Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

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Advanced Industrial and Engineering Polymer Research

journal homepage: http://www.keaipublishing.com/aiepr

Advanced characterization techniques for nanostructured materials in

biomedical applications
Praveenkumara Jagadeesh, Sanjay Mavinkere Rangappa*, Suchart Siengchin
Natural Composites Research Group Lab, Department of Materials and Production Engineering, The Sirindhorn International Thai-German Graduate School
of Engineering (TGGS), King Mongkut's University of Technology North Bangkok (KMUTNB), Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Recent advancements in nanostructured materials have found widespread application across many
Received 16 December 2022 domains, particularly in the biomedical field. Before using nanostructured materials in clinical applica-
Received in revised form tions, many important challenges, especially those related to their uses in biomedicine, must be resolved.
6 March 2023
Biological activity, compatibility, toxicity, and nano-bio interfacial characteristics are some of the major
Accepted 7 March 2023
problems in biomedicine. We may therefore investigate the nanostructured materials for biomedical
applications with the aid of modern characterization techniques. This overview article illustrates the
Keywords:
present state of nanostructured materials in the biomedical field with uses and the importance of
Nanostructures
Characterization techniques
characterization methods through the use of cutting-edge characterization techniques. In this article, the
Biomedical applications techniques for analysing the topology of nanostructures, including Field Emission Scanning Electron
Spectroscopy Microscopy (FESEM), Dynamic Light Scattering (DLS), Scanning Probe Microscopy (SPM), Near-field
Microscopy Scanning Optical Microscopy (NSOM), and Confocal microscopy, are described. In addition, the internal
Morphology structural investigation techniques X-ray diffraction (XRD), Transmission Electron Microscopy (TEM), and
Magnetic Resonance Force Microscopy (MRFM) are discussed. In addition, composition analysis tech-
niques such as X-ray Photoelectron Spectroscopy (XPS), Energy Dispersive X-ray spectroscopy (EDS),
Auger Electron Spectroscopy (AES), and Secondary Ion Mass Spectroscopy (SIMS) have been discussed.
The essence of the nanomaterials as they relate to physics, chemistry, and biology is thoroughly
explained in this overview along with characterization techniques through case studies. Additionally, the
constraints and difficulties with specimen and analysis that are related to comprehending nano-
structured materials have been identified and addressed in this study.
© 2023 Kingfa Scientific and Technological Co. Ltd. Publishing services by Elsevier B.V. on behalf of KeAi
Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction fertilizers, and farming. It is not astonishing that nanostructures

can be modified and incorporated into biomedical tools, given that
In the recent decade, nanotechnology in material science and the majority of biological systems, notably virus, membranes, and
engineering has advanced dramatically in numerous disciplines. protein complexes, possess inherent nanostructures [3e5]. Medi-
NANO is the science discipline there at heart of developing tech- cine and biomedical technology are one of the most intriguing and
nologies with a nanomaterial size of 1e100 nm with variety of difficult applications of nanostructures at present. The continuous
shapes and morphologies [1,2]. It has been demonstrated that growth and advancements in nanostructures lead to different
nanostructured materials with extremely tiny grain sizes have biomedical applications, such as tissue engineering scaffolds,
unusually high strength and toughness, higher diffusivity, benefi- implant engineering, cell culture, and drug delivery applications.
cial sintering capabilities, and other features. The dimensions and Nanotechnology comprises many steps, including the design, pro-
shapes of the materials lend themselves to a multitude of scenarios duction, characterization, and use of functional devices at the
in biomedicine, energy generation, storage systems, coatings, nanoscale, taking advantage of their distinct chemical, physical, and
biological characteristics compared to those of individual sub-
stances [6]. Researchers have studied nanomaterials in areas of
medicine including bio-imaging and biosensors as a result of ad-
* Corresponding author.
E-mail address: [email protected] (S.M. Rangappa).
vances in characterization techniques and their unique features,

https://doi.org/10.1016/j.aiepr.2023.03.002
2542-5048/© 2023 Kingfa Scientific and Technological Co. Ltd. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

resulting in the creation of a number of critical instruments for multiplexing, short reaction times, a wide dynamic range, and the
diagnostic purposes [7]. Proteins adhere non-specifically to the capacity for ongoing analysis are some of the rising demands for
material surface, a phenomenon that is considerably more promi- molecular diagnostic devices that nanostructured sensors are
nent on nanostructures with a high surface-to-volume ratio, which ideally suited to address. In addition to diagnostic instruments,
may exhibit more active areas for future interaction with body cells. sensors are required to investigate biological systems, anticipate
Adhered proteins are called as opsonins, which alter the surface's the progression of illness, and logically create superior therapeutics
chemical compositions and condition it. Before examining all of for the patients [15]. The nanoparticles' safety, which remains a
these interfacial features and applications, it is necessary to char- serious issue, is one of the primary constraints limiting their
acterize nanostructures using advanced techniques [8]. Practically, application. The worry increases when these substances are
all characterization techniques are not suitable for all types of intended to reach the human body, whether on purpose or by
nanostructured materials (metals, carbon based materials, poly- mistake. One may be quite hopeful about the potential advantages
mers, biomolecules, etc.) due to material sensitivity and different of nanomaterials, which highlights the dangers connected with
kinds of handling techniques. Majorly, the selection of particular their usage. There is a rising concern over the possible toxicity and
technique is highly dependent on materials nature and surface detrimental effects of nanostructures on human health, as well as
characteristics. Especially, the care must be taken while character- the absence of a standard approach for assessing the toxicology of
izing the biological sample to keep up the alive nature. The bio- these substances. It was also reported that the small and bare
logical samples imaging is critical and challenging task because of nanostructures possesses more toxicity as compared to bulk and
possibilities in morphology changes [9]. Several nanostructured modifies nanostructures respectively. With their high surface area
substances have already been investigated for biomedical uses. and nanoscale dimensions, nanomaterials have extensive uses in
Different forms of carbon, silica, and metals are the most widely medication delivery. Due to their ability to pass the blood-brain
investigated substances in various rod, tube, cylinder, and sphere barrier, nanoparticles are now the leading study topic in medica-
shapes. These major factors like toxicity and biocompatibility rely tion supply to the brain [16,17]. The overall importance gained by
on a number of variables, including their size, surface area, carboxyl nanoparticles in recent days in different biomedical branches is
groups, quantity, and dose [10]. The topological studies (spatial illustrated in Fig. 1. This illustration also delivers the fabrication
relation, geometric properties) of nanostructures is possible to methods of nanoparticles, their unique characteristics, and appli-
analyse using techniques like Field Emission Scanning Electron cability in various biomedical and health sectors.
Microscopy (FESEM), Dynamic Light Scattering (DLS), Scanning
Probe Microscopy (SPM), Near-field Scanning Optical Microscopy 3. Significance of characterization techniques
(NSOM), and Confocal microscopy. Whereas the internal structure
investigation takes place with the help of techniques like X-ray Nanomaterials exhibit different form, size, constitution, surface
diffraction (XRD), Transmission Electron Microscopy (TEM), and characteristics, molecular weight, solubility, and sustainability in
Magnetic Resonance Force Microscopy (MRFM). Additionally, the terms of their physicochemical qualities. These characteristics are
composition analysis is possible with X-ray Photoelectron Spec- the major critical parameters that defines the physiological per-
troscopy (XPS), Energy Dispersive X-ray spectroscopy (EDS), Auger formance. Consequently, the characterization of nanomaterials is
Electron Spectroscopy (AES), Secondary Ion Mass Spectroscopy required to ensure the safety and reliability of nano-medicines
(SIMS), and X-ray Photoelectron Spectroscopy (XPS) techniques. In during the development phase. Nanomaterials have distinct
this review, it was focused on detailed study on above mentioned physico-chemical properties that influence their in vivo circulation
techniques with their case studies, also incorporated the some and behaviour, respectively, hence it is crucial to identify their
applications of nanostructured materials in biomedical field. distinguishing characteristics. The physical interactions of body
cells and tissues with nanostructures are completely or partially
2. Current scenario of nanostructured materials in different from conventional medicines, hence it is necessary to
biomedical sector utilize compatible and robust characterization techniques for
nanomaterials which helps in providing a route for controlling
In the past five decades, material scientists have conducted toxicity, quality, and safety [18,19]. The main important factor in
substantial research into the use of nanoparticles and nano- subjecting nanomaterials to biomedical field is the bio-
structures in many medicinal and therapeutic applications. compatibility, hence biocompatibility study is much needed
Recently, the synthesis of nanostructures was also established in through potential characterization techniques. The assessment of
advanced level with higher efficiency to increase its applicability in nanostructured materials in various forms such as thin films,
biomedical field. Colloidal inorganic nanocrystals have attracted nanoparticles, and bulk nanostructures necessitates the use of
substantial attention in the biomedical domain due to their inter- specialized techniques that provide the structural and composi-
esting size-subordinate qualities, such as their quantum control tional study of these materials with a suitable spatial resolutions.
impact and confined surface plasmonic impact, which are not Therefore, in addition to the basic procedures accessible in mate-
noticed in their bulk form [11,12]. Recent studies have centred on rials research, additional special characterization methods are
mesoporous nanostructures for delivering therapeutic drugs to necessary for biomedical sector [20,21]. These novel nanomaterials
tumour cells with little medication leakage into active cells. Mes- exhibit optical, electrical, thermal, and structural characteristics
oporous nanostructured materials possesses higher porosity, sur- that are size, form, and structure dependent. For the investigation
face functionality, and tiny pore size provide regulated release of of these innovative nanomaterials, characterization techniques are
the drug and effective drug loading at the designated location [13]. indispensable. The understanding of individual nanomaterials
In other part, the magnetic nanoparticles were gained much more strength and limitations is possible with advanced characterization
importance due to continuous improvements in image guided techniques and possible to get pre
cised results. The in-depth
therapies such as X-Ray CT, magnetic resonance etc. Moreover, structural analysis, molecular interactions, surface purity analysis,
these nanoparticles are also popular in advanced cancer theranosis and elemental identifications are easily performed with different
due to their favourable properties including absorption of X-rays, characterization techniques. Majority of the characterization tech-
high energy transfer efficiency, and generation of reactive oxygen niques are non-destructive, direct measurement tools, reproducible
constituents [14]. Extremely high sensitivity, discrimination, measurements, and possesses maximum spatial resolution in
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Fig. 1. Recent subject on nanoparticles and nanostructured materials and their uses in numerous health and biomedical sectors [12].

