Pulsed Laser Based Synthesis of Metallic Nanoparticles and Their Possible Applications
Abstract
The pulsed laser ablation in liquid (PLAL) is a relatively new emerging technique in the field of nanoparticle synthesis. This technique has evolved as an alternative-novel method to successfully overcome the challenges of synthesizing pure nanoparticles using conventional wet chemical synthesis techniques. PLAL can prepare a vast variety of pure nanoparticles, using a robust experimental setup and simple-clean reaction. By tuning different laser and solvent parameters one can easily control the properties of synthesized nanoparticles. Surfactant-free silver (Ag) nanoparticles were synthesized by a nanosecond Nd-YAG laser (1064 nm wavelength) by ablating a pure Ag target immersed in a liquid medium (D.I. water). To evaluate the effect of different laser parameters on the productivity, size, and stability of silver nanoparticles, different diagnostic tools were used such as UV-Vis spectroscopy and scanning electron microscopy. It was observed that the concentration of silver nanoparticles significantly increases with increasing laser energy. However, a decrease in the nanoparticle concentration was observed when the laser energy was > 40 mJ. Moreover, the increase in the target ablation time increases the concentration of synthesized nanoparticles. Furthermore, the SEM analysis revealed that pure spherical silver nanoparticles with a diameter ranging from 30 to 100 nm were synthesized using PLAL.
Metallic nanoparticles are contemplated as the most promising nanomaterial with antibacterial properties. Among all the metal NPs, Ag NPs are the most effective against bacteria, viruses, and other micro-organisms. Silver was also used most widely in medical and consumer goods, such as household antiseptic sprays and medical device antimicrobial coatings. The synthesized Ag nanoparticles were used for the evaluation of antimicrobial susceptibility of Ag nanoparticles against pathogenic bacteria, Staphylococcus aureus and Escherichia coli. Silver nanoparticle as the perfect candidate was therefore used against both S. aureus and E. coli bacteria. However, it was determined that nanoparticles with a concentration lower than 100’s of microgram do not show significant susceptibility. Therefore, the concentration of the synthesized nanoparticles by pulsed laser ablation in liquid needs to be increased.
Chapter 1 Introduction
Mankind has repeatedly wished for the smallest and the greatest things. Such a vision created wonders such as the Eiffel Tower and the Pyramids. The quest for smaller on the other hand contributed to the growing interest in the field of nanotechnology. The term “Nanotechnology” was first coined by a Japanese scientist Taniguchi, in 1974 [1]. The US National Nanotechnology Initiative (NNI) in 2000 described nanotechnology and nanoscience as “Development at the atomic levels in the length scale of approximately 1-100 nanometer range, to provide a fundamental understanding of phenomena and materials at the nanoscale and to create and use structures, devices, and systems that have novel properties and functions”. According to the International Organization for Standardization (ISO), nanomaterials are defined as “material with any external dimension in the nanoscale or having the internal structure or surface structure in the nanoscale”. Whereas nanoparticles refer to spherical particles with all three dimensions in nanoscale. The term Nano, a Greek word meaning “dwarf”, refers to size reduced to 10-9 meter. That is one-billionth of a meter, approximately 100,000 times smaller than the diameter of a human hair.
Nanomaterials being the cornerstone of nanotechnology have fascinated mankind since ancient times, due to their brilliant colors that could be used to dye glass and fabrics. The most ancient use of colloidal silver and gold can be traced back to be in BC by Egyptian alchemists. Eventually, catching the Mesopotamian artisans’ attention, in the 9th century due to their ability to create glittering effects on the surface of pots. Which was caused by the homogeneously dispersed silver and copper nanoparticles in the outer layer of the glaze. By the 10th century AD, they were extensively used to color cathedrals glass throughout Europe.
However, the scientific era of nanomaterials began in 1875 when Michal Faraday synthesized the first colloidal solution of gold nanoparticles called the “activated gold” [2]. He concluded, “The introduction into a ray of separate particles…the gold is reduced in exceedingly fine particles which becoming diffused, produce a beautiful fluid…the various preparations of gold, whether ruby, green, violet, or blue…consist of that substance in a metallic divided state known phenomena appeared to indicate that a mere variation in the size of its particles gave rise to a variety of resultant colors”. He recognized that light was scattered due to its interaction with small metallic particles. Thus, Faraday is seen as the first researcher to enter the realm of nanotechnology and nanoscience. On 29th December 1959, in his visionary lecture which is considered as the origin of the nanotechnology paradigm, Richard Feynman stated: “There is plenty of room at the bottom” indicating the endless hidden possibilities in the smallest of dimensions.
Nanomaterials have unique characteristics and properties which are completely different from their bulk, macroscopic counterparts. These unique properties arise due to their small size and large surface to volume ratio. This results in the dominance of surface atoms over the internal atoms.
- Optical properties
Nanomaterials have unusual optical properties that depend on the size, composition, and shape of the nanomaterial. By reducing the dimension to the nanoscale, energies of the valance band and the conduction band are prominently affected. Consequently, affecting the optical absorption and emission that is completely dependent on the transitions between these two states. Therefore, nanomaterial exhibit different colors from their bulk material, depending on the size. In general, it affects the photocatalysis, photoconductivity, and photoemission of the particles.
In the case of noble nanomaterials such as silver gold and copper, the brilliant bright color is the result of collective oscillation of free electrons in the conduction band, known as surface plasmonic resonance (SPR). Fig 1.1. [3], shows how varying the size and shape of colloidal gold affects its color. Since the frequency of the SPR is in the visible range of these noble nanomaterials, they are used in several optical applications, such as biosensors.
- Thermal properties
Unlike bulk material, the thermal properties of nanomaterial depend on the surface properties and the interatomic spacing of the particles. That is affected by the size reduction from micro to nano-scale. Therefore, the melting point of nanomaterials is less than that of the bulk material. Interestingly, as the size of the nanomaterials is further reduced, their melting point further decreases. For example, as illustrated in Fig 1.2. [4], even though the melting point of bulk gold is 1064° C, it decreases to 1000° C when the size of the nanoparticles is 10 nm. It is further reduced to 900° C and 700° C when the size is decreased to 4 nm and 2 nm, respectively.
- Catalytic properties
Catalysis is a process by which the rate of reaction of a chemical process is accelerated by adding a substrate called a catalyst. Only a small amount of the catalyst is enough to alter the rate of reaction as it is not consumed during the reaction.
Many materials when in bulk state, exhibit no catalytic properties, such as gold which is inert in its bulk state. However, surface-free energies of the particles significantly increase when the size is reduced, due to the increase in surface-to-volume ratio. As a result, nanomaterials present a significantly greater chemical reactivity that is dependent on the size of the synthesized particle. Such as, in the reduction of 4-nitrophenol by NaBH4, 3.4 nm gold particles are more catalytically reactive than 8.2 nm [5].
1.1 Noble Metal Nanoparticles
Among the vast range of nanomaterials, noble metal nanoparticles have attracted a lot of attention due to their peculiar physical, optical, chemical, and biological properties. This has allowed researchers to develop devices for diverse applications. For instance, for biotechnological and biochemical applications due to their sharp plasmonic peaks being in the visible region. They are used for drug delivery, treatment of cancerous tumors, and skin diseases, as disinfectants, as chemical and biological sensors, and as catalysts. Due to their bright properties and Raman scattering SERS in the visible range, it is possible to enhance Raman scattering of various molecules. They are also used to develop new and improved optical devices with high data storage capabilities and fast data processing. They are used to improve the characteristics of solar panels and photodetectors. Additionally, to further utilize noble nanoparticles, numerous other areas are being actively researched.
1.2 Nanoparticle Synthesis Techniques
All fields of technology cover current and possible applications of nanomaterials and nanoparticles. It has been predicted that the field of material synthesis can be revolutionized by increased control of the material behavior combined with the need for fewer resources. Synthesis techniques for nanomaterials can be approached from two different directions. One route is to use small nano factories that can assemble individual atoms to build materials, which is also known as the bottom-up approach. In this method, the physical forces operating at the nanoscale are exploited to combine basic units into larger, stable structures, as shown in Fig 1.3. The bottom-up approach includes green methods, wet-chemistry techniques such as the Turkevich method, and several other techniques. The second approach for material synthesis is known as the top-down approach. In this approach, the starting material is macroscopic bulk materials and they are carved and broken down to produce nanomaterials. The synthesis techniques which can be categorized as the top-down approach are pulsed laser deposition, pulsed laser ablation in liquids, mechanical milling, sputtering, etc.
1.3 Laser based synthesis techniques
1.3.1 Pulsed laser deposition (PLD)
Pulsed laser deposition is a laser based vapor deposition technique which involves ablation of a target material inside an ultra-high vacuum or in the presence of diluted background gas, as shown in Fig 1.4. [6] By focusing a high-density laser pulse on the target surface, a perpendicularly propagating, direction plasma plume is produced. As it cools down small clusters are formed that deposit on a substrate where they condense to form a thin film. Over the years PLD has attracted a lot of attention to produce a variety of nanoparticles and thin-films by simply changing the gas composition and pressure, target material, and laser parameters, such as pulse duration, wavelength, and laser fluence. It is considered as a perfect technique to create a thin film as no chemical reagents are used, it has a relatively lower cost than other techniques and various thin films can be created on several substrates, with high crystalline quality. Such as nitrides, metallic multilayers, and many single or polycrystalline films [7], [8]. Those are used in different applications, such as chemical and biological sensors, electrodes, and semiconductor devices.
Figure 1.4. A schematic diagram of the pulsed laser deposition setup [6].
It is possible to remove the nanoparticles deposited on the substrate and form colloidal solution but a significant number of nanoparticles are damaged; losing their colloidal dispersity and size uniformity, in the process. Thus it is not well suited to synthesize colloidal nanoparticles, which limits the possible applications of the synthesized nanoparticles by PLD.
1.3.2 Pulsed laser ablation in liquid (PLAL)
Pulsed laser ablation in a liquid is a relatively new technique that can directly synthesize pure; ligand-free, and well-dispersed colloidal nanoparticle. Ever since it was first used by Fojkit et al. in 1993 [9], it has been gaining immense attention because of which several reviews are available [10]–[13]. This method uses a simple and robust experimental set-up, as illustrated in Fig 1.5. To produce nanoparticles it employs a hybrid mechanism, consisting of both top-down and bottom-up methods. A high-density laser pulse induces the matter breakdown of the solid target immersed in the liquid medium. Producing a hot-dense, expanding plasma plume that is rapidly quenched by the surrounding liquid. Resulting in the fast condensation of the ablated material and the collisions of the plume and liquid species at the interface between plasma and liquid. Subsequently, initiating nucleation followed by slow growth that forms pure crystallized nanoparticles. By simply varying the target material and the liquid medium it is possible to produce a variety of pure nanoparticles; such as metal, noble metals, oxides, and core-shell structures, etc.
Figure 1.5. Schematic diagrams of the experimental setup and PLAL basic mechanism.
Many researchers demonstrated how by changing different laser parameters it is possible to produce diverse nanoparticles. Such as Tsuji et al. reported the influence of focusing conditions and different laser parameters, such as wavelength; 1064, 532, 355 nm, and fluence, on the ablation efficiency of silver and copper targets [14]. They found that the relation of ablation efficiency and wavelength changed with the laser fluence. It was possible to achieve high ablation efficiency at low fluence and shorter wavelength or high fluence and longer wavelengths. They also reported that the mean particle size decreased by decreasing the wavelength. Due to the fragmentation of particles by self-absorption of the incoming laser pulse. Zheng et al. also reported that the size and shape of the nanoparticles can be controlled by laser wavelength and power [15].
Moreover, the surrounding liquid medium that confines and cools the plasma and in some cases providing reactive species, affects the surface charge of the nuclei and controls the size and the aggregation of nanoparticles. Mafune et al. synthesized silver nanoparticles in water and Sodium Dissolve Sulfate (SDS) by using a high number of laser pulses (50000) and 532 nm laser wavelength [16]. They concluded that by increasing the concentration of SDS, the size distribution of NPs shifted to a smaller size. Tarasenko et al. prepared silver nanoparticles in acetone to study the effect of laser irradiation at different laser wavelengths; 532, 266, 400, and 800 nm [17]. Their experiment led to surface modification of silver and produced spherical NPs. Zamiri et al. synthesized silver nanoparticles in a natural polymer which improved the stability of the nanoparticle [18].
