).</li>
	</ol>
	</li>
	<li>Set the Nd:YAG laser to operate at the fundamental wavelength of 1,064 nm, with a pulse duration of 5 ns, and a pulse repetition rate of 10 Hz. Measure the energy per pulse with a laser power and energy meter. The energy required is 240-250 mJ.</li>
	<li>Focus the beam beneath the target on the ablation stage using a 250 mm focal length converging lens (NA = 0.05). 
	NOTE: The incoming beam has a radius of 4.025 mm and a lens height of 161 mm is required to attain the desired spot size. The optimal spot size is determined empirically. A larger spot size is utilized to reduce the effect of shielding by NPs present in solution. This is balanced with the fact that increasing spot size requires higher energy to maintain adequate fluence.</li>
	<li>Determine the spot size by placing a metal target (see section 2) on the stage and ablating with several laser pulses. View the ablated target together with a micrometer slide on a CCD camera-equipped light microscope (4X objective) to measure the spot size (Figure 1A). 
	NOTE: For the apparatus here, the ablation system yields a spot size with a mean area of 5.51 mm2. The spot size remains in this range for each ablation.</li>
	<li>Calculate fluence by dividing the pulse energy by the spot area. For the apparatus here, the fluence is 4.80 J/cm2.</li>
</ol>
2. Synthesis of Silver Nanoparticles by Pulsed Laser-ablation in Liquid
<ol>
	<li>Weigh a flat silver target using a microbalance to obtain the pre-ablation mass.</li>
	<li>Adhere the silver target to the porous stage using double-sided carbon tape. Add 40 mL of ablation liquid to the beaker (Figure 1A). The liquid height above the target is 11 mm. 
	NOTE: Typical ablation liquids are aqueous solutions containing either 60 mM SDS or 2 mM PVP to enhance monodispersity.</li>
	<li>Under constant stirring, move the computer-controlled motorized xy-stage in an elliptical pattern (dimensions: major axis = 2.09 cm, minor axis = 0.956 cm, area = 1.57 cm2) at a speed of 0.42 cm/s and ablate the target for 20-40 min. 
	NOTE: The concentration of NPs increases with longer ablation times. Ensure that the stirring is sufficiently vigorous to keep the NP concentration uniform throughout the solution to minimize shielding effects7.</li>
</ol>
3. Characterizing Metal Nanoparticles
<ol>
	<li>Collect the nanoparticle solution from the beaker by decanting. Confirm the presence of nanoparticles by measuring their UV-visible light spectra (200-1,100 nm). 
	NOTE: The NPs have a peak-absorption at the surface plasmon resonance (SPR) wavelength. For silver, the SPR is centered at 400 nm. Highly concentrated NP solutions require dilution prior to measuring the UV-VIS spectrum to ensure that the absorbance readings remain within the linear range of the spectrophotometer.</li>
	<li>Measure the hydrodynamic diameter of the NPs by dynamic light scattering (DLS) utilizing a number distribution analysis method29.
	<ol>
		<li>Dilute the NP solution 1:40 in the ablation solution and pipette 1 mL into a 1 cm plastic cuvette. Utilizing a measurement angle of 180&#176;, measure the light scattering at a wavelength of 633 nm to determine the NP diameter according to the Stokes-Einstein equation: 
		<img alt="Equation 1" src="/files/ftp_upload/55416/55416eq1.jpg" /> 
		where d is the hydrodynamic radius, k is Boltzmann&#39;s constant, T is absolute temperature, &#951; is viscosity, and D translational diffusion coefficient or velocity of Brownian motion.</li>
	</ol>
	</li>
	<li>Confirm NP size and shape using transmission electron microscopy (TEM)30. 
	NOTE: The hydrodynamic diameter measured using DLS is larger than the size measured using TEM due to the solvent layer surrounding the NPs.
	<ol>
		<li>Dilute the NP solution 1:40 in double-distilled water to remove any excess additives (e.g. SDS or PVP) that may interfere with imaging. Drop 2 &#181;L of the solution onto a copper TEM grid pre-coated with lacey/thin carbon film (commercially available; see the Materials List) and dry overnight at room temperature under vacuum in a desiccator.</li>
		<li>Image the NPs to assess size and shape as described in reference30.</li>
	</ol>
	</li>
	<li>To calculate the NP concentration, dislodge any loosely attached NPs from the ablated metal target (step 2.3) by placing the target in a sonicating water bath containing distilled water for 1 min.
	<ol>
		<li>Dry the target under a stream of compressed air for 1 min. Measure the mass of the target on a microbalance. Quantify the mass of NPs in solution as the difference in weight before and after ablation, which is assumed to be the result of ejection of metal nanoparticles into the solution.</li>
	</ol>
	</li>
</ol>
4. Post-irradiation
<ol>
	<li>Dilute the NPs to a maximum concentration of 100 &#181;g/mL in the same ablation solution used in 2.2. This concentration limit is critical to ensure uniform irradiation.</li>
	<li>Transfer 15-17 mL of the diluted NPs to a quartz cuvette containing a stir bar (Figure 1B). Place the cuvette on a magnetic stir-plate aligned parallel with the incoming laser.</li>
	<li>Use a Nd:YAG laser system to produce 25 ps 532 nm laser pulses and a 75 mm focal-length lens to focus the laser on the center of the solution. Irradiate the solution for 30 min to multiple hours, depending on the desired size. 
	NOTE: The total energy delivered depends on the concentration of the solution and time of irradiation and can range from 0.5 mJ to 3.5 mJ. For the apparatus here, 30 min of post-irradiation of a transparent, low concentration sample (&#60;50 &#181;g/mL) yields silver NPs with a diameter of 10 nm.</li>
</ol>
5. Measuring the Antibacterial Properties of the Nanoparticles
NOTE: The toxicity of silver NPs against both Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) was tested31. The method is easily adapted to any species; however the efficacious dose of nanoparticles may vary considerably and must be determined empirically. Here, E. coli is used as the model system for the description of the method.
<ol>
	<li>Grow E. coli cultures (strain MG1655) overnight at 37 &#176;C in Luria Broth (LB) containing 10 g/L Bacto tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride. Dilute the overnight cultures to an optical density (&#955; = 600 nm) of 0.01 in fresh LB.</li>
	<li>If the NPs were synthesized in ablation media containing additives (e.g. SDS or PVP), add the respective chemical to the LB such that the concentration remains constant upon adding the NPs. 
	NOTE: For example, in a typical experiment a silver target is ablated into a 60 mM SDS solution to yield a 100 &#181;g/mL solution of NPs. If the final concentration of NPs in the culture media is 10 &#181;g/mL, prepare LB containing 6 mM SDS (i.e. 1/10 the concentration of SDS in the ablation liquid). There is no negative effect on the bacteria&#39;s growth when using these concentrations. This is shown in the -AgNP control in Figure 3.</li>
	<li>Add the NPs to the diluted cultures at concentrations ranging from 0-50 &#181;g/mL and grow the cultures with shaking at 37 &#176;C for an additional 2 h. As a positive control for toxicity, treat E. coli with an antibiotic (e.g. 30 &#181;g/mL kanamycin).</li>
	<li>After the 2 h incubation, serially dilute the culture samples 1:10 into fresh LB and spot 10 &#181;L drops of each dilution onto LB agar plates. Typically, 104-108 fold dilutions are sufficient to see individual colonies.</li>
	<li>Once the droplets have been absorbed, incubate the plates overnight at 37 &#176;C and count colony forming units (cfu) the following morning.</li>
</ol>

<ol>
	<li>Amendola, V., Meneghetti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+controls+the+composition+and+the+structure+of+nanomaterials+generated+by+laser+ablation+in+liquid+solution?.">What controls the composition and the structure of nanomaterials generated by laser ablation in liquid solution?.</a> Phys Chem Chem Phys. 15 (9), 3027-3046 (2013).</li><li>Anastas, P., Eghbali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+chemistry:+principles+and+practice.">Green chemistry: principles and practice.</a> Chem Soc Rev. 39 (1), 301-312 (2010).</li><li>Mafune, F., Kohno, J. Y., Takeda, Y., Kondow, T., Sawabe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Gold+Nanoparticles+by+Laser+Ablation+in+Aqueous+Solution+of+Surfactant.">Formation of Gold Nanoparticles by Laser Ablation in Aqueous Solution of Surfactant.</a> J Phys Chem B. 105 (22), 5114-5120 (2001).</li><li>Rao, S. V., Podagatlapalli, G. K., Hamad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+laser+ablation+in+liquids+for+nanomaterials+and+applications.">Ultrafast laser ablation in liquids for nanomaterials and applications.</a> J Nanosci Nanotechnol. 14 (2), 1364-1388 (2014).</li><li>Tsuji, T., 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=Preparation+of+silver+nanoparticles+by+laser+ablation+in+polyvinylpyrrolidone+solutions.">Preparation of silver nanoparticles by laser ablation in polyvinylpyrrolidone solutions.</a> Appl Surf Sci. 254 (16), 5224-5230 (2008).</li><li>Zeng, H., 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=Nanomaterials+via+laser+ablation/irradiation+in+liquid:+a+review.">Nanomaterials via laser ablation/irradiation in liquid: a review.</a> Adv Funct Mater. 22 (7), 1333-1353 (2012).</li><li>Naddeo, J. 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. V., Meunier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+colloidal+nanoparticles+during+femtosecond+laser+ablation+of+gold+in+water.">Synthesis of colloidal nanoparticles during femtosecond laser ablation of gold in water.</a> J Appl Phys. 94 (12), 7941-7943 (2003).</li><li>Nichols, W. T., Sasaki, T., Koshizaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+of+a+platinum+target+in+water+I.+Ablation+mechanisms.">Laser ablation of a platinum target in water I. Ablation mechanisms.</a> J Appl Phys. 100 (11), 114911 (2006).</li><li>Povarnitsyn, M. E., Itina, T. E., Levashov, P. R., Khishchenko, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+nanoparticle+formation+by+ultra-short+laser+ablation+of+metals+in+liquid+environment.">Mechanisms of nanoparticle formation by ultra-short laser ablation of metals in liquid environment.</a> Phys Chem Chem Phys. 15 (9), 3108-3114 (2013).</li><li>Mortazavi, S. Z., Parvin, P., Reyhani, A., Golikand, A. N., Mirershadi, S. <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+Laser+Wavelength+at+IR+(1064+nm)+and+UV+(193+nm)+on+the+Structural+Formation+of+Palladium+Nanoparticles+in+Deionized+Water.">Effect of Laser Wavelength at IR (1064 nm) and UV (193 nm) on the Structural Formation of Palladium Nanoparticles in Deionized Water.</a> J Phys Chem C. 115 (12), 5049-5057 (2011).</li><li>Kim, J., Reddy, D. A., Ma, R., Kim, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+laser+wavelength+and+fluence+on+palladium+nanoparticles+produced+by+pulsed+laser+ablation+in+deionized+water.">The influence of laser wavelength and fluence on palladium nanoparticles produced by pulsed laser ablation in deionized water.</a> Solid State Sci. 37, 96-102 (2014).</li><li>Mortazavi, S. Z., Parvin, P., Reyhani, A., Mirershadi, S., Sadighi-Bonabi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+various+carbon+nanostructures+in+water+using+IR/UV+laser+ablation.">Generation of various carbon nanostructures in water using IR/UV laser ablation.</a> J Phys D: Appl Phys. 46 (16), 165303 (2013).</li><li>Hunter, B. M., 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=Highly+active+mixed-metal+nanosheet+water+oxidation+catalysts+made+by+pulsed-laser+ablation+in+liquids.">Highly active mixed-metal nanosheet water oxidation catalysts made by pulsed-laser ablation in liquids.</a> J Am Chem Soc. 136 (38), 13118-13121 (2014).</li><li>Momma, C., 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=Short-pulse+laser+ablation+of+solid+targets.">Short-pulse laser ablation of solid targets.</a> Opt Commun. 129 (1), 134-142 (1996).</li><li>Sonntag, S., Roth, J., Gaehler, F., Trebin, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+laser+ablation+of+aluminium.">Femtosecond laser ablation of aluminium.</a> Appl Surf Sci. 255 (24), 9742-9744 (2009).</li><li>Yamashita, Y., Yokomine, T., Ebara, S., Shimizu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+transport+analysis+for+femtosecond+laser+ablation+with+molecular+dynamics-two+temperature+model+method.">Heat transport analysis for femtosecond laser ablation with molecular dynamics-two temperature model method.</a> Fusion Eng Des. 81 (8), 1695-1700 (2006).</li><li>Zhigilei, L. V., Lin, Z., Ivanov, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomistic+modeling+of+short+pulse+laser+ablation+of+metals:+connections+between+melting,+spallation,+and+phase+explosion.">Atomistic modeling of short pulse laser ablation of metals: connections between melting, spallation, and phase explosion.</a> J Phys Chem C. 113 (27), 11892-11906 (2009).</li><li>Schmidt, M., 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=Lasers+in+Manufacturing+2011+-+Proceedings+of+the+Sixth+International+WLT+Conference+on+Lasers+in+Manufacturing+Metal+Ablation+with+Short+and+Ultrashort+Laser+Pulses.">Lasers in Manufacturing 2011 - Proceedings of the Sixth International WLT Conference on Lasers in Manufacturing Metal Ablation with Short and Ultrashort Laser Pulses.</a> Physics Procedia. 12, 230-238 (2011).</li><li>Azam, A., Ahmed, A. S., Oves, M., Khan, M. S., Memic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-dependent+antimicrobial+properties+of+CuO+nanoparticles+against+Gram-positive+and-negative+bacterial+strains.">Size-dependent antimicrobial properties of CuO nanoparticles against Gram-positive and-negative bacterial strains.</a> Int. J. Nanomed. 7, 3527-3535 (2012).</li><li>Ivask, A., 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=Size-dependent+toxicity+of+silver+nanoparticles+to+bacteria,+yeast,+algae,+crustaceans+and+mammalian+cells+in+vitro.">Size-dependent toxicity of silver nanoparticles to bacteria, yeast, algae, crustaceans and mammalian cells in vitro.</a> PLoS One. 9 (7), e102108 (2014).</li><li>Kim, T. H., 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=Size-dependent+cellular+toxicity+of+silver+nanoparticles.">Size-dependent cellular toxicity of silver nanoparticles.</a> J Biomed Mater Res A. 100 (4), 1033-1043 (2012).</li><li>Raghupathi, K. R., Koodali, R. T., Manna, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-dependent+bacterial+growth+inhibition+and+mechanism+of+antibacterial+activity+of+zinc+oxide+nanoparticles.">Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles.</a> Langmuir. 27 (7), 4020-4028 (2011).</li><li>Plech, A., Kotaidis, V., Gresillon, S., Dahmen, C., von Plessen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+heating+and+melting+of+gold+nanoparticles+studied+by+time-resolved+x-ray+scattering.">Laser-induced heating and melting of gold nanoparticles studied by time-resolved x-ray scattering.</a> Phys Rev B. 70 (19), 195423 (2004).</li><li>Takami, A., Kurita, H., Koda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-Induced+Size+Reduction+of+Noble+Metal+Particles.">Laser-Induced Size Reduction of Noble Metal Particles.</a> J Phys Chem B. 103 (8), 1226-1232 (1999).</li><li>Kamat, P. V., Flumiani, M., Hartland, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picosecond+Dynamics+of+Silver+Nanoclusters.+Photoejection+of+Electrons+and+Fragmentation.">Picosecond Dynamics of Silver Nanoclusters. Photoejection of Electrons and Fragmentation.</a> J Phys Chem B. 102 (17), 3123-3128 (1998).</li><li>Yamada, K., Tokumoto, Y., Nagata, T., Mafune, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+laser-induced+size-reduction+of+gold+nanoparticles+as+studied+by+nanosecond+transient+absorption+spectroscopy.">Mechanism of laser-induced size-reduction of gold nanoparticles as studied by nanosecond transient absorption spectroscopy.</a> J Phys Chem B. 110 (24), 11751-11756 (2006).</li><li>Pecora, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Light+Scattering+Measurement+of+Nanometer+Particles+in+Liquids.">Dynamic Light Scattering Measurement of Nanometer Particles in Liquids.</a> J. Nanoparticle Res. 2 (2), 123-131 (2000).</li><li>Pyrz, W. D., Buttrey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+size+determination+using+TEM:+a+discussion+of+image+acquisition+and+analysis+for+the+novice+microscopist.">Particle size determination using TEM: a discussion of image acquisition and analysis for the novice microscopist.</a> Langmuir. 24 (20), 11350-11360 (2008).</li><li>Ratti, M., 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=Irradiation+with+visible+light+enhances+the+antibacterial+toxicity+of+silver+nanoparticles+produced+by+laser+ablation.">Irradiation with visible light enhances the antibacterial toxicity of silver nanoparticles produced by laser ablation.</a> Appl Phys A. 122 (4), 1-7 (2016).</li><li>Sajti, C. L., Sattari, R., Chichkov, B. N., Barcikowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gram+Scale+Synthesis+of+Pure+Ceramic+Nanoparticles+by+Laser+Ablation+in+Liquid.">Gram Scale Synthesis of Pure Ceramic Nanoparticles by Laser Ablation in Liquid.</a> J Phys Chem C. 114 (6), 2421-2427 (2010).</li><li>Streubel, R., Barcikowski, S., Gokce, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+multigram+nanoparticle+synthesis+by+high-power,+high-repetition-rate+ultrafast+laser+ablation+in+liquids.">Continuous multigram nanoparticle synthesis by high-power, high-repetition-rate ultrafast laser ablation in liquids.</a> Opt Lett. 41 (7), 1486-1489 (2016).</li><li>Streubel, R., Bendt, G., Gokce, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot-scale+synthesis+of+metal+nanoparticles+by+high-speed+pulsed+laser+ablation+in+liquids.">Pilot-scale synthesis of metal nanoparticles by high-speed pulsed laser ablation in liquids.</a> Nanotechnology. 27 (20), 205602 (2016).</li></ol>

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><tbody><tr><td>Nanosecond Nd:YAG laser</td><td>Ekspla</td><td>NL303</td><td></td></tr><tr><td>Motorized xy scanning stage</td><td>Standa</td><td>8MTF</td><td></td></tr><tr><td>UV-VIS spectrophotometer</td><td>Agilent</td><td>Cary 60</td><td></td></tr><tr><td>Dynamic light scattering unit</td><td>Malvern</td><td>Zetasizer ZS 90</td><td></td></tr><tr><td>Microbalance</td><td>Maktek</td><td>TM 400</td><td></td></tr><tr><td>Transmission electron microscope</td><td>Zeiss</td><td>EM 902</td><td></td></tr><tr><td>Silver foil target</td><td>Alfa Aesar</td><td>12127</td><td></td></tr><tr><td>250 mm focal length lens</td><td>Edmund Optics</td><td>69-624</td><td></td></tr><tr><td>Copper TEM grids</td><td>Pacific Grid-Tech</td><td>Cu-400LD</td><td>Lacey/thin film coated grid</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; MG1655</td><td>ATCC</td><td>47076</td><td></td></tr><tr><td>Bacto tryptone</td><td>BD Biosciences</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD Biosciences</td><td>212750</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP3581</td><td></td></tr><tr><td>Bacto agar</td><td>BD Biosciences</td><td>214010</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Fisher Scientific</td><td>BP166-100</td><td></td></tr><tr><td>Polyvinylpyrrolidone</td><td>Fisher Scientific</td><td>BP431-100</td><td></td></tr><tr><td>Stainless steel disc (for ablation stage)</td><td>Metal Remnants, Inc.</td><td>N/A</td><td>1.5 inch diameter, 16 gauge</td></tr><tr><td>Beaker</td><td>Fisher Scientific</td><td>02-540G</td><td></td></tr><tr><td>Magnetic stir bar</td><td>Fisher Scientific</td><td>14-513-57</td><td></td></tr><tr><td>Magnetic stir plate</td><td>Fisher Scientific</td><td>11-100-49SH</td><td></td></tr><tr><td>Laser energy and power meter</td><td>Coherent</td><td>1098579</td><td></td></tr><tr><td>Carbon tape</td><td>Shinto Chemitron Co. Ltd.</td><td>STR Tape</td><td></td></tr><tr><td>Sonicating water bath</td><td>Branson</td><td>1510</td><td></td></tr><tr><td>Air compressor</td><td>GMC</td><td>Syclone 3010</td><td>For drying ablation target</td></tr><tr><td>75 mm focal length lens</td><td>Edmund Optics</td><td>34-096</td><td>Focusing lens for post-irradiation</td></tr><tr><td>Quartz cuvette</td><td>Precision Cells Inc</td><td>21UV40</td><td>50 mm light path (for post-irradiation)</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td></td></tr><tr><td>Light microscope</td><td>Nikon</td><td>50i</td><td>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><td>CCD camera</td><td>AmScope</td><td>MT5000-CCD</td><td></td></tr><tr><td>Micrometer slide</td><td>Ted Pella</td><td>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...

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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 ...

production-metal-nanoparticles-pulsed-laser-ablation-liquids-tool-for

Research

JoVE Journal

Bioengineering

15.0K Views.  Rutgers University-Camden.  De antimicrobiële eigenschappen van metalen zoals koper en zilver zijn al eeuwen herkend. Dit protocol beschrijft gepulseerde laser ablatie in vloeistoffen, een methode voor het synthetiseren van metalen nanodeeltjes die het vermogen biedt om de eigenschappen van deze nanodeeltjes te verfijnen om hun antimicrobiële effecten te optimaliseren. 

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

A Method to Fabricate Disconnected Silver Nanostructures in 3D

The standard nanofabrication toolkit includes techniques primarily aimed at creating 2D patterns in dielectric media. Creating metal patterns on a submicron scale requires a combination of nanofabrication tools and several material processing steps. For example, steps to create planar metal structures using ultraviolet photolithography and electron-beam lithography can include sample exposure, sample development, metal deposition, and metal liftoff. To create 3D metal structures, the sequence is repeated multiple times. The complexity and difficulty of stacking and aligning multiple layers limits practical implementations of 3D metal structuring using standard nanofabrication tools. Femtosecond-laser direct-writing has emerged as a pre-eminent technique for 3D nanofabrication.1,2 Femtosecond lasers are frequently used to create 3D patterns in polymers and glasses.3-7 However, 3D metal direct-writing remains a challenge. Here, we describe a method to fabricate silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm. The method enables the fabrication of patterns not feasible using other techniques, such as 3D arrays of disconnected silver voxels.8 Disconnected 3D metal patterns are useful for metamaterials where unit cells are not in contact with each other,9 such as coupled metal dot10,11or coupled metal rod12,13 resonators. Potential applications include negative index metamaterials, invisibility cloaks, and perfect lenses.
In femtosecond-laser direct-writing, the laser wavelength is chosen such that photons are not linearly absorbed in the target medium. When the laser pulse duration is compressed to the femtosecond time scale and the radiation is tightly focused inside the target, the extremely high intensity induces nonlinear absorption. Multiple photons are absorbed simultaneously to cause electronic transitions that lead to material modification within the focused region. Using this approach, one can form structures in the bulk of a material rather than on its surface.
Most work on 3D direct metal writing has focused on creating self-supported metal structures.14-16 The method described here yields sub-micrometer silver structures that do not need to be self-supported because they are embedded inside a matrix. A doped polymer matrix is prepared using a mixture of silver nitrate (AgNO3), polyvinylpyrrolidone (PVP) and water (H2O). Samples are then patterned by irradiation with an 11-MHz femtosecond laser producing 50-fs pulses. During irradiation, photoreduction of silver ions is induced through nonlinear absorption, creating an aggregate of silver nanoparticles in the focal region. Using this approach we create silver patterns embedded in a doped PVP matrix. Adding 3D translation of the sample extends the patterning to three dimensions.

Femtosecond-laser direct-writing is frequently used to create three-dimensional (3D) patterns in polymers and glasses. However, patterning metals in 3D remains a challenge. We describe a method for fabricating silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm.

The standard nanofabrication toolkit includes  techniques primarily aimed at creating 2D patterns in dielectric media. Creating  metal patterns on a ...

Engineering

Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition

Nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas allows the deposition of metal oxides with tunable morphology, structure, density and stoichiometry by a proper control of the plasma plume expansion dynamics. Such versatility can be exploited to produce nanostructured films from compact and dense to nanoporous characterized by a hierarchical assembly of nano-sized clusters. In particular we describe the detailed methodology to fabricate two types of Al-doped ZnO (AZO) films as transparent electrodes in photovoltaic devices: 1) at low O2 pressure, compact films with electrical conductivity and optical transparency close to the state of the art transparent conducting oxides (TCO) can be deposited at room temperature, to be compatible with thermally sensitive materials such as polymers used in organic photovoltaics (OPVs); 2) highly light scattering hierarchical structures resembling a forest of nano-trees are produced at higher pressures. Such structures show high Haze factor (&gt;80%) and may be exploited to enhance the light trapping capability. The method here described for AZO films can be applied to other metal oxides relevant for technological applications such as TiO2, Al2O3, WO3 and Ag4O4.

We describe the experimental method to deposit nanostructured oxide thin films by nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas. By using this method Al-doped ZnO (AZO) films, from compact to hierarchically structured as nano-tree forests, can be deposited.

Nanosecond Pulsed  Laser Deposition (PLD) in the presence of a background gas allows the deposition  of metal oxides with tunable morphology, structure, ...

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Immunology and Infection

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Studying the Effects of Temperature on the Nucleation and Growth of Nanoparticles by Liquid-Cell Transmission Electron Microscopy

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron microscopy. In this paper, we describe how to prepare an in situ heating experiment for studying the effect of temperature on the formation of gold nanoparticles driven by radiolysis in water. The protocol of the experiment is fairly simple involving a special liquid cell with uniform heating capabilities up to 100 &#176;C, a liquid-cell TEM holder with flow capabilities and an integrated interface for controlling the temperature. We show that the nucleation and growth mechanisms of gold nanoparticles are drastically impacted by the temperature in liquid cell. Using STEM imaging and nanodiffraction, the evolution of the density, size, shape and atomic structure of the growing nanoparticles are revealed in real time. Automated image processing algorithms are exploited to extract useful quantitative data from video sequences, such as the nucleation and growth rates of nanoparticles. This approach provides new inputs for understanding the complex physico-chemical processes at play during the liquid-phase synthesis of nanomaterials.

Temperature control during liquid-phase electron microscopy experiments opens up new perspectives of studying the dynamic of nanoparticles in liquid environments mimicking their formation or application media. Using recently developed heating liquid cells, we directly observed the influence of temperature on the nucleation and growth processes of gold nanoparticles in water.

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron ...

Chemistry

Synthesizing Nanoparticles and Nanostructures via Laser Ablation

Ultrafast Laser-Ablated Nanoparticles and Nanostructures for Surface-Enhanced Raman Scattering-Based Sensing Applications

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields such as sensing, catalysis, and medicine. The exceptional feature of this technique is the formation of nanoparticles (colloids) and nanostructures (solids) in a single experiment with ultrashort laser pulses. We have been working on this technique for the past few years, investigating its potential using the surface-enhanced Raman scattering (SERS) technique in hazardous materials sensing applications. Ultrafast laser-ablated substrates (solids and colloids) could detect several analyte molecules at the trace levels/mixture form, including dyes, explosives, pesticides, and biomolecules. Here, we present some of the results achieved using the targets of Ag, Au, Ag-Au, and Si. We have optimized the nanostructures (NSs) and nanoparticles (NPs) obtained (in liquids and air) using different pulse durations, wavelengths, energies, pulse shapes, and writing geometries. Thus, various NSs and NPs were tested for their efficiency in sensing numerous analyte molecules using a simple, portable Raman spectrometer. This methodology, once optimized, paves the way for on-field sensing applications. We discuss the protocols in (a) synthesizing the NPs/NSs via laser ablation, (b) characterization of NPs/NSs, and (c) their utilization in the SERS-based sensing studies.

Author Spotlight: Advancements and Applications in Nanoparticle Synthesis Through Laser Ablation in Liquids 

Ultrafast laser ablation in liquid is a precise and versatile technique for synthesizing nanomaterials (nanoparticles [NPs] and nanostructures [NSs]) in liquid/air environments. The laser-ablated nanomaterials can be functionalized with Raman-active molecules to enhance the Raman signal of analytes placed on or near the NSs/NPs.

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

 Zinc Oxide Nanoparticles Synthesis and Analysis of its Antibacterial Effects

Preparation of Zinc Oxide Nanoparticles and the Evaluation of their Antibacterial Effects

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant bacterial strains in hospitals has been attributed to the extensive and varied use of antibiotics, further exacerbating the problem of antibiotic resistance. Metal nanomaterials have been widely studied as an alternative solution for eradicating antibiotic-resistant bacterial cells. Metallic nanoparticles attack bacterial cells through various mechanisms, such as the release of antibacterial ions, generation of reactive oxygen species, or physical disruption, against which bacteria cannot develop resistance. Among the actively researched antimicrobial metal nanoparticles, zinc oxide nanoparticles, which are FDA-approved, are known for their biocompatibility and antibacterial properties. In this study, we focused on successfully developing a precipitation method for synthesizing zinc oxide nanoparticles, analyzing the properties of these nanoparticles, and conducting antimicrobial tests. Zinc oxide nanoparticles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet/visible spectroscopy, and X-ray diffraction (XRD). Antibacterial tests were conducted using the broth microdilution test with the multidrug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. This study demonstrated the potential of zinc oxide nanoparticles in inhibiting the proliferation of antibiotic-resistant bacteria.

Author Spotlight: Exploring the Antibacterial Effects of Zinc Oxide Nanoparticles in Overcoming Antibiotic Resistance

In this study, zinc oxide nanoparticles were synthesized using a precipitation method. The antibacterial effect of the synthesized particles was tested against multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa bacterial strains.

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant ...

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

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