atomic scale, hence these are strengths for the characterization of the surface geometry of the substance. The secondary electrons are
nanostructures for biomedical sector. captured by a detector, which then generates an electrical signal.
Finally, every signal is amplified and converted to a display or a
4. Characterization techniques for nanostructures topology digital picture that may be processed and stored. Organs and nu-
and case studies cleus of cells, polymers, and coats are a few examples of items that
are researched using a FESEM in practice [23,24].
4.1. Field emission scanning electron microscopy (FESEM) An interesting work by Nindawat et al. reveals the character-
ization of biosynthesized silver nanoparticles with the help of
The presence of field emission cathode segment in the electron Arnebia hispidissima extracts for the HeLa cancer cells treatment.
gun of a SEM produces narrower probing beams, hence enhancing The FE-SEM images revealed triangular, pyramidal, trigonal bipyr-
spatial resolution and reducing sample charge. These devices are amid, circular, and rod-shaped nanostructures ranging in size from
known as field emission scanning electron microscopes (FESEM). 20 to 200 nm, however spheres were the predominate form [25].
The FESEM resembles a cylinder column attached to a desk. The The synthesized ZnO nanoparticles for antimicrobial activity have
beam of electrons is supported by the column. The analysis of equal size and shape when refluxing with diethylene and tri-
micellar surfaces with the help of scanning electron micrographs ethylene alcohols, both in the presence and absence of sodium
was attempted, and it exhibited with lack of resolution issues acetate medium. The FESEM image shows that the addition of so-
because of earlier technologies with the need of metal layer over dium acetate, the use of various polyols, and the increase in reflux
the sample [22,23]. Recent exposure to a cold field-emission duration from 2 h to 3 h result in a shift in morphologies from oval
extreme high resolution scanning electron microscope (FESEM) to rod-shaped particles with a mean particle size of 15e100 nm
helps to examine the surface features of casein micelles in deeper [26]. Mondal et al. [27] synthesized hollow hydroxyapatite (H-HAp)
level than previously possible. In conventional SEM with therm- nanostructures for the improvements in drug delivery applications.
ionic emission, the electron beam is generated by heating a fila- The FE-SEM analysis revealed that the H-HAp nanomaterials
ment, which is typically a tungsten hairpin. The dimensions of the (Fig. 3(a) and b) are much bigger than the HAp nanostructures
subsequent electron beam is therefore constrained by the filament (Fig. 3 c and d). The typical morphology of H-HAp materials is close
geometry that in turn restricts pixel density and resolving power. A to spherical, while only HAp nanomaterials have variable forms. An
cold field emission source generates electrons by delivering a high innovative work reported by Ahmed et al. reveals the character-
voltage to a very narrow pointed tip and then retrieving the elec- ization of synthesized metallic nanoparticles blended with ε-pol-
trons straight from the point through a tunnels without heating. ycaprolactone (PCL) and cellulose Acetate (CA) for wound
The FESEM module is illustrated in Fig. 2 [24]. The FESEM is a mi- disinfection purpose. The morphology observations of CA/PCL pure
croscope that utilizes negatively charged electrons as opposed to blend through FESEM reveals the non-directional distribution of
light. These electrons are able to scan the objects in the zig-zag fibrous scaffolds with a blend diameters within the range of
pattern. Electrons are freed by an emission source and propelled 0.5e1.1 mm with more porosity ratio. The ZnONPs@CA/PCL blended
by a strong electric field gradient. Inside the section of high vac- configuration follows the neat blended one morphology with a
uum, these primary electrons are concentrated and diverted by scaffold diameters range of 0.45e0.9 mm. Whereas the
electronic lenses to generate a tiny scan beam that assails the item. CuONPs@CA/PCL exhibited random distribution of fibers with
As a consequence, each area on the surface emits secondary elec- diameter ranges from 1.8 to 6.5 mm. The fibrous scaffold's texture
trons. Angle and speed of these secondary electrons are related to seems rough and adorned with spherical [28].

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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

Fig. 2. Illustration of FESEM module [24]. (Reused with Elsevier's kind consent (Lic. No. 5502860079452)).

Fig. 3. FESEM analysis of nanostructured (a, b) hollow hydroxyapatite (H-HAp), and (c, d) hydroxyapatite (HAp) [27]. (Reused with Elsevier's kind consent (Lic. No.
5502860437518)).

4.2. Dynamic light scattering (DLS) measurements requiring only minutes. DLS can differentiate be-
tween a homogeneous monodisperse sample and an aggregation
Dynamic light scattering (DLS) methods have been chosen as sample. It has achieved such market dominance that several nano-
the major approach for measuring the size distribution of nano- colloid researchers depend only on it for size characterization [29].
particles in liquid solutions on the bench. DLS is a reasonably quick The approach is employed for particle characterization in dyes,
approach for determining the size of biomolecules in solution, with paint, and, more significantly, biological diagnosis and medical

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intervention systems, as well as in several other sectors where the permit the quasi-continuous modification of the scattering angle
particle size is crucial. However, the approach has certain applica- across a certain range. In addition to particle size analysis, these
tion limitations, such as in case of working capacity and data con- multiangle dynamic light scattering (MA-DLS) systems are often
tent. DLS is often used to measure the Brownian movement of used for other applications [34,35].
discrete particles in liquids for particle size characterization. In Mani et al. utilized the DLS technique (HORIBA SZ-100 equip-
contrast to different microscopy, the calibrated size is not only a ment) to analyse the synthesized particles size from Solanum sur-
feature of the element core size in air, as well as the hydrodynamic attense leaf for e therapeutic application and also analysed the
diameter, along with the impacts of fluid stabilizing agents, wetting nanoparticles dissolution in colloidal solution. The obtained mean
agents, and the electrical double layer thickness [30,31]. In practice, particle size was 52.27 nm, and silver nanoparticles achieved a zeta
DLS measures a greater size distribution than microscopy methods potential of 20.4 mV. The size of the particles determined by DLS
(TEM and SEM), which need nano-colloids to be transferred to a is slightly larger than the particle size obtained by SEM and TEM
medium and dehydrated. DLS works on the theory of time-resolved micrographs, which is due to utilization hydrodynamic radius
monitoring of coherent light dispersed by scattering objects such as technique for size calculations [36]. The green synthesis of iron
big macromolecules or tiny particles, which is shown in Fig. 4. The oxide nanoparticles from the extracts of dried fruit (Ficus carica)
signals obtained after scattering are analysed based on the fluctu- utilized DLS technique for hydrodynamic diameter analysis in
ations that are obtained from different origins. DLS handles these colloidal solutions. DLS analysis determined the amplitude mean
oscillations, which are induced by the thermal motion of the diameter of nanoparticles as 475 nm, which is the diameter of a
dispersion particles and exist for extremely short time periods. DLS core and the materials surrounding it [37]. The AgNPs extracted
may, for instance, measure the vibrations in nanoparticle connec- from Camellia sinensis through biogenic synthesis was subjected to
tions, allowing for the investigation of phase transformations in DLS equipment with 173
angle and in an aqueous solution at 25
C.
aggregates and the quantification of the elastic characteristics of The DLS tests observed that the mean hydrodynamic diameter size
gels. The most prevalent use of DLS is the measurement of of AgNPs was 34.68 ± 4.95 nm, while the mean PDI value was
submicron-sized particles [32,33]. 0.28 ± 0.01 and the mean zeta potential was 35.50 ± 3.50 mV [38].
Throughout its operation, samples are exposed to a coherence The DLS study was incorporated for the analysis of biosynthesized
monochromatic polarized laser beam light source and detector is copper nanoparticles from C. halicacabum leaves extract for
placed at certain position (typically 173
) to receive the fluctuated biomedical application, and the mean particles size was in the
light with different intensities. This scattered amplitude is depen- range of 30e40 nm. These values are in good agreement with TEM
dent on particle diameter, solute molar mass, and the difference and FESEM surface analysis results. The diverse size distribution of
between the refractive indices of solvent and solute particles. DLS Cu NPs may be attributable to the varying reduction capacities of
functions on the idea that the diffusion coefficient (D) of a particle is several eco-friendly natural compounds abundant in the aqueous
depending on its size, as described by the Stokes-Einstein equation CHL extracts [39]. Another interesting work reported by Patil et al.
[32]: also reveals the metallic nanoparticles extracted from marine mi-
croorganisms show larger particle size in DLS as compared to size
KB T observed in TEM equipment [40].
D¼ (1)
6phRS
4.3. Scanning probe microscopy (SPM)
where KB - Boltzmann constant, T-colloid temperature, h-viscosity
of solvent, RS e radius of sphere. This essentially describes how Scanning probe microscopy (SPM), a crucial innovation in
light reflects at varying rates based on size of particles. The DLS nanoscience, has been used in a vast array of fundamental and
concept can be implemented in several instrumental configura- applied domains at this point. Its application to fundamental
tions. Regarding optical setup, signal modification, signal process- physics resulted in a paradigm shift in the comprehension and
ing, and sensor geometries, they differ. Conventional DLS devices acumen of matter at both the atomic and nanoscopic ranges. By
have a scattered angle that is either sideward (90
) or rearward (at incorporating nanosensors on the tip or linking different electro-
or near 180
). There are also devices with a small number of fixed magnetic waves to the tipesample interface, a vast family of SPM
scattering angles (3e5), allowing for the selection of the optimal techniques has evolved as variants of STM and AFM. SPM-based
angle q for a specific sample. A small number of DLS sensors even techniques can photograph living biological structures with an

Fig. 4. DLS working principle [32]. (Reused with Elsevier's kind consent (Lic. No. 5502860893899)).

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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

astonishingly reduced signal-to-noise ratio in comparison to success. A STM image of DNA nanotubes showed a highly organized
traditional microscopies. In quite often scenarios, the complexity pyrolytic graphite surface with exceptional resolution [47].
exist in biological structures or materials led to use of other fluo- Atomic Force Microscopy (AFM) has been utilized in a number of
rescence microscopes for the clear analysis of morphology through scientific disciplines, notably physics, biology, and engineering
integrated images [41,42]. Scanning probe microscopy is an um- field. Atomic force microscopy (AFM)-based technologies have
brella term for a number of methods, including scanning tunneling developed into a potent nanoscopic platform, allowing for the
microscopy (STM) and atomic force microscopy (AFM). Scanning analysis of a vast array of biological and synthetic bioactive com-
tunneling microscopy is not as necessary as a traditional micro- ponents, including organs, enzymes, films, protein, nucleic acids,
scope since the picture is not able to magnify. The scientists Gerd and functional biomaterials. AFM utilizes a cantilevered system
Binnig and Heinrich Rohrer invented STM and won the Nobel Prize with a molecularly sharpened stylus to raster scan and contour a
in 1986. It is composed of a fine, highly conductive tip (consisting of specimen's surface. That molecularly perforated specimen con-
a single atom) that passes just over the surface of an electrically touring diverts the cantilever and modifies the location of a laser
conducting nanoparticle, and is illustrated in Fig. 5 (a). When the beam that is reflected from the rear of the cantilever onto a
voltage is passed through the tip, nanoparticle electrons tunnel location-sensitive photodiode. These height measurements of the
through the gap between the tip and specimen. The movement of stylus at distinct scan locations are shown as a recreation of the
electrons creates a detectable electric current. As the tip goes over specimen's topology [48]. More often than the STM, the AFM is
the substrate, its location is modified to maintain a consistent utilized to assess surface roughness, which is incredibly beneficial
distance between the tip and the specimen surface. This maintains when working with thin membranes and their development.
the constant flow of current. The mobility of the STM tip is followed Observe that, however unlike STM, the AFM may be utilized on a
and documented. The characteristics are shown visually on a wide range of surfaces, including semiconductors, metal, and
computer monitor. The continuous repeated tip movement gener- insulating compounds, because it monitors force [49]. The atomic
ates a 1-D profile that related to surface height, and is illustrated in force microscope is illustrated in Fig. 6. Vorobjova and team worked
Fig. 5 (b) [43,44]. Krupski utilized STM technique for growth anal- on the synthesis and characterization of a Ni-alumina nano-
ysis of ultra-thin gold films over Mo (110) surface in different di- composite that was synthesized through the electrodeposition of
rections. Examination of STM data reveals that the development of dc-Ni onto a porous anodic alumina template (PAAT). It is evident
the initial two Au layers occurs layer by layer. The alteration in from such images and profiles that upon electro-deposition of Ni
surface morphology was observed when the material was post into the PAAT, pores and dense, equidimensional Ni nanopillars
annealed at 800 K. This results in film rearrangements and both the with a smooth surface were observed. The free development of a
elements forms AueMo alloy [45]. Another interesting study vi- NiNPs array in current phase demonstrates that the Ti coating is
sualizes the development of perovskites over SrTiO3 (001) using conductive and indicates that the PAAT porosity is exposed to the Ti
STM equipment because of the ability to identify the real-space surface [50]. Gomes et al. employed atomic force microscopy (AFM)
structural distribution on the solid thin surfaces. Determining the to evaluate a novel chimeric protein composed of the common
atomic-scale development processes of perovskite thin films ne- repeat for Nephila clavipes spider dragline protein and bone sialo-
cessitates a combination of oxide film formation and STM tech- protein (6mer þ BSP). In the advanced AFM technology, it is
nologies. STM enables instant access to the regional structural and possible to assess the films elasticity and mechanical characteristics
electrical characteristics of a surface via atomically precise surface of soft polymers. Tapping mode AFM imaging revealed that the
topography and spectroscopic evidence, which is important for the majority of the 6mer, when soluble in HEPES 0.1 mM (pH 7.4),
study of TMOs. For contrast, the unrivalled spatial resolution of STM appeared as massive, spherical, unstructured protein groups. The
has presented empirical accessibility to atomic-range images of the diameter and lengths of the aggregates ranged from 0.489 to
nucleation step in film formation [46]. Rafati and co-worker char- 0.736 mm and 122.7e134.7 nm, respectively [51]. AFM has been
acterized the extracted ultrastructure DNA nanotubes using high used in the development of several drugs in recent years, according
resolution STM equipment. This image clearly depicted well orga- to several study papers. The ability of AFM to provide structural
nized nanotemplates, also, the sticky extremities of the main information about molecules at the level of a single molecule al-
nanotemplates could be connected to the DNA nanotubes with lows for a thorough analysis of their behaviour in what are thought

Fig. 5. Scanning tunneling microscope (STM) (a) Schematic illustration, (b) creation of 1-D and 2-D profiles due to variation in height [43]. (Reused with Elsevier's kind consent (Lic.
No. 5502861466667)).

