Characterization of Nanoparticles in Antimicrobial Coatings for Medical Applications—A Review

Abstract

This review discusses relevant topics concerning the understanding of the characterization of antimicrobial coatings due to powerful antimicrobial nanoparticles in their composition. These coatings are utilized in the surface modification of yarns and materials designed for use in medical and dental applications. Various physical and chemical methods are employed to create these coatings, ensuring the development of efficient, homogeneous, and uniform layers on diverse surfaces and materials. The primary objective is to confer antimicrobial and/or antiviral properties upon these materials. For these coatings to be effective, they must incorporate active compounds that can combat a wide array of microorganisms, including those that have developed resistance to antibiotics. Examples of such active compounds include metallic nanoparticles such as silver, copper, and gold, as well as nanoparticles of metal oxides such as zinc, titanium, and aluminum. Upon the application of these coatings to medical materials, extensive testing and characterization procedures are undertaken, which will be thoroughly detailed in this review. It is crucial to emphasize that the absence of proper characterization and testing of nanoparticles in antimicrobial coatings could lead to the absence of standards, norms, or procedures necessary to safeguard human health and the environment. Despite their widespread application in the medical field, concerns have been raised regarding the potential toxicity of nanoparticles to living organisms. Consequently, this paper provides a comprehensive overview of the current state-of-the-art methodologies for characterizing nanoparticles in antimicrobial coatings, specifically focusing on materials with varying roughness and structures. Additionally, it outlines the issues associated with the potential accumulation of antimicrobial nanoparticles within the human body.

1. Antimicrobial Coatings with Metal and Metal Oxide Nanoparticles

Characterizing antimicrobial metal and metal oxide nanoparticles using state-of-the-art instrumental methods is a pivotal component in their research and development, particularly in the realm of medical applications. This comprehensive protocol involves a series of fundamental steps, beginning with identification and progressing to the quantification of various attributes, including morphological, physical, chemical, biological, and protective properties of these nanoparticles. The wealth of information garnered through this thorough characterization is indispensable for gaining insights into their behavior within biological systems. It provides valuable insights into their interactions with living organisms and microorganisms, offering crucial data on their antimicrobial potency, potential toxicity, efficacy, and stability under diverse conditions. In essence, this meticulous characterization process serves as the foundation for advancing our understanding of antimicrobial nanoparticles and their potential contributions to the realm of medical coatings.

Monitoring the antimicrobial effects of coatings with metal and metal oxide nanoparticles heavily relies on their chemical and morphological properties. Not only do the chemistry, concentration, and quantity affect the particular antimicrobial potential, but the size and morphology, such as sphere, needle-like, or star-shaped structures, can also make a substantial difference. Small nanoparticles in low concentrations often exhibit stronger antimicrobial effects than those in higher concentrations. Additionally, the agglomeration of nanoparticles in some samples leads to changes in their antimicrobial effects. To accurately monitor antimicrobial effects, it is crucial to conduct a thorough characterization of coatings containing metal and metal oxide nanoparticles, linking antimicrobial properties to a sound physical, chemical, and morphological foundation.

Furthermore, nanoparticles can accumulate in the human or animal body and the environment, leading to toxic, genotoxic, and other undesirable effects. Preserving human health and other living organisms necessitates the meticulous formulation of coatings with nanoparticles using current state-of-the-art methodologies. Therefore, this manuscript underscores the importance of proper instrumental characterization of antimicrobial coatings with nanoparticles. Analytical procedures must be sensitive, robust, and accurate enough to detect minute samples and distinguish them within the complex matrices of polymeric-coated materials. Prior to validation, including the determination of limits of detection and limits of quantification, robustness and optimization, and the measurement uncertainty of the particular methodology, a comprehensive understanding of the antimicrobial and toxic mechanisms of antimicrobial nanoparticles (NPs) is essential.

Antimicrobial coatings serve multifaceted purposes, encompassing protection, aesthetics, decorativeness, and more. They are meticulously engineered with the overarching goal of inhibiting bacterial proliferation, surface colonization, and the formation of biofilms (refer to Figure 1).

Antimicrobial coatings have gained extensive traction in the realms of medicine and dentistry, largely owing to their notable attributes such as powerful antibacterial properties, antimicrobial capabilities, and resilience against water, alongside various other protective features. However, despite their broad usage and diverse applications, effectively monitoring their potential adverse impacts remains a challenging task. While the development of legal frameworks is currently underway, their efficacy will ultimately hinge on the precision of the testing procedures implemented.

The ongoing investigation emphasizes that among all the materials enumerated in

Table 1. Nanoparticles in antimicrobial coatings. The antimicrobial activity of the nanoparticles depends strongly not only on the chemical composition of the particular component but also on the size of the nanoparticle. Not all compounds have the good activity. Those with the highest potential are small particles that can easily interact with the microorganism cell (Figure 2 and Figure 3).

Metal	Diameter Minimal	Diameter Maximal
Ag	1.5 nm	35 nm
Al	18 nm	80 nm
Au	50 nm	150 nm
Co	28 nm	50 nm
Cr	50 nm	100 nm
Cu	25 nm	500 nm
MgO	20 nm	100 nm
Mn	30 nm	60 nm
Mo	70 nm	100 nm
Ni	20 nm	50 nm
TiO2	30 nm	150 nm
ZnO	80 nm	130 nm

[1,2,3], silver nanoparticles (AgNPs) are leading the way in commercialization. This trend persists particularly in the fields of medicine and dentistry, where AgNPs find extensive use due to their robust antibacterial properties [4]. Significantly, within the industry, the segment associated with AgNPs stands as one of the fastest-growing product sectors [5].

The significance of nanoparticles (NPs) is further highlighted by their capacity to modify the physical and chemical characteristics of coatings. This alteration encompasses the enhancement of stain and water resistance, improvement in the absorption capabilities of materials, and adjustments in wettability based on surface energy and roughness. These versatile properties cater to a wide spectrum of applications, extending across both medical and non-medical domains [5]. Notably, silver nanoparticles (AgNPs) have demonstrated their effectiveness through substantiated antimicrobial and antibacterial effects, as delineated in Table 1.

Considering that a substantial portion of medical materials often encounters prolonged and direct contact with microorganisms, including those highly perilous to human health, it becomes crucial to examine a specific group of potentially harmful substances: metallic and metal oxide antimicrobial nanoparticles (NPs) (as illustrated in Table 1). While recent research primarily focuses on their beneficial properties, it is essential to address other aspects as well. Therefore, the principal objective of this manuscript is to underscore the intricate task of selecting appropriate characterization methods that facilitate the precise and accurate determination of nanoparticles present in antimicrobial coatings.

1.1. Silver Nanoparticles in Antimicrobial Coatings

Silver nanoparticles (AgNPs) offer distinct advantages over molecular antimicrobials, mainly due to their facile integration into polymers, facilitating the creation of functional antimicrobial coatings. This attribute is particularly notable because of the controlled release capabilities of AgNPs, which enable them to maintain their antimicrobial potency over prolonged periods. Furthermore, specific encapsulation can even extend the release duration. Once encapsulated, the active compounds remain preserved until they come into contact with microorganisms on human skin, wounds, the oral cavity, or any other targeted area. Following this initial contact, the nanoparticles are released. Moreover, if these nanoparticles are loaded with drugs or other active agents, their potential medical applications could significantly broaden. Figure 2 illustrates the release of active compounds from the hollow nanoparticles embedded within the antimicrobial coating.

Several researchers have delved into the antimicrobial efficacy of materials coated with AgNPs. When comparing the effectiveness of silver/polyamide 6 systems with nanoparticles at the nanometer scale to those at the micrometer scale, it was observed that nanocomposites with lower silver content exhibited superior efficacy against Escherichia coli compared to microcomposites with significantly higher silver content. Notably, even after 100 days of immersion in water, polyamide 6 infused with 2 wt. % AgNPs remained effective against E. coli.

The effectiveness of these materials is contingent on surface roughness, as rough surfaces provide a larger area for the release of silver ions, resulting in higher antimicrobial activity than smoother surfaces. Additionally, factors influencing silver ion release rates, such as the degree of polymer crystallinity, type of filler, matrix hydrophobicity, and particle size, can impact antimicrobial activity.

A coating based on AgNPs demonstrates effectiveness against a broad spectrum of bacteria and fungi, with a relatively higher efficacy against Gram-negative compared to Gram-positive bacteria. Despite significant progress in the use of silver nanostructures for medicine applications, further research is required to elucidate the key factors influencing not only the benefits, but also the disadvantages and toxicity of such coatings.

1.2. Titanium Oxide Nanoparticles in Antimicrobial Coatings

Titanium dioxide (TiO₂) is widely recognized for its application as a photocatalytic disinfecting agent in surface coatings. The development of TiO₂ coatings has demonstrated the capability to deactivate Escherichia coli in vitro when exposed to UV light.

In the field of nanoparticle-based antimicrobial research, substantial evidence has accumulated, confirming the antimicrobial properties of titanium dioxide nanoparticles (TiO₂ NPs), despite ongoing discussions concerning their interaction with UV light. These antimicrobial characteristics make TiO₂ NPs effective in combating bacterial infections. It is crucial to acknowledge that a significant portion of the findings supporting their antimicrobial effectiveness has been derived from in vitro studies often conducted in the presence of various additional nanomaterials. Various methodologies have been employed to assess these properties, contingent on the specific objectives of the research. Researchers have utilized a variety of techniques, including the determination of minimum inhibitory concentrations, quantification of cell counts, and the application of disk and well diffusion assays. Notably, cell count assessment has emerged as the predominant and most prevalent criterion for evaluating the antimicrobial activity of TiO₂ NPs [6].

Biodegradable poly(butylene adipate-co-terephthalate) (PBAT) nanocomposite films incorporating titanium dioxide (TiO₂) nanoparticles were characterized through Fourier-transform infrared spectroscopy, X-ray diffraction analysis, scanning electron microscopy, and transmission electron microscopy. Interestingly, the PBAT/TiO₂ nanocomposite films exhibited remarkable antimicrobial activity against both Gram-positive and Gram-negative food-borne pathogenic bacteria, particularly Escherichia coli and Staphylococcus aureus. These results underscore the significance of filler–polymer interactions and highlight the role of TiO₂ as a reinforcing component in these nanocomposites, ultimately contributing to their enhanced antimicrobial properties [7].

1.3. Zinc Oxide and Magnesium Oxide in Antimicrobial Coatings

More recently, the antimicrobial attributes of nano-sized zinc oxide (ZnO) and magnesium oxide (MgO) have come to the forefront. In contrast to nano-silver, ZnO and MgO nanoparticles are anticipated to provide more cost-effective coating solutions. Unlike nanocomposites loaded with TiO₂, which necessitate UV light, materials containing nano-ZnO-based photocatalysts can sterilize in indoor lighting. Both ZnO and MgO showcase antibacterial properties that amplify with diminishing particle size.

The potential impact of metallic nanoparticles on the horizontal transfer of resistance genes has become a subject of investigation, attributable to their toxicity to bacterial cells and the mechanisms of metal-induced co-selection. An examination of the toxicity of ZnO nanoparticles on the E. coli DH5α laboratory strain was conducted, alongside an assessment of the influence of ZnO nanoparticles on the transfer of resistance genes. The study revealed that even at concentrations as low as 10 mg L⁻¹, ZnO nanoparticles brought about a 14.6% reduction in the survival rate of E. coli cells. Intriguingly, these nanoparticles also resulted in an almost 1.8-fold increase in the frequency of transformation of resistance genes. Furthermore, an evaluation of the environmental impact in the soil environment, where ZnO nanoparticles were present at a concentration of 1000 mg kg⁻¹, showed a significant impact on the overall abundance of bacteria, leading to a decrease in the copy number of the rRNA gene. These collective findings underscore the role of metallic nanoparticles in facilitating the dissemination of both antibiotic and metal resistance genes [8].

Furthermore, the implications of this research extend broadly to environmental concerns, suggesting that the inevitable pollution of environments with nanoparticles may exacerbate the spread of antibiotic resistance, potentially exerting a significant influence on public health. These findings emphasize the need for heightened awareness and proactive measures in managing nanoparticle pollution to mitigate its potential impact on the dissemination of resistance genes and, consequently, public health [8].

1.4. Chromium Oxide NPs in Antimicrobial Coatings

Chromium (Cr) has long been recognized as a hazard to the environment and its associated biota, particularly when its levels surpass permissible thresholds. Extensively utilized across various industries, the toxicological implications of Cr have undergone comprehensive scrutiny. However, the emerging applications of nano-sized chromium (nano-Cr) or chromium oxide nanoparticles (Cr₂O₃-NPs) have not received as much attention in the existing literature, particularly concerning their phytotoxic effects.

A recent study sought to bridge this research gap by investigating the morpho-physiological impacts of both macro- and nano-sized Cr on Hordeum vulgare L. plants. The findings indicated that the escalated accumulation and translocation of Cr, regardless of its form (macro or nano), disrupted cellular metabolism, resulting in the inhibition of germination, growth, and interference with photosynthesis in the plants. Importantly, the phytotoxicity was more pronounced when exposed to Cr nanoparticles compared to their macro-sized counterparts. This heightened phytotoxicity in the presence of Cr nanoparticles likely stemmed from the increased bioavailability of Cr ions, a phenomenon supported by observations related to total Cr content, mobility, and the factor toxicity index.

To reinforce these findings, synchrotron X-ray techniques were utilized to identify Cr-containing compounds within plant tissues. The X-ray spectra revealed that the dominant crystalline phase corresponded to Cr₂O₃. Consequently, this research contributes to a deeper comprehension of the mechanisms underlying the phytotoxic effects induced by both macro- and nano-sized Cr at the tissue, cellular, and sub-cellular levels. These insights shed light on the potential environmental impacts of nano-Cr and underscore the importance of considering nanoparticle-specific effects in environmental and biological studies [9].

2. Antimicrobial Coatings on Polymers

A variety of physical surface modifications of polymers, including treatments such as flame, corona discharge, UV, gamma-ray, electron beam, ion beam, plasma, and laser techniques, have proven effective in instilling antimicrobial properties. For instance, the antimicrobial potential of polyamide films subjected to UV irradiation has been well-documented. It is believed that the antimicrobial activity arises from an increase in the concentration of amines on the surface of the film.

While the antimicrobial effects of nanoparticles offer numerous advantages, concerns persist regarding potential adverse toxic effects in living organisms [5,6,7,8,9,10,11,12,13]. Both coated and uncoated nanoparticles have the capacity to induce DNA double-strand breaks and cell death in various types of mammalian cells, while also impeding the binding of molecules through non-covalent interactions [10]. This underscores the critical necessity for comprehensive assessments of the potential harm to humans resulting from the commercial applications of AgNPs. Several parameters contribute to the potential toxicity of nanoparticles, encompassing biocompatibility, biodistribution, biodegradation, inflammation, and their interference with normal organ function. These parameters are intricately tied to the size, shape, composition, and reactivity of engineered nanoparticles (ENPs) [10,11,12,13,14,15,16,17,18]. Casals et al. [19] have attributed the biological activity and biokinetics of ENPs to factors extending beyond size, shape, and chemistry, including crystallinity, surface properties, agglomeration state, bio-persistence, and dosage. Among these factors, surface properties such as area, porosity, charge, surface modifications, and coatings emerge as particularly crucial [19].

Exposure to nanoparticles (NPs) from coatings on antimicrobial materials can occur through various pathways, including ingestion, particularly in children, as well as inhalation and skin absorption [20,21,22]. Workers who experience prolonged contact with antimicrobial NPs are at heightened risk due to their extended and continual exposure to substantial quantities of NPs. The primary routes for NPs to enter the body are through the skin and respiratory tract. Once inside the body, they have the potential to accumulate in organs such as the liver, kidneys, bone marrow, and spleen [19]. A conceptual model illustrating the potential pathways of NPs within the organism and their associated toxic and harmful effects is depicted in Figure 3.

The antimicrobial effects associated with nanoparticles extend to various materials, encompassing titanium dioxide powder, aluminum, gold, and silver particles, with documented impacts on organs such as the kidneys, throat, skin, and brain [23]. It is worth noting that oxidative stress is often identified as a key toxicity mechanism resulting from exposure to nanoparticles, as emphasized by Green and Howman [24]. Currently, the majority of nanotoxicity studies primarily focus on in vitro models, with in vivo studies remaining limited.

Emerging legal frameworks are now shifting their focus towards identifying the long-term effects of nanoparticles (NPs), with the primary objective of mitigating health risks associated with exposure to antimicrobial coatings. Maynard and colleagues [25] have outlined significant strategies to drive research efforts in this direction. Their proposal advocates for the development of instruments, validated methodologies, and strategic programs aimed at facilitating research, with a specific focus on risk assessment.

Furthermore, it is essential to note that high concentrations of nanoparticles originating from antimicrobial coatings are being added to the cumulative pool of nanoparticles found in everyday products such as cosmetics, food, and various other samples. This intricate intermingling poses challenges in estimating the true extent of exposure and uptake [25]. Numerous studies examining the fate of nanoparticles once they enter the environment have assumed that these materials can accumulate in living organisms.

3. Toxic Effects of Antimicrobial NPs from Coatings

Once nanoparticles (NPs) enter the body, they tend to accumulate in specific organs, including the liver, kidneys, bone marrow, and spleen [19]. To comprehend the toxicokinetics of nanomaterials, extensive research efforts are currently underway. NPs, being smaller than 100 nm, have the capability to readily penetrate cells. Their toxicity varies based on properties such as size, shape, charge, surface energy, and chemical composition, and can also depend on the genetic makeup and DNA coverage of different organisms [26].

The investigation into the accumulation effects of antimicrobial nanoparticles reveals a pattern within cells, where they tend to accumulate within organelles, typically without export. In cases where these nanoparticles exhibit slow degradation within the cell, which is a common occurrence among engineered nanoparticles, their accumulation continues until they are divided between daughter cells during cell division. This phenomenon occurs even in cells with slow division rates, such as those in the brain. Notably, nanoparticles conjugated with antibiotics display extended retention times within the body compared to free drugs alone. Furthermore, nanoparticles equipped with targeting biomolecules, such as antibodies, proteins, or DNA, exhibit improved membrane penetration capabilities, enabling the specific targeting of particular tissues, cells, or organs.

Antimicrobial nanoparticles represent a unique approach in combating infectious diseases, particularly those caused by antibiotic-resistant pathogens. Their high surface area-to-volume ratio leads to the emergence of novel mechanical, chemical, electrical, optical, magnetic, electro-optical, and magneto-optical properties. Studies indicate that metal-based nanoparticles possess non-specific toxicity mechanisms against bacteria. This characteristic not only makes it challenging for bacterial resistance to develop but also broadens the spectrum of antibacterial activity. For example, ZnO nanoparticles have demonstrated sensitivity to certain Gram-positive bacteria, such as Bacillus subtilis and Staphylococcus aureus.

Silver nanoparticles (AgNPs) have undergone extensive study for their antimicrobial properties. They attach to cell membranes, inducing alterations in the lipid bilayer, subsequently leading to increased membrane permeability, damage, and cell death. This potent antibacterial effect appears to be more pronounced when smaller-sized nanoparticles are utilized. In summary, the combination of antimicrobial nanoparticles, antibiotic conjugation, and targeting biomolecules holds promise for improving antimicrobial therapy, addressing infectious diseases, and combating antibiotic resistance. A schematic overview of the potential pathways of antimicrobial NPs within the human body is presented in Figure 4.

Skin absorption stands as one of the most concerning routes of exposure to nanoparticles (NPs) from various sources. The skin, with its abundant blood supply, tissue macrophages, lymph vessels, dendrites, and nerve endings, serves as an excellent medium for absorption [19]. Research by Tinkle et al. [27] has revealed that flexed and moving skin can be even more permeable to NPs. This becomes particularly pertinent when NPs are integrated into materials utilized in sportswear, socks, and underwear [28].

Despite growing concerns expressed by various research groups regarding NPs and the European Union’s willingness to evaluate the risks associated with their use, there is currently no specific regulation governing the safety assessment of NPs [29]. Presently, guidelines from organizations such as the European Food Safety Authority (EFSA) and the Organization for Economic Cooperation and Development (OECD) serve as the primary reference points for safety assessments related to nanomaterials [29]. Casals et al. [19] have stressed the need for a comprehensive approach to risk assessment, taking into account factors such as dosage, duration of exposure, cellular damage, and toxicity.

Numerous researchers have highlighted the scarcity of studies related to risk assessment and toxicology concerning NPs, underscoring the substantial work that still remains. Consequently, comprehensive methods for gathering data on the physical and chemical behavior, occurrence levels, fate, and transport of NPs from sample materials into the environment are still lacking. Current legislative efforts primarily focus on addressing the most harmful properties of samples, including pH control, formaldehyde content, carcinogenic dyes, dyes that can break down into carcinogenic aryl amines, extractable harmful metals, halogenated carriers, and contamination with substances like pentachlorophenol and pesticides. Sample products are categorized based on their use, such as products for babies, products in direct contact with the skin, products without direct skin contact, and decorative materials. The limits for toxic and allergenic metals and chemicals vary depending on the degree of contact between the fabric and the consumer’s skin, as well as the toxicity of the heavy metals involved. These limits do not pertain to the total amount of compounds present in the fabric but rather to the portion that can be extracted. A similar approach should be developed for assessing the toxicity of NPs in samples, given that their impact on human health and the environment currently remains unpredictable.

4. Separation Methods of Antimicrobial NPs in Coatings

Before analysis, it is essential to separate NPs from the coating materials. Various extraction procedures exist, including liquid–liquid extraction, Soxhlet extraction, solid-phase extraction (SPE), and centrifugation, with established protocols and methods available for some of these techniques. However, the analytical challenge arises because coatings, when used as analytical samples, have highly complex matrices. Therefore, it is often necessary to develop an individualized extraction method for each specific sample.

To address this challenge, analytical specialists have proposed a range of techniques for separating NPs from liquid samples, emphasizing the need for sensitivity and non-destructiveness. These techniques include size exclusion chromatography (SEC), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), hydrodynamic chromatography (HDC), and field-flow fractionation (FFF).

It is worth noting that NPs tend to exhibit instability in water-based media, leading to agglomeration or encapsulation when they come into contact with other components. This issue is particularly pronounced in emulsions, such as gels, cosmetic antimicrobial mixtures, and others, which often contain organic agents, ligands, dyes, complexing agents, metal ions, salts, and various other compounds. Consequently, each antimicrobial sample requires the development of a suitable approach for the extraction and characterization of nanoparticles.

Real-time aerosolization methods in conjunction with membrane filtration techniques (including membrane filtration, ultrafiltration, and nanofiltration) can determine the size and quantity of dissolved and suspended colloid NPs in samples. Validation of the results can be achieved using transmission electron microscopy (EM, various manufacturers) equipped with energy-dispersive X-ray spectroscopy (TEM-EDS) analysis. This approach could prove valuable for the separation of NPs from antimicrobial coatings, serving as a crucial preparatory step before characterizing them using spectroscopic, chromatographic, or microscopic methods.

Capillary electrophoresis (CE) emerges as a highly suitable procedure for the separation of water-soluble and charged NPs, where particle mobility depends on the zeta potential of the NPs. Lo et al. [30] have successfully developed an effective CE technique for separating negatively charged, polydisperse, water-soluble gold monolayer-protected clusters. Liu [31] has also proposed the application of CE for the analysis of non-protected gold NPs, in conjunction with other liquid chromatography (LC) techniques such as FFF, size exclusion chromatography (SEC), HPLC [32,33,34], and ion-exchange chromatography (IEC), all of which are valuable for NP analysis.

SEC is a commonly employed method for the fractionation of nanoparticles. However, its efficiency depends on the pore size of the stationary phase, and, due to the polydispersity of NPs in liquid samples, proper pretreatment may be required when using SEC. An alternative approach that does not rely on stationary phases is FFF. F. Kammer et al. [35] have identified FFF as one of the most promising techniques for achieving relevant characterization of NPs. For nanoparticles, asymmetric flow field-flow fractionation (AF4) is often more beneficial due to its higher resolution and enhanced efficiency for sample preconcentration. AF4 size separation is more efficient when coupled with in-line dynamic light scattering (DLS), as well as liquid chromatography with atmospheric pressure photoionization-mass spectrometry (LC-APPI-MS).

In cases where only information regarding the total quantity of NPs on a material is needed, it is advisable to employ total digestion procedures (such as open vessel digestion, microwave-assisted digestion, and other routine methods) prior to characterization and detection. This approach allows for a comprehensive assessment of NP content. In the future, much more complete assessments will be performed for Safety by Design, which is implemented in industry to address all possible challenges from the beginning [36].

5. Instrumental Analysis of Antimicrobial NPs in Coatings

In the realm of nanoparticle analysis and characterization, the separation of water-soluble and charged NPs presents a fascinating challenge. In a laboratory where scientists are delving into the depths of these minuscule entities, seeking to unravel their secrets, among the tools at their disposal, capillary electrophoresis (CE) emerges as a powerful instrument. Within the confines of a laboratory, CE allows researchers to discern the mobility of particles. In the recent literature, the work of Lo et al. [30] stands as a testament to the effectiveness of CE. These researchers have meticulously crafted a CE technique that skillfully separates negatively charged, polydisperse, water-soluble gold monolayer-protected clusters, shedding light on the intricacies of NP behavior.

Meanwhile, in the vast landscape of nanoparticle analysis, another luminary, Liu [31], has illuminated a path forward. In this scholarly expedition, CE is joined by an ensemble of other LC techniques, each offering its unique perspective on the enigmatic world of NPs. FFF, size exclusion chromatography (SEC), secondary ion mass spectroscopy (SIMS), photon correlation spectroscopy (PCS), ultraviolet–visible spectroscopy (UV-VIS), infrared spectroscopy (IR), X-ray photoelectron spectrometry (XPS), X-ray powder diffraction (XRPD), HPLC [37,38,39], and IEC collectively serve as compass points, guiding researchers through the labyrinthine terrain of NPs. Moreover, SEC is utilized as a well-trodden path for fractionating nanoparticles. Yet, there are challenges to overcome. The efficiency of SEC is intricately linked to the pore size of the stationary phase, a detail that underscores the need for careful pretreatment, especially when navigating the tumultuous waters of samples.

