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

Summary

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.

Abstract

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.

Introduction

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 – PLAL) provides an environmentally friendly route for NP synthesis that satisfies all 12 of the Principles of Green Chemistry^1,2. In PLAL, a submerged metal target is irradiated by repeated laser pulses. As the laser ablates the target, a dense plum....

Protocol

1. Focusing the Nanosecond Laser and Measuring Fluence

1. 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-f....

Results

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 µg/mL. In assessing the antimicrobial toxicity o.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nanosecond Nd:YAG laser	Ekspla	NL303
Motorized xy scanning stage	Standa	8MTF
UV-VIS spectrophotometer	Agilent	Cary 60
Dynamic light scattering unit	Malvern	Zetasizer ZS 90
Microbalance	Maktek	TM 400
Transmission electron microscope	Zeiss	EM 902
Silver foil target	Alfa Aesar	12127
250 mm focal length lens	Edmund Optics	69-624
Copper TEM grids	Pacific Grid-Tech	Cu-400LD	Lacey/thin film coated grid
E. coli MG1655	ATCC	47076
Bacto tryptone	BD Biosciences	211705
Yeast extract	BD Biosciences	212750
Sodium chloride	Fisher Scientific	BP3581
Bacto agar	BD Biosciences	214010
Sodium dodecyl sulfate	Fisher Scientific	BP166-100
Polyvinylpyrrolidone	Fisher Scientific	BP431-100
Stainless steel disc (for ablation stage)	Metal Remnants, Inc.	N/A	1.5 inch diameter, 16 gauge
Beaker	Fisher Scientific	02-540G
Magnetic stir bar	Fisher Scientific	14-513-57
Magnetic stir plate	Fisher Scientific	11-100-49SH
Laser energy and power meter	Coherent	1098579
Carbon tape	Shinto Chemitron Co. Ltd.	STR Tape
Sonicating water bath	Branson	1510
Air compressor	GMC	Syclone 3010	For drying ablation target
75 mm focal length lens	Edmund Optics	34-096	Focusing lens for post-irradiation
Quartz cuvette	Precision Cells Inc	21UV40	50 mm light path (for post-irradiation)
Kanamycin	Fisher Scientific	BP906-5
Light microscope	Nikon	50i	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.
CCD camera	AmScope	MT5000-CCD
Micrometer slide	Ted Pella	2280-70