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one of the greatest challenges in the comprehension or subsequent

quantitative analysis of NSOM-obtained optical data. This impact is
readily apparent when scanning the material's steep slopes or
corners (here, it may be clearly predicted) or when scanning a
surface with variable roughness. Topographical distortions often
can affect or totally hide the optical information included in the
dataset, owing to the fact that differences in the specimen refrac-
tive indices and topographical fluctuations can have the same
impact on the NSOM data [59].
Measurements using a near-field scanning optical microscope
(NSOM) are a proven method for approaching the nanometric
features of a complex picture. The current notion of a “cloaked
sensor” applied to an NSOM apparatus in collecting mode,
demonstrating how a correct plasmonic cover deployed to the
NSOM fiber tip might significantly enhance its total measuring
potential [60]. Zhang and colleagues examined the cell endocytosis
of iron Fe2O3 nanoparticles by typical breast epithelial cells
(MCF10A) and cancer breast epithelial cells (MCF7) in the absence
of radioactive identification with the help of NSOM and AFM hybrid
technologies. This dual AFM/NSOM mapping approach is capable of
simultaneously capturing topographical and near-field micro-
graphs, which give complementing morphological and optical as-
sessments of the cellular surface and inside. Without any of the
Fig. 6. Illustration of Atomic Force Microscopy (AFM) [49]. (Reused with Elsevier's kind requirement for radioactive tagging on nanoparticles, the interface
consent (Lic. No. 5502870193410)).
among Fe2O3 and cells may be simply investigated [61]. According
to an intriguing study, high-resolution reflection and topographical
to be the main environments. One of the most useful features of characteristics can be used in other biological uses by using hybrid
AFM is its capacity to watch various physiological and chemical fiber optic NSOM/AFM sensors for live cell fluorescence imaging.
processes in modeled environments in real time while modifying Those recorded NSOM live HASM cell images were taken under
the factors or substance of the liquid medium used. Probably the stabilized conditions [62]. It is a feasible and potential approach to
most complex application of AFM involves observing a molecule's use quantum cascade laser (QCL) and NSOM hybrid technologies for
activity in its native environment [52e54]. (See Fig. 7) the infrared imaging of cancer cells. Observing changes in the
chemical makeup of an OE33 cell through the analysis of pictures
captured at different wavelengths ranging within the Amide I band
4.4. Near-field scanning optical microscopy (NSOM) demonstrates the apparatus potential to provide intuition into
human illness. This device is capable of producing pictures with a
Near-field scanning optical microscopy (NSOM) is an effective spatial resolution much superior to the deflection limit while uti-
technique for optically photographing surfaces with a nano-scale lizing a convenient, economically affordable benchtop source and
magnification. The exposure of NSOM was done during 1980s by benefiting from the QCL's smaller frequency range [63]. Combining
utilizing visible light. Using this innovative optical microscopy IR microscope with AFM, scattering scanning near-field optical
technique, an optical sensitivity of a fraction of the transmitted microscopy (s-SNOM) enables IR nanospectroscopy further than
excitation wavelength is attainable, with its accuracy primarily the percolation threshold of light. Lopez et al. utilized s-SNOM for
dependent on the tip size and tip-specimen distance. Since the the analysis of hydroxyapatite nanofilms morphology for the
advent of NSOM, substantial work has been devoted to the creation biomedical applications. According to Fig. 9(b) and 10(b), the s-
of nanoprobe designs that are particularly advantageous. Near the SNOM studies of the 66 and 198 nm dimensioned films were
tip of a scanning device, the objective is to produce a single, very conducted on the tops of island-shaped nanoparticles and
confined, elevated-intensity near-field point [55]. The primary el- throughout the troughs among the particles. At the same time, the
ements of an NSOM consist of the scanner tip, the feedback system, surface topography analysis of the nanofilms with above
the piezoelectric actuator, and light source. Moreover, the tip point mentioned dimensions are illustrated in Figs. 9(a) and 10(a) [64].
that scans the substrate and retains a consistent height utilizing
piezoelectric steering parts. Fig. 8 depicts a simplified design of an 4.5. Confocal microscopy
NSOM setup featuring light irradiation from the top and light col-
lecting under the sample surface. The sample is illuminated by a Confocal microscopy is one of the potential technique for
light beam via optical cable. The fiber tip is in tapered shape and enhancing the contrast of microscope pictures, particularly for
metal-coated; light pressurizes through a subwavelength-sized objects with a substantial thickness. It offers various benefits over
opening at the fiber tip. The NSOM fiber architecture and circuits traditional optical microscopy, such as controlled depth of focus,
are comparable to those of previous SPM systems, with the improved picture quality, and the capacity to gather optical sec-
exception of a metal film-coated, conical optical fiber replacing the tions from thick materials. Over fifteen manufacturers currently
metal tip. Associated piezoelectric devices regulate the location of provide confocal microscope equipment, including Zeiss, Biorad,
the fiber tip, while the detector monitors changes in the scattered Leica, and Nikon. Confocal microscopy has gained widespread
light caused by minute surface characteristics [56,57]. By exceeding acceptance, particularly for the imaging of biological structures and
the Abbe limit using a relatively close probe, it is possible to capture membrane activity. The confocal microscopy functions more effi-
optical pictures well beyond traditional resolution limitations of ciently than a conventional microscope by lighting just a tiny region
optical microscopy, i.e. beyond nearly 150 nm [58]. The relationship of the material with the resolution limit of the focus and re-imaging
between NSOM optical data and the local surface morphology is light from that region via a pinhole filter bank. The principle of
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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

Fig. 7. Displays the surface SEM images (a,b) and AFM profiles (c,d) of test sample following Ni and PAAT dc-deposition mode at 1.8 V during 5 min [50].

Fig. 8. (a) Primary elements of NSOM setup, (b) Fiber tip and light scattering mechanism [56]. (Reused with Elsevier's kind consent (Lic. No. 5502870624848)).

operation is illustrated in Fig. 11. Structures beyond the lighted be used to create a 3-D picture stack. In another form, the reflec-
region or beyond the depth of the focal plane are not detected. tance confocal microscopy (RCM) was utilized for skin disease
Designs may be derived from any one of the direction, either an imaging and also to identify cellular alterations associated with
upward or downward microscope. Projected laser sources are diseases. In circumstances where resources are limited, this RCM
typically modelocked oscillators having a wavelength approxi- has the potential to enable precise, quick identification of skin
mately 800 nm and a pulse time in the range of 100 fs [65,66]. In disorders. RCM application in low-resource regions has proven
most cases, inverted microscopes are very helpful for cell culture challenging, primarily because of the high cost and limited move-
observation, whereas upright microscopes are preferred for imag- ment of the devices [67].
ing uncovered objects. If the sample has a thick, shaped formation, Confocal fluorescence microscopy is an effective technique for
a sequence of optical segments captured at distinct focus points can observing biological processes. Bednarkiewicz and team worked on

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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

Fig. 9. s-SNOM measurement of a ZnHA film deposited over the course of 60 min (66 nm-thick) showing: a) AFM topographies of the film surface and regions R1 and R2 and b)
nano-FTIR at locations R1 and R2 [64]. (Reused with Elsevier's kind consent (Lic. No. 5502870824216)).

Fig. 10. s-SNOM measurement of a ZnHA film deposited over the course of 180 min (198 nmthick) showing: a) AFM images of the film surface and regions R1, R2, and R3 and b)
nanoFTIR of regions R1, R2, and R3 [64]. (Reused with Elsevier's kind consent (Lic. No. 5502870824216)).

analysing the high resolution images of photon avalanche in or outer cell membranes, according to confocal microscopy analysis
lanthanide doped nanoparticles especially for biomedical applica- [71]. Kihara et al. studied the possibility of nanozeolