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 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 ...

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

Research

JoVE Journal

Bioengineering

14.9K Views.  Rutgers University-Camden. 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.

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

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

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

Summary

Explore More Videos

Chapters in this video

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Viral Nanoparticles for In vivo Tumor Imaging

Harmonic Nanoparticles for Regenerative Research

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles