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 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.
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 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 'long' and 'ultra-short' 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 (λ = 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.
1. Focusing the Nanosecond Laser and Measuring Fluence
2. Synthesis of Silver Nanoparticles by Pulsed Laser-ablation in Liquid
3. Characterizing Metal Nanoparticles
4. Post-irradiation
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.
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 of silver NPs, 15 µg/mL strongly inhibits E. coli growth (Figure 3).
Figure 1: Apparatus configurations. (A) For the PLAL process, the Nd:YAG laser operating at a wavelength of 1,064 nm is focused through a 250 mm focal length lens to produce a spot size of 5.51 mm2 on the target stage. The spot size image is captured using a CCD camera coupled with an optical microscope. The ablation target is set on a porous stage with ten 0.65 cm diameter holes and six 0.50 cm diameter holes. An additional 3 holes are tapped for set screws which function as legs to support the stage above the stir bar. (B) For post-irradiation, the Nd:YAG laser output is set to 532 nm and focused through a 75 mm focal-length lens onto the center of a quartz cuvette containing NPs. Please click here to view a larger version of this figure.
Figure 2: Characterization of silver nanoparticles. (A) The UV-VIS spectrum of silver NPs shows a characteristic peak at the SPR wavelength (400 nm). (B) The size distribution of the silver NPs before post-irradiation was measured by TEM. The inset shows a representative TEM image of AgNPs (85,000X magnification, Scale bar = 100 µm). Please click here to view a larger version of this figure.
Figure 3. Antimicrobial effects of silver nanoparticles. E. coli cells were treated for 2 h with varying concentrations of silver NPs. Serial dilutions of cultures were plated on LB-agar to determine bacterial viability. Cells were treated with 30 µg/mL kanamycin as a positive control. Note that the cells not receiving AgNPs (-AgNP sample) were grown in the presence of 6 mM SDS to ensure that the surfactant did not independently result in toxicity. The figure is a composite of colonies on two plates from the same experiment and is a representative result (n = 5). Please click here to view a larger version of this figure.
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 process to yield the optimal fluence and liquid height over the target. Since the size of the nanoparticles can be adjusted by post-irradiation, the efficiency of particle production can be optimized through target mass-loss and optical density measurements.
In contrast to conventional chemical synthesis methods, laser ablation in liquids has the advantage of producing nanoparticles in either pure water or a surfactant solution. There are no precursor compounds to contaminate the cultures and act as interferents. Researchers can produce the nanoparticles in their laboratory in a short period of time for immediate use. The technique is highly reproducible and does not require specialized training or personnel. However, it is important to note the limitations of this method. First, the diversity in shapes of the nanoparticles that are produced by laser ablation in liquids can vary over a wide range. If one is required to target particular aspect ratios or other shape parameters, highly iterative processing would be required if it is possible at all. Perhaps the most significant limitation of this technique is that experiments which require large masses of nanoparticles will be difficult. Gram-scale synthesis can occur, but it is challenging and requires specialized laser equipment32,33,34.
It is important to note that many metal NPs are light-sensitive. Indeed, irradiation of silver nanoparticles with visible light results in increased antibacterial toxicity31. The enhanced efficacy is due to an increase in silver ion release from the NPs. Therefore, it is important to consider whether to perform the PLAL method and store the resulting NPs protected from light.
Lastly, the choice of surfactants and volume-excluders (e.g. SDS and PVP) to decrease NP size is critical when studying the antimicrobial potency of NPs. It is important to perform control experiments to ensure that the additives are not toxic on their own. For example, E. coli tolerates SDS at concentrations up to 10 mM; however, B. subtilis is much more sensitive31. Therefore, when working with B. subtilis, non-toxic concentrations of PVP (2 mM) can be added to the ablation liquid to obtain 25 nm particles.
Laser ablation in liquids along with post-irradiation can be used to produce nanoparticle distributions with a range of dispersities and sizes. This will facilitate studies with different bacteria, metals, and even alloys. The use of PLAL for nanoparticle synthesis provides a novel method for developing antimicrobial NPs to combat the ever-growing challenge of antibacterial resistance.
The authors have nothing to disclose.
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.
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 |