In this quest for understanding, an unconventional guide emerges—FFF. F. Kammer et al. [35] have heralded FFF as one of the most promising techniques, a beacon of hope for achieving a comprehensive grasp of NPs. As the research caravan advances, hydrodynamic forces, orchestrated by an external field, propel it forward. Within the FFF framework, the AF4 variant often takes center stage. Its higher resolution and enhanced efficiency for sample preconcentration make it a valuable tool for discerning the intricacies of NPs. And yet, there is more to this narrative. In the work of Isaacson and Bouchard, the story takes an unexpected twist. Here, AF4 size separation is paired with in-line DLS, a conjunction that yields deeper insights into NP behavior. The ensemble is completed with liquid chromatography, which, when coupled with atmospheric pressure photoionization-mass spectrometry (LC-APPI-MS), provides a harmonious symphony of analytical capabilities.

In the annals of scientific exploration, the journey to decipher the enigmatic world of NPs continues. As researchers navigate this captivating realm, each technique and method serves as a lantern, illuminating a path that was once shrouded in darkness. Together, they unveil the secrets of NPs, inch by inch, page by page, in the ever-expanding book of scientific knowledge.

6. Microscopical Investigation of Antimicrobial NPs in Coatings

Microscopy techniques hold special significance in the comprehensive characterization of NPs residing upon coated substrates. This exposition delineates the pivotal role of EM in the assessment of NP attributes, encompassing shape, dimensions, chemical composition, morphology, and topological attributes, thereby underscoring its routine application in this domain. After EM imaging, requisite computational software is judiciously employed to process and analyze acquired data, thus affording a granular understanding of NP attributes.

Surface analysis of coated specimens earmarked for scrutiny entails a multifaceted approach. X-ray photon spectroscopy, Raman spectroscopy (RS), potentiometric titration, and atomic force spectroscopy (AFS) are among the arsenal of techniques invoked to scrutinize surface elements. This methodological repertoire extends further to encompass chemical composition determination, a facet buttressed by energy-dispersive X-ray spectroscopy (EDS), inductively coupled plasma–optical emission spectrometry (ICP-OES), inductively coupled plasma mass spectrometry (ICP-MS), secondary ion mass spectrometry (SIMS), and analogous modalities.

Of notable import is scanning electron microscopy (SEM), a veritable linchpin in sample NP scrutiny. However, its efficacious application hinges upon judicious sample preparation, e.g., pre-coating, to forestall the inadvertent loss of NPs within the vacuum milieu. SEM coupled with EDS (SEM-EDS) confers a non-destructive dimension on the analysis, affording the luxury of sample reinstatement to its pristine state post-examination. It is imperative to note, however, that SEM-EDS outcomes proffer the average chemical composition limited to a specific demarcated segment of the sample surface, a limitation not shared by more comprehensive analytical methods such as laser ablation ICP-MS (LA-ICP-MS).

In the sphere of transmission electron microscopy (TEM), its prowess finds application in the sizing and morphological appraisal of silver nanoparticles (AgNPs) adorning samples. TEM, renowned for its ability to discern particles within the size bracket of 1 nm to 5 μm, supplements the SEM modality. While SEM presents expediency and provides 3D representations, it is tempered by comparatively lower resolution vis-à-vis TEM. To determine hydrodynamic size and the zeta potential of AgNPs in solution, DLS and laser Doppler velocimetry (LDV) are invaluable techniques. Frequently, DLS is co-opted in conjunction with size exclusion chromatography (SEC), alongside other techniques such as voltammetry, multi-angle laser light scattering (MALLS), and ICP-MS for enhanced analytical insights. It is pertinent to observe that dissimilarities in results between TEM and DLS analyses, as observed by Ahamed et al., can be ascribed to the divergent principles underlying these analytical methodologies. Specifically, TEM interrogates dried layers on a specialized grid, while DLS elucidates the proclivity of particles to form clusters rather than exist as discrete entities. The time-intensive nature of TEM, especially in the context of a sample harboring a sparse population of NPs, is a salient drawback, as reported by Blasco et al. There is also the option to correlate TEM result with X-ray powder diffraction (XRPD), as reported by Biljan et al. [40].

In summation, the gamut of microscopy techniques elucidated herein plays an instrumental role in the meticulous characterization of NPs on samples. These modalities collectively furnish invaluable insights into NP attributes and spatial distribution, facilitating comprehensive understanding and analysis tailored to the specific research objectives and nature of the coated samples under investigation.

In the realm of nanomaterial analysis, researchers have leveraged various analytical techniques to evaluate the purity and characteristics of polymer nanoparticles. Harča et al. [36] employed nuclear magnetic resonance (¹H NMR) spectroscopy to assess the purity of these nanoparticles, shedding light on the presence of monomer residues and oligomers. Concurrently, gel permeation was utilized to determine the molecular weight. Doucet et al. [41] embarked on a comprehensive investigation, employing a combination of environmental scanning electron microscopy (ESEM, from various manufacturers), scanning electron microscopy (SEM), analytical electron microscopy (AEM), and atomic force microscopy (AFM). Their study aimed to evaluate the efficiency of cross-flow filtration (CFF) in size fractionating colloids and particles within water samples. The results demonstrated that, when utilizing ESEM and SEM, the estimated size cut-offs were generally smaller than the nominal pore size of 0.45 μm membranes, yet reasonably accurate for membranes with 0.1 μm pore sizes. Remarkably, AFM revealed the existence of colloids smaller than 50 nm, signifying that CFF fractionation is not entirely quantitative and relies on factors beyond size alone.

Evidently, EM techniques, including TEM, SEM, and AFM, along with others, such as confocal laser scanning microscopy (CLSM), near-field scanning optical microscopy, analytical electron microscopy (AEM), and auger electron microscopy, remain the preferred choices for researchers. These techniques offer not only visual imaging of samples but also comprehensive characterization capabilities, rendering them invaluable for monitoring NPs on samples.

Beyond microscopic techniques, a plethora of spectroscopic methods, such as acoustic spectroscopy (AS), can be harnessed for NP analysis. Following the complete digestion of NPs, flame atomic absorption spectrometry (F-AAS), graphite furnace atomic absorption spectrometry (GF-AAS), ICP-OES, ICP-MS, and ultraviolet–visible spectroscopy (UV-VIS) emerge as viable options for analyzing both sample materials. Integration of UV-VIS with ICP-MS is efficient in monitoring protein adsorption and cellular uptake studies of NPs [42]. ICP-OES, among other methods, has proven highly suitable as a multi-elemental technique for NP monitoring due to its low detection limits, high precision, and extensive linear range. However, a limitation of ICP-OES is its inability to distinguish between NPs and solvated ions in the sample. Colloid and sludge samples, characterized by complex matrices, sometimes favor the use of UV-VIS over ICP-OES, as it is less susceptible to matrix effects stemming from elevated concentrations of mineral acids and electrolytes. During testing by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and laser desorption/ionization-time-of-flight mass spectrometry (LDI-TOF-MS), efficient nanoparticle characterization from antimicrobial coatings is achieved [43,44,45,46,47,48,49].

Emerging methods poised for wider adoption in NP analysis on samples encompass various light scattering techniques, including laser-induced breakdown detection (LIBD), static light scattering (SLS), photon correlation spectroscopy (PCS), and DLS. Weinberg et al. [45] introduced LIBD as a novel and sensitive method for non-invasively determining NP concentration and diameter, boasting remarkably low detection limits. The authors advocate for combining LIBD or FFF with spectroscopy techniques, biosensors, or microscopy analyses as the most promising avenue for comprehensive NP information.

Brar and Verma [48] conducted a comparative evaluation of DLS and SLS, ultimately favoring DLS for its rapid analysis, lack of calibration requirements, sensitivity to NPs, and tolerance for non-pristine samples. Additionally, they underscored the simplicity, sensitivity, selectivity, and user-friendliness of light scattering methods. Therefore, the combination of different spectroscopic, microscopic, and chromatographic methods enables precise, accurate, and reliable analysis of nanoparticles in very small amounts (

Table 2. Methods for characterization of NPs in antimicrobial coatings [42,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88]. There is a wide variety of methodologies by which the efficient characterization of NPs is enabled. Many of those methods still have many drawbacks and limitations, so the best routine approach is to combine one or more methods during the analysis of nanoparticles.

Method	Name Abbreviation	References
Acoustic spectroscopy	AS	[51,52,53]
Analytical electron microscopy	AEM	[54,55]
Asymmetric flow field-flow fractionation	AF4	[56,57]
Atmospheric pressure photo-ionization mass spectrometry	APPI-MS	[58,59]
Atomic force microscopy	AFM	[60,61]
Capillary electrophoresis	CE	[62,63]
Confocal laser scanning microscopy	CLSM	[64,65]
Cross-flow filtration	CFF	[66,67]
Dynamic light scattering	DLS	[68,69]
Electron microscopy	EM	[70,71]
Energy dispersive X-ray spectroscopy	EDS	[72,73]
Environmental scanning electron microscopy	ESEM	[74,75]
Field-flow fractionation	FFF	[76,77]
Flame atomic absorption spectrometry	F-AAS	[78,79]
Graphite furnace atomic absorption spectrometry	GF-AAS	[80,81]
High-performance liquid chromatography	HPLC	[42,82]
Hydrodynamic chromatography	HDC	[83,84]
Infrared spectroscopy	IR	[85,86]
Inductively coupled plasma mass spectrometry	ICP-MS	[87,88]

).

Microscopical analysis plays a pivotal role in characterizing nanoparticles within coatings, offering valuable insights into their purity and composition. Utilizing an electron microscope, analytical electron microscopy (AEM) facilitates the comprehensive characterization of nanoparticles. This technique involves the use of a nanometer-scale electron probe directed towards the target area for analysis. Upon incidence, the electrons give rise to X-rays, Auger electrons, and secondary electrons through the process of secondary emission. Analyzing the energy of these secondary emissions enables precise elemental analysis and measurement of various physical properties. AEM offers the unique advantage of simultaneously providing elemental analyses and structure analysis through high-resolution imaging and electron diffraction, with the capability to examine areas as small as 5 nm [54].

As mentioned before, SEM, TEM, and AFM are tools that cannot be avoided in the analysis of nanomaterials in coatings. Due to this, the special sample preparation might include coating of nonconductive samples, or even preparation of the sample crosscut, in which the sample is placed within the resin and, after polishing, enables a clear overview of all layers within the sample. Among the techniques employed for this purpose, microscale thermogravimetric analysis (micro-TGA) stands out. Micro-TGA allows researchers to assess the surface ligand coverage of gold nanoparticles and confirm the presence of a poly-(ethylene glycol) coating on SiO₂ nanoparticles. This approach provides crucial information about the surface properties and functionalization of nanomaterials [56,57,58,59,60,61].

The utilization of asymmetric-flow field-flow fractionation (AF4) has become a crucial methodology in the characterization of particle size, polydispersity, drug loading, and stability assessment for nano-pharmaceuticals. However, the efficacy of this technique is contingent upon the implementation of robust and high-quality standard operating procedures (SOPs), particularly considering the inherent polydispersity of complex drug nano-formulations. Several case studies exemplify how MD-AF4 has been effectively employed for the analysis of crucial quality attributes, including particle size, shape, drug loading, and stability of intricate nanomedicine formulations. The outcomes underscore the versatility of MD-AF4 in providing vital information on particle size distribution and polydispersity and qualitative insights into drug loading. Moreover, the capacity to conduct analyses in complex physiological matrices stands out as a significant advantage of MD-AF4 over other analytical techniques commonly used in stability studies. The collaborative experience between different research institutes highlights the successful implementation of MD-AF4 within the stringent quality standards set by regulatory authorities for the pre-clinical safety assessment of nanomedicines [56,57].

Atomic force microscopy (AFM) serves as a powerful method for the three-dimensional characterization of nanoparticles, offering sub-nanometer resolution. Its utility extends to the analysis of nanoparticles and nanomaterials within environmental samples, enabling precise determination of particle size, chemical composition, and concentrations. In comparison to dynamic light scattering, electron microscopy, and optical characterization methods, AFM boasts several advantages. Notably, it can effectively measure nanoparticles with varying geometries, further enhancing its versatility and applicability in diverse research contexts. While AFM and other scanning probe microscopy techniques are commonly employed for nanoparticle measurement, challenges arise when dealing with nanoparticles situated on rough substrates or when they are not isolated. In such scenarios, employing a straightforward model for realistic simulations of nanoparticle deposition can prove beneficial. This approach facilitates the modeling of nanoparticles on uneven substrates, taking into account different modeling conditions such as coverage and relaxation after deposition. Additionally, the convolution with different tip shapes allows for the generation of a comprehensive range of virtual AFM nanoparticle images, akin to those encountered in practical applications. Furthermore, statistical parameters of nanoparticles are subject to analysis using various data processing algorithms. This analysis serves to reveal any systematic errors, enabling the estimation of uncertainties inherent in the atomic force microscopy analysis of nanoparticles under non-ideal conditions. Such systematic evaluations are pivotal for refining the accuracy and reliability of AFM analyses, particularly for challenging experimental scenarios [60,61].

Another valuable technique for nanoparticle analysis is nanoparticle tracking analysis (NTA). NTA is particularly useful for measuring the size distribution of nanoparticles dispersed in a liquid medium. It leverages the principles of light scattering and Brownian motion to obtain a comprehensive nanoparticle size distribution profile. This technique is instrumental in assessing the size and distribution of nanoparticles in suspension, aiding in various applications, including drug delivery systems and nanotoxicology studies.

Inorganic nanoparticles find wide-ranging applications in chemical surface coatings, encompassing processes like electroless plating, electroplating, silane treatment, and chemical conversion coatings. These nanoparticles bring unique properties and functionalities to coatings, enhancing their performance and durability in diverse industrial and research applications.

In the realm of analytical chemistry, there exists a fervent pursuit to meet the ever-growing demands for monitoring and analyzing substances. Nanoparticle-tracking analysis (NTA) measures nanoparticles size distributions and concentrations within liquid samples. It shines particularly bright when dealing with samples entangled in complex matrices, such as colloid NPs or coatings. Moreover, for the elucidation and assessment of NPs in the context of sample materials and the often-challenging milieu of samples, an invaluable compendium of methods for separation, digestion, and characterization is presented in Table 2. This table likely serves as a concise yet comprehensive reference, offering insights into the diverse techniques available to researchers and analysts grappling with the intricacies of NPs within sample-related domains. Both the development and validation of separation and characterization techniques are crucial in the context of NPs in sample materials. The use of certified reference materials is essential for accurate results in these processes. However, it is worth noting that, except for some rare NPs, such reference materials are currently unavailable.

7. Conclusions

The application of nanoparticles (NPs) represents a promising advancement in enhancing the quality and safety of antimicrobial coatings infused with metal and metal oxide nanoparticles. These cutting-edge materials, with their exceptional properties, stand poised to transform the landscape of antimicrobial substances. Nonetheless, the growing exposure of individuals to antimicrobial nanoparticles, driven by emerging contaminants and nanomaterials, is occurring without sufficient awareness and regulatory constraints regarding their toxicological data. Numerous research initiatives have emphasized the urgent need for more cautious and comprehensive evaluations concerning the commercial use of nanoparticles.

In the sphere of medical applications, it is crucial to strike a delicate balance between the protective benefits and potential risks posed by antimicrobial nanoparticles. In the foreseeable future, we can expect the implementation of legal regulations and procedural frameworks aimed at facilitating the safer and more informed deployment of antimicrobial coatings. These regulatory efforts will heavily rely on robust characterization methodologies that deliver precise and accurate results, as elucidated in this paper.

Consequently, the development and safe implementation of novel characterization protocols for coating materials demand an interdisciplinary and multidisciplinary approach. Such an approach is not only imperative for protecting human health but also for preserving the global environment. Through collaborative endeavors, we can effectively and responsibly harness the full potential of these materials, ensuring that their promising advantages do not compromise our well-being or the integrity of our planet.