Furthermore, it is possible to reshape and resize the synthesized nanoparticles by using laser fragmentation and melting techniques. Tsuji et al. demonstrated that it is possible to synthesize submicron-sized spherical gold particles while using a NaCl stabilizer by laser-induced melting in liquid [19]. Likewise, Pyatenko et al. reported the mechanism of pulse laser interaction with colloidal nanoparticle to control the laser processing of different nanomaterials [20]. They showed the particle heating-melting and evaporation model which can also be applied to other complicated processes that arise while laser irradiation of material, such as particle-size reduction and spherical growth.
Extensive work done in the field of PLAL in the last decade has proved that by using a simple single-step process it is possible to fabricate a variety of colloidal solutions, without using any reagents thus no by-products are created. But still, there are many reservations regarding batch production, industrialization, and cost-effectiveness of laser based nanoparticle synthesis techniques.
1.4 The economic aspect of pulsed laser ablation in liquid
As discussed above, wet-chemical reduction and pulsed laser ablation in liquid technique both are capable enough to synthesize nanoparticles and both methods also possess similar physicochemical properties of nanoparticles. Though these two techniques are quite similar in delivering the final product, they are quite different when the overall cost of the synthesis is evaluated. The overall manufacturing cost can be distributed into four major contributors, labor cost, investment, material cost, and energy cost. The detailed composition of manufacturing costs including labor, energy, material, and investment costs are exemplarily shown in Fig 1.6. [21] for a nanoparticle mass concentration of 100 mg/L and a batch size of 100 mg corresponding to a liquid volume of 1 L. Fig 1.6. (a), depicts a higher labor cost for wet-chemistry is than that of pulsed laser ablation. The high labor cost in the case of wet- chemistry can be attributed to the extra number of steps involved in the experiment. To synthesize 1 gram of nanoparticles, steps in wet chemistry include heating, the addition of reagents, cooling-down, and centrifugation. After which supernatant needs to be removed and the obtained nanoparticles are re-dispersed. Whereas, in the case of pulsed laser ablation it is possible to synthesize 1 gram of pure colloidal nanoparticles in a single step process. Further, the running cost of the source materials for expensive metals, such as Au, Ag, and Pt, contributes significantly to the overall cost. As seen in Fig 1.6. (b), the material cost in the case of laser ablation based technique is 49% less as compared to the wet-chemistry technique. The reason is evident, as laser based technique only used a small piece of a metal target whereas metal precursors are used in wet-chemistry. The manufacturing cost of a metal target is solely that of actual metal cost whereas synthesis of the metal precursor is more resource consuming resulting in higher cost of metal precursors.
Figure 1.6. The detailed composition of manufacturing cost; (a) labor, (b) material, (c) investment, and (d) energy cost per gram for chemical and laser synthesized gold nanoparticles [21].
Other significant costs that are added to be the total costs of synthesizing nanoparticles arise from the experimental equipment used during synthesis. Laser equipment is 30 % more expensive than the equipment required for wet chemical synthesis. While the absolute investment costs for the chemical synthesis are therefore lower than for the laser-based method, the relative investment costs per gram of colloidal gold nanoparticles are 61% higher, as shown in Fig 1.6. (c) due to high synthesis time, including centrifugation, in case of wet-chemistry as compared to the time required in the single-step synthesis technique of laser ablation. Fig 1.6. (d), compares the energy cost calculated from the energy consumption of the required equipment. The energy costs for the wet chemical gold nanoparticle and laser ablation synthesis of gold nanoparticles 1 g are US$4.7 and US$3.7 respectively. The low average energy consumption of both methods corresponds to a marginal 1% share of the overall cost of synthesis.
Chapter 2 : Pulsed laser ablation in liquid
Over the last two decades, pulsed laser ablation has evolved into a thriving field of research due to its impact on all areas of technology. The basic mechanism of PLAL is based on laser-matter-interaction. In which the amount of material removal depends on the target’s properties, liquid medium, and laser parameters such as, pulse duration, wavelength, laser fluence, and inter-pulse distance. To gain a better understanding of the fundamental mechanism involved in the process of synthesizing nanoparticles it is instructive to describe the temporal evolution of the ablation processes that take place when a single pulse hits the target material.
2.1 Temporal evolution of a single pulse during the laser ablation process
The process of laser ablation starts with the transfer of energy of the incoming photon of the laser pulse, to the electrons and the lattice of the target material. Some of this energy is reflected, depending on the material’s properties and the incoming laser wavelength. While the majority of energy deposited on the material causes heating to critical temperature and photoionization that leads to material removal as ionized vapors. These ionized vapors containing electrons and ions expand into a dense plasma plume that is constantly and instantaneously transferring its energy to the liquid at the point of solid-liquid interference. Subsequently creating a vapor layer that expands in the liquid and confines the plasma plume, causing the plume to cool down and form aerosol particles. However, several physicochemical mechanisms that occur with the progress of temporal sequence during laser ablation are discussed as follows
2.1.1 For t < 0 s: laser penetration in liquid
As illustrated in Fig 2.1. (a) the laser pulse penetrates the liquid medium before reaching the target material. Laser-liquid interaction has to be avoided to deliver maximum energy of the laser pulse onto the target. Or else due to liquid breakdown the laser energy reaching the target is significantly decreased, which results in decreased ablation efficiency of the process. Therefore, careful selection of the liquid medium is essential for optimum ablation by pulsed laser ablation in liquid. For this reason in the case of water mostly 1064 nm and 532 nm wavelengths are favored since their absorption is negligible, as shown in Fig 2.1. (b) [22].
2.1.2 For t = 0 to t = 10-9 s: material detachment
As illustrated in Fig 2.2. at t = 0 the laser pulse interacts with the target material till the pulse duration (tpulse) of few nanoseconds. At the point of the laser-matter interface due to the high density of photons, several processes take place, depending on the laser pulse intensity and the material’s skin depth.
Laser induced heating and melting
In the case of nanosecond laser, due to the linear and non-linear absorption of the high density of photons, electron-lattice thermalization takes place. Leading to a rise in the target’s temperature and activation of processes like melting or evaporation. The heat distribution due to the energy that is propagated through the material is governed by the heat conduction equation:
| [1] |
Where is the density, is the specific heat, is the thermal conductivity, and is the temperature. The second term on the right side of the equation represents the laser energy absorbed by the material. Where represent, depth at which energy is absorbed, surface reflectivity, laser irradiance, and absorption coefficient, respectively.
Explosive boiling
When the laser fluence is equivalent to or higher than the fluence threshold, material detachment takes place at the surface of the target. The fluence threshold is defined as the minimum fluence required to ablate a material. Such material detachment can be described by exploiting classical thermodynamics. The target material is superheated to its thermodynamically critical temperature, causing the surface to undergo rapid phase transition by decomposing the superheated liquid is into a mixture of vapors and liquid droplets. Experimental results are present for the well-defined fluence threshold that starts the process of droplet ejection and increases the rate of ablation. These results provide evidence of the transition from normal vaporization to phase explosion during laser ablation [14], [23].
Evaporation
The decomposed material containing highly ionized species moves away from the surface and forms an initial plasma plume, (as shown in Fig 2.3.) at an extremely high temperature and pressure. The rate of vaporization is determined by the surface temperature and is given by the equation:
| [2] |
Where represents the number of atoms per unit volume, and in the subscript represent liquid and vapor, respectively. is the latent heat of vaporization, is the temperature of the vapor, is the atomic mass, is the Boltzmann constant and is the probability of the vapor atom being reabsorbed by the liquid surface. Overall the evaporation rate from a liquid surface is represented by the first term, whereas the condensation rate of the molecules back to the liquid surface is represented by the second term.
Figure 2.3. (a) The detachment of ablated material, and (b) formation of the hot dense plasma plume.
Plasma formation
Most of the irradiating laser energy is used to heat the target surface to a very high temperature. Consequently, ablating the surface target and reducing the energy transfer to the interior of the target material. As shown in Fig 2.3. (a) the ablated material consisting of highly ionized ions and electrons expands into non-equilibrium plasma plume. Moreover, the recoil pressure of the ablated material generates a shockwave that propagates in the target and the liquid medium with supersonic velocity. Subsequently, heating the liquid and target which further promotes material detachment from the target surface. Fig 2.3. (b) shows the fully formed plasma plume at about 10-9 seconds. With high temperature and pressure in the order of 103 K and 1010-109 Pa, respectively, and an ion density of 1016-1018 ions/cm3 [24], [25].
2.1.3 For t = 10-9-10-7 s: plasma confinement
Solid exfoliation
Thermal expansion and stress in the target material are caused by the fast heating and cooling of the target due to the fast heat exchange with the surrounding liquid. Resulting in the removal of melted droplets and solid fragments that are large in size and irregular in shape. The presence of these exfoliated materials in the plasma prevents it from reaching thermodynamic equilibrium. This kind of removal is prominent in brittle materials such as silicon, glass, and other single-crystal materials.
Plasma shielding
A single laser pulse interacts with the target material until the pulse duration () of a few nanoseconds. During this time there is a temporal and spatial overlap of the laser pulse with the hot dense plasma plume which is depicted in Fig 2.4. The plasma absorbs the energy of the incoming laser pulse and increases its temperature and lifetime. Subsequently, reducing the amount of laser energy directly delivered to the target material.
Figure 2.4. Laser induced plasma shielding effect.
Formation of the vapor layer
Due to the low compressibility of the liquid medium, the plasma is confined by the surrounding liquid buffer that slows down its expansion. At the point of plasma-liquid interference, fast heat exchange takes place due to the high heat capacity of the liquid medium. Consequently, the liquid undergoes ionization and pyrolysis as it is heated up to the temperature of the plasma plume; 103 K. Instigating the formation of a thin vapor layer that surrounds and confines the expanding plasma plume, as illustrated in Fig 2.5.
Figure 2.5. Formation of thin vapor layer.
2.1.4 For t = 10-7-10-4 s: expansion of the vapor layer and quenching of plasma plume
Formation of the cavitation bubble
The high energy release of the plasma to the surrounding liquid expands the vapor layer in all directions inside the liquid medium. In a short timescale of 10-7-10-6 s a spherical cavitation bubble is created, as illustrated in Fig 2.6.
Figure 2.6. Plasma quenching and formation of seeds.
Nucleation
Nucleation is the first process in the formation of nanoparticles through self-assembly of the ablated species. Due to the supersaturation state of the hot plasma specie and the confinement of the liquid medium ionic seeds of a few atoms are formed that are portrayed in Fig 2.6. By the end of the plasma phase at about 10-7 s, these seeds diffuse into the cavitation bubble and interact with its molecules. Subsequently, the rate of collision is immensely increased due to the saturation of plasma and liquid species inside the cavitation bubble. Causing the seeds and ions to rapidly combine and grow into crystal particles.
Expansion and collapse of the cavitation bubble
The cavitation bubble due to its high temperature and pressure expands at supersonic speed up to the maximum radius in the order of a few millimeters. That is estimated through fast shadowgraph technique and the lifetime of the cavitation bubble strongly depends on the laser pulse energy, laser wavelength, pulse duration, and the focusing conditions. During the expansion highly energetic ions and electrons of the extinguishing plasma diffuse into the cavitation bubble and interact with its ionized molecules. With the increase in the rate of the collision, plasma and liquid species combine to form nano-crystals. To reduce their surface area and surface charge these nano-crystals further grown and agglomerate to form nanoparticles, as depicted in Fig 2.7. (a). Hypothetically, this occurs in a short period of 10-6 to 10-4 s. Consequently, during the expansion of the cavitation bubble the concentration of the plasma and liquid species rapidly decreases while the concentration of the nanoparticles is maximized. Fig 2.7. (b) illustrates the expansion and the existence of nanoparticles inside the cavitation bubble. Furthermore, during the expansion, the internal temperature and pressure are constantly decreasing, until dropping to a value lower than that of the surrounding liquid. As a result, surrounding compresses the cavitation bubble with the high concentration of nanoparticles occupying most of its volume. The compressed bubble is unable to drag the nanoparticles along with it thus it passes over the nanoparticles clouds and hence the nanoparticles are released into the liquid medium. As the surface tension of the bubble breaks due to the continuous compression, the cavitation bubble collapses while emitting a shockwave.
2.1.5 For t = 10-4 s: nanoparticle dispersion
The shockwave generated at the collapse of the cavitation bubble disperses the synthesized nanoparticles throughout the liquid medium. As the system establishes a steady physical and chemical state, NPs no longer undergo any further modification. If the dispersed nanoparticles are not stable then they aggregate and form precipitation. Additionally, depending on the laser wavelength the dispersed nanoparticles in front of the target interact and shield the second laser pulse from reaching the target. As a result, nanoparticles undergo modification and breakdown.
2.2 Effect of laser parameters
Pulse laser ablation in liquid consists of a complex physicochemical mechanism that controls the composition, morphology, and productivity of the synthesized nanoparticles. Under the same experimental conditions just by changing a single laser parameter, the synthesized nanoparticles are immensely affected. The parameters with the most significant influence on the productivity of the synthesized nanoparticles are discussed as follows:
2.2.1 Laser wavelength
The laser wavelength is one of the most prominent parameter in PLAL as the ablation rate of the target material is strongly dependent on the energy provided by a single photon. As illustrated in Fig 2.8. [26], shorter wavelengths have higher productivity due to their larger absorption coefficient. For instance, UV is effectively absorbed by the metal targets due to high photon energy. Uniform absorption of the photons by the interband transition induces a regular ablation of the irradiated spot. Therefore, a higher amount of material is ablated and the productivity of the nanoparticles is increased, as can be seen in Fig 2.8. (a). Whereas in the case of longer wavelengths such as near infra-red, the low energy photons are preferentially absorbed by the defects and the impurities of the target material. This absorption generates irregular and rugged ablation of the irradiated spot, as a result, the removal rate of material and the productivity of the nanoparticles is lower. Furthermore, at shorter wavelength multiphoton absorption and photoionization process are favored thus more reactive plasma species are formed which increases the rate of the chemical reaction between the plasma and liquid species. Hence have higher productivity by UV wavelength than by near infra-red wavelength.
However, as shown in Fig 2.8. (b), the overlap of the synthesized nanoparticles and laser wavelength decreases the productivity of the nanoparticles. Since the laser pulse is absorbed by the nanoparticles, chemical and structural changes are induced and the overall energy reaching the target for the ablation process is significantly reduced. This self-absorption process is dominant at shorter wavelengths, while it can be avoided at longer wavelengths due to their small absorption coefficient. Therefore, under ordinary experimental conditions, longer wavelengths are more preferred than the shorter wavelengths. Since they have higher comparable productivity when the colloidal concentration is high, as can be seen in Fig 2.8 (c).
2.2.2 Laser fluence
The second prominent parameter that significantly affects the productivity of the nanoparticles is laser fluence. Defined as the energy delivered per unit area, the fluence is dependent on the energy and the spot size of the laser pulse. Fig 2.9 illustrates the typical effect of varying laser fluence on the productivity of the nanoparticles [26].
The laser pulse energy determines the number of photons per pulse that irradiates the target material. The amount of energy delivered by the laser pulse is directly proportional to the amount of material ablated. Therefore, the productivity of the nanoparticles linearly increases with the increase in the laser energy, as seen in Fig 2.9. (a). Additionally, a high amount of ablated material implies a higher concentration of plasma specie thus there is an increase in the average size and the size distribution of the synthesized nanoparticles. In detail, with an increase in the laser energy various mechanisms of material detachment; fragmentation, explosive boiling, and vaporization are simultaneously induced. However, this tread is only applicable until the optimum fluence is achieved. Further increase of the laser energy causes stagnating (shown in Fig 2.9. (c)) or decrease in the productivity of synthesized nanoparticles due to the self-focusing and filamentation effect in the liquid medium. That results in the formation of a secondary plasma at the air-liquid interface, which absorbs most of the laser energy and significantly reduces the amount reaching the target material.
Generally, the high laser fluence is achieved by increasing the laser energy or by decreasing the spot size of the laser pulse. A larger amount of energy is delivered per unit area when the spot size is reduced hence the amount of ablated material is considerably increased, which is illustrated in Fig 2.9. (b). However, if the spot size becomes too small the productivity drops as the penetration depth of the laser pulse into the matter is dependent on the materials’ absorption properties. As reported by Shugaev et al. with smaller spot size the laser pulse interaction with the defects of the materials is reduced as a result of which the material has a higher ablation threshold [27].
2.2.3 Inter-pulse distance
Inter-pulse distance is defined as the distance between the spots where two successive pulses irradiate the target material. Fig 2.10. portrays the effect of short and appropriate inter-pulse distance on the productivity of nanoparticles [26]. With frequency above the frequency threshold of kHz, the lifetime of the cavitation bubble which is in the order of 10-4 s exceeds the inter-pulse delay that is necessary to avoid cavitation bubble shielding phenomena. That is, with a short inter-pulse distance laser pulse and the cavitation bubble generated by the previous pulse interact, as illustrated in Fig 2.10. (a). Resulting in the scattering of laser light due to the discontinuity of the refractive index of the cavitation bubble. Subsequently, the energy reaching the target is reduced hence productivity of the synthesized nanoparticles significantly drops. Therefore, it is of utmost importance to have a higher repetition rate of the laser pulse or have different techniques to spatially and temporally bypass the cavitation bubble to have higher nanoparticle productivity, as shown is in Fig 2.10. (b). Additionally, for longer pulses with a significant impact on the thermalization of the ablated material the productivity is maximized at an optimum inter-pulse distance.
Chapter 3 : Experimental methods
This particular chapter is divided into two sections; the first part contains a detailed description of the materials used, and the experimental procedure followed for nanoparticle synthesis which includes laser ablation and the characterization of nanoparticles. The second part contains the experimental proceedings for evaluating the antibacterial activity of the synthesized nanoparticles.
3.1 Materials and experimental methods
Target
Bulk silver metal target was used to conduct all the experiments for this thesis, to synthesize nanoparticles using the PLAL method. The silver metal was favored due to the bright color change that occurs when synthesizing silver nanoparticles that conveniently and immediately confirms the success of the experiment. The pure silver plate (99.99%) was polished, cleaned using an ultrasonic bath for 20 minutes, and then carefully washed with ultra-pure deionized water. The same silver plate was used to carry out all the experiments thus before each experiment it was thoroughly rinsed with deionized water.
Solvent
To form pure, ligand-free nanoparticle it is of utmost importance to use highly pure water. Generally, water is a mixture of ions, such as calcium sodium, chlorides, etc. which can interfere with the experiment by causing contamination. Therefore deionized water; water with all of its ions removed was used to conduct all the experiments. The DI water was prepared in the university lab by the ion exchange process. For each experiment, 10 mL of DI water was used to immerse the target material in a glass beaker. After each experiment, the glass beaker was washed with DI water to remove the residue of the previously synthesized nanoparticles.
Pulsed laser ablation in liquid
Fig 3.1. shows the typical experimental set-up of pulsed laser ablation in a liquid of a metal target at room temperature and pressure. An Nd-YAG laser with a pulse duration of 6 ns and a repetition rate of 10 Hz was used for the ablation process. All the experiments were conducted using a laser wavelength of 1064 nm, to prevent liquid breakdown. The laser pulse was redirected vertically using a mirror inclined at 45° and focused using a double convex lens of 70 cm focal length to irradiate the metal target, with a laser spot diameter of 1 mm. The defocusing condition was used instead of fully focused one to increase productivity. The laser energy and ablation time were varied from 10 mJ to 50 mJ, and 10 min to 30 min, respectively. During the experiment, the glass beaker containing the pure silver target was mounted on a rotating platform of a D.C. motor which was then fixed on an XY-translation stage to provide continuous rotational and translational motion. Thus, ensuring that uniform ablation of the target takes place without aging a single spot.
UV-visible spectroscopy
UV-visible absorption spectroscopy is one of the most popular characterization technique that is used to get a quantitative and qualitative analysis of the optical properties of the synthesized nanoparticles. Using a relatively simple, fast, and inexpensive method instant results are obtained. The method consists of measuring the intensity of the absorbed and transmitted light at various wavelengths through the sample. Fig 3.2. [28], shows the simple set-up of the basic components of a double beam UV-vis spectrometer, consisting of a light source, monochrometer, beam splitter, sample compartment, and a detector.
Generally in a UV-vis spectrometer, for the light source, a combination of two lamps is used as it is difficult to maintain a wide range of wavelengths with high and uniform brightness distribution, which is required for the spectrometer. A deuterium lamp is used for the UV spectrum and a tungsten lamp is used for the visible spectrum, ranging from 190 nm to 400 nm and 350 nm to 2500 nm, respectively. The monochrometer, an assembly of an entrance slit, a prism, and an exit slit, is used to disperse polychromatic light from the light source into individual wavelengths. Which passes through the beam splitter that splits the incoming rays of light into two equal beams that enter the sample compartment containing the reference and sample cell. The reference cell contains only the pure solvent used in the experiment, whereas the sample cell contains the colloidal solution that needs to be tested. The light transmitted through the sample compartment is collected and converted by the detector into electronic signals, which are processed to give a quantitated graph of the intensity of the absorbed light.
For the characterization in this thesis, Lambda 950 UV-visible spectrometer producing light in the range of 240 nm to 1100 nm was used to obtain the absorption spectra of the synthesized colloidal nanoparticles at different laser energy and ablation time. Furthermore, various spectra were obtained over irregular intervals to check the stability of synthesized nanoparticles which were kept in the same conditions; at room-temperature without any interaction with direct or diffused light. All spectra were obtained at room temperature by measuring the colloidal solution in a quartz cuvette with the optical path of 1 cm.
Scanning electron microscopy
Scanning electron microscopy (SEM) provides high-resolution and clear images of the nanoparticles by scanning the surface of the sample with a highly focused electron beam. The interaction of electron and the sample produces various signals that are analyzed to obtain information about the size and shape of the nanoparticles. Furthermore, it is combined with energy-dispersive X-ray spectroscopy (EDX) to give an elemental analysis of the sample. Fig 3.3. [29], shows the schematic diagram of the scanning electron microscope which includes an electron gun, condenser lens, detectors, and a sample stage inside a vacuum chamber.
Figure 3.3. Schematic diagram of the scanning electron microscope [29].
In principle due to the short wavelength of electrons; 1.23 nm, they are used in SEM instead of photons to form images. The electrons are emitted by heating a thin tungsten or lanthanum hexaboride cathode. These electrons are attracted by the metal anode that compresses them into a collimated beam. The beam of electrons is further accelerated through the condenser system; made of electromagnetic lenses to adjust the width and the intensity of the beam reaching the sample target, and the objective lens to focus the incoming electron beam onto the sample inside a vacuum chamber. As the high energy electron beam interacts with the sample several phenomena take places, such as absorption, the reflection of primary electrons, emission of secondary electrons, and emission of characteristic X-rays. The secondary electrons and the reflected primary electrons, also called the backscattered electrons are readily collected by a detector with some applied bias. The detector converts these electrons into electrical signals that are used to produce an image of the sample. Additionally, the emitted X-rays are collected by an EDX detector to obtain an elemental analysis of the sample.
The image for this thesis was obtained by using TESCAN-MAIA3 ultra-high resolution scanning electron microscope. Additionally, it was equipped with EDX for the elemental analysis of the sample. The obtained image and the X-ray spectra were analyzed to obtain the size distribution and the elemental composition of the synthesized nanoparticles.
3.2 In vitro evaluation of the antibacterial activity
Silver has been incorporated in humans’ daily lives as a powerful tool against various infectious diseases caused by microbial since ancient times. In the last few years, due to the high bacterial resistance against antimicrobials, silver has once again immersed as an effective therapeutic option. With the advancement in nanotechnology, the antimicrobial activity of silver has been further enhanced by decreasing its size to the nanoscale. All experiments for the in vitro evaluation of the antibacterial activity of nanoparticles were conducted under sterilized conditions inside a laminar flow hood. Furthermore, all media culture, glassware, falcon tube, and pipette tips were sterilized in an autoclave at 120° C, for more than 15 minutes.
Bacterial culture and strain
Two separate strains of gram-positive; Staphylococcus aureus and gram-negative; Escherichia coli were cultured in LB broth and incubated at 37° C for 3 hours in a shaking incubator at 250 rpm. The inoculum suspension was then used to measure their optical density (OD) at 600 nm and was adjusted to 0.6 for S. aureus corresponding to 5 × 108 CFU/ml, and 0.1 for E. coli corresponding to 1.5 × 108 CFU/ml. The inoculum suspensions were further diluted to 106 CFU/ml by double dilution.
Evaluating bacterial resistance
Bacterial resistance has increased with time due to the prolonged use of antimicrobial. That can be demonstrated by in vitro evaluation of antimicrobial using the agar disk diffusion method. As shown in Fig 3.4. the bacterial lawn was prepared by depositing 100 µl of the diluted E. coli inoculum with the help of a pipette, in the center of an agar plate. The inoculum was evenly spread on the plate by using a sterilized L-shaped glass spreader, and at the same time by carefully rotating the agar plate underneath it. With the help of sterilized tweezers three disks; control and ampicillin antibiotic, were carefully placed and pressed on the agar plate. The inverted plate was then incubated at 37° C for 24 hours.
Figure 3.4. Spread-plate method for the agar disk-diffusion evaluation.
Micro-dilution method
The PLAL synthesized nanoparticles were mixed in the liquid growth medium (broth) and dispensed in a 96-well microtiter plate that is illustrated in Fig 3.5. Each well was then inoculated by the prepared bacterial inoculum suspension. Additionally, control of diluted silver nanoparticles in culture medium without bacteria and a positive control (antibiotic; ampicillin) was prepared in deionized water. Having the same volume of 200 µl. After proper mixing, the 96-well microtiter plate was incubated at 37° for 24 hours.
Figure 3.5. Preparation of 96-well microtiter plate for antimicrobial susceptibility testing.
Determining the MIC value
The lowest concentration of the antimicrobial that inhibits the growth of bacteria after 24 hours of incubation is known as the minimum inhibitory concentration (MIC). That is defined as the minimum concentration of the antimicrobial agent that completely inhibits the growth of bacterial cells present in a growth medium. For the silver nanoparticle used against the S. aureus and E. coli strain, the MIC value was determined by reading the microtiter plate, as demonstrated in Fig 3.6. [30].
Chapter 4 : Results and discussion
Pulsed laser ablation in a liquid of bulk material, has recently gained much attention due to its simplicity and versatility. By tuning different laser parameters such as laser energy, wavelength, pulse duration, and ablation time, it is possible to control the ablation efficiency and the characteristics of nanoparticles. However, it is important to change only one parameter at a time, to correctly evaluate its impact.
This chapter presents the result of the conducted experiments. In particular, the first part will be focused on the effects of laser parameters on the shape, size, and stability of Ag nanoparticles. Different diagnostic techniques such as UV–Vis spectroscopy and scanning electron microscopy, were used to characterize the synthesized nanoparticles. In the second part, synthesized Ag NPs were applied to well-known four human pathogenic bacteria like Staphylococcus aureus and Escherichia coli
4.1 Effect of laser parameters
4.1.1 Pulse laser energy
Fig 4.1. (a and b) shows the absorption spectra of silver nanoparticles, obtained by UV-visible spectroscopy and the photograph of the colloidal solution, by varying the pulse laser energy. Each ablation process was carried out for 10 mins, by using an Nd-YAG laser with 1064 nm wavelength and 1 mm spot size. The pulse laser energy of 10, 20, 30, 40, and 50 mJ was used to ablate a pure sliver metal target immersed in DI water. With each conducted experiment the solution turned yellow, though with different color intensity, which is consistent with previous studies [31]. A significant increase was observed in the absorption peak, along with a slight change in the bandwidth and maximum wavelength of the SPR spectrum. Fig 4.1. (a) shows that all absorption peaks occurred around 406 nm, which is a characteristic signature peak of silver nanoparticles, with its broad tail extending towards the infra-red region. The broad tail in the absorption spectra is normally attributed to the large particle size distribution of the synthesized nanoparticles. By increasing the pulse laser energy from 10 mJ to 40 mJ an increase in the intensity of the absorption peak and the intensity of the color of the colloidal solution was observed, indicating the increase in the concentration of the nanoparticles in the solution. due to the increase in the amount of the ablated material per laser pulse.
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However, when the energy is further increased from 40 mJ to 50 mJ the intensity of the absorption peak and the intensity of the color of the colloidal solution significantly decreased. When the energy is increased by more than 40 mJ it results in the formation of a secondary plasma on the air-water interface as depicted in Fig 4.2. The formation of the secondary plasma in the beam path absorbs most of the pulse laser energy therefore resulting in low laser energy available for target ablation. Subsequently decreasing the ablated material per pulse, thus decreasing the concentration of the synthesized nanoparticles. As the energy is further increased the dominance of the secondary plasma phenomenon creates splashes at the surface of the liquid medium. Hence the maximum energy is limited to 50 mJ to avoid the splashing of the liquid medium.
4.1.2 Ablation time
Fig 4.3. shows the absorption spectra of silver nanoparticles synthesized at different ablation times. An Nd-YAG laser with 1064 nm wavelength and 1 mm spot size was used to ablate the silver metal target with 20 mJ pulse laser energy. The ablation time was varied from 10 to 30 minutes. The absorption spectra demonstrate the dependence of nanoparticles concentration on the ablation time. The rise in the absorption peak of the spectra is attributed to the increase in the concentration of the nanoparticles in the solution. The increase in the ablation time corresponds to an increase in the number of laser pulses used to ablate the target material. Consequently, increasing the amount of ejected material which increases the concentration of the produced nanoparticles. The lowest concentration is achieved in 10 minutes while the highest is achieved in 30 minutes. Moreover, the characteristic peak of silver nanoparticles near 400 nm does not shift from its position significantly, thus indicating that the average nanoparticle size remained constant by only varying the ablation time.
4.1.3 Aging time/ stability studies
Fig 4.4. illustrates the absorption spectra obtained over time to evaluate the stability of the synthesized nanoparticles. As nanoparticles stability is one of the most prominent factors in all subsequent applications. The instability of the nanoparticles is caused by the higher relative surface area, surface atoms, and excess energy of the small-sized nanoparticles. Such particles are subjected to the phenomena of dangling bonds due to each atom having unsaturated and vacant coordinates. To overcome this, nanoparticles tend to aggregate by forming bonds between adjacent particles and therefore, reduce their surface area and surface energy.
The nanoparticles were prepared by using 20 mJ pulse laser energy for 30 mins, and UV-visible spectroscopy was performed over the course of 8 days to obtain the illustrated spectra. The results show that the characteristic absorption peak of Ag nanoparticles, near 400 nm, does not shift from its position, and also no change was observed in the width of the peak. Indicating that no aggregation of the silver nanoparticles took place over time. Giorgetti et al. hypothesized that a thin oxide layer; 1 to 2 nm, is formed, which insulates the silver nanoparticles and prevents them from aggregating [32]. Therefore, the obtained results verify that the nanoparticles synthesized by the PLAL method are highly stable.
4.1.4 Shape and the size distribution
Fig 4.5. (a and b) shows the SEM micrograph and the corresponding size distribution obtained by scanning electron microscopy of the synthesized nanoparticles. The pulse laser energy of 20 mJ was used to produce silver nanoparticles by ablation of pure silver plate in DI water. To prepare the sample for SEM, a single drop of synthesized colloidal silver nanoparticles was dried on a polished silicon substrate.
The typical SEM micrograph illustrated in Fig 4.5. (a) clearly shows that mostly spherical nanoparticles were synthesized. With diameter ranging from approximately 30 to 100 nm which is shown by the histogram in Fig 4.5. (b). The average diameter of the synthesized nanoparticles was calculated to be 50.4 nm with a standard deviation of 13.9 nm. Furthermore, the purity of the synthesized nanoparticle was confirmed by using an energy dispersive x-ray spectrometer (EDX) that is incorporated in the SEM system. From the EDX spectra illustrated in Fig 4.6. highly pure nanoparticles were synthesized with no contamination or impurity.
Figure 4.6. Energy-dispersive X-ray spectra of the synthesized nanoparticles deposited on a silicon substrate.
4.2 Antibacterial evaluation
4.2.1 Preparation of bacterial strain
As shown in Fig 4.7. (a) for the inoculation of both gram-negative and gram-positive bacteria, the streaking plate method was effectively used to isolate bacterial colonies. Indicating the successful dilution of bacteria obtained from an inactive strain. A single colony can thus be used to ensure that genetically identical bacteria growth after inoculation in the culture medium. Making it easier for antimicrobial evaluation and microscopic examination.
Furthermore, after preparing the inoculum suspension, it was diluted to 106 CFU/ml by double dilution method. Further dilution to 70 CFU/ml was carried out for the confirmation of colonies formation. By using the spread-plate method the diluted inoculum showed the desired results as illustrated in Fig 4.7(b).
4.2.2 Antibacterial resistance
The number of effective antimicrobials has been limited to only a few over the years as a consequence of bacteria developing intrinsic and acquired resistance against them. Bacteria such as E. coli develop resistance by altering their cellular genes by attaining resistive genes from the environment or by different mechanisms. Such as, they can develop the ability to neutralize, rapidly pump out, or change the attack site of the antimicrobial agent [33], [34]. Fig 4.8. shows that the antimicrobial agent ampicillin did not have a prominent effect in inhibiting the growth of E. coli bacteria. It can be concluded that E. coli developed resistance over time as its bacterium with the ability to escape the effect of antimicrobial survived and multiplied due to its rapid reproducing ability. Those particular bacteria replaced all the bacteria that were susceptible to the ampicillin. Additionally, the susceptible bacteria could have acquired resistance through mutation to their cellular genes or by acquiring DNA code from other bacteria with resistance properties.
Conclusion
There were two aims of this thesis; to investigate the effect of laser parameters on the shape, size, and stability of silver nanoparticles synthesized by pulsed laser ablation in liquid, and to check the antibacterial susceptibility of silver nanoparticles against pathogenic bacteria. Regarding the effect of laser parameters, it was observed that it is possible to improve the productivity of silver nanoparticles by accurately tuning different laser parameters. In particular, by using optimum laser energy and longer ablation time it is possible to significantly increase the productivity of silver nanoparticles. It is important to note that further increase of laser energy or too high ablation time can reduce the concentration of silver nanoparticles due to the formation of secondary plasma and interaction of laser pulse with the high concentration of synthesized nanoparticles present in the solution, respectively. Moreover, our results demonstrated no aggregation of silver nanoparticles occurs over a long course of time, therefore, the synthesized nanoparticles by pulsed laser ablation in liquid possess high colloidal stability, which is necessary for several subsequent applications. Additionally, it was evident by the SEM micrograph and EDX results that a clean and contamination-free process was conducted as pure spherical silver nanoparticles were synthesized. It was possible to achieve and interpret these results due to our better understanding of the fundamental mechanisms involved in the process of laser ablation in liquid and due to our insight on how each laser parameter affects the productivity of synthesized nanoparticles. Therefore, we can conclude that we succeeded in evaluating the effect of laser parameters on the size, shape, and stability of silver nanoparticles synthesized by the pulsed laser ablation process. However, other studies must be carried out to investigate the effect of other parameters that significantly affect the productivity of silver nanoparticles. To, best exploit the pulsed laser ablation in liquid technique for synthesizing various pure and stable nanoparticles.
The second part of this thesis aimed to investigate the antimicrobial susceptibility of silver nanoparticles against two pathogenic bacteria that are the cause of various human infections. The conducted experiment verified the bacterial resistance developed due to the prolonged use of antimicrobial agents. Due to which the development of new antimicrobial agents is necessary. Although the antimicrobial susceptibility could not be verified, by our results, we concluded that if the concentration of the silver nanoparticles can be increased to 100’s of micrograms then we will be able to observe good nanoparticle antimicrobial activity against various bacteria.
The concentration of nanoparticles synthesized by pulsed laser ablation in liquid can be increased by using the continuous flow method. As by constant volume flow, the saturation of colloidal liquid is avoided. Therefore, it is possible to increase the productivity of nanoparticles from batch to industrial scale. As mentioned in section 1.4 mass production by pulsed laser ablation in a liquid is to be more economical than conventional wet-chemistry due to its lower labor, material, energy, and absolute investment cost. Hence, once optimized the pure nanoparticles synthesized by pulsed laser ablation in liquid can be used for endless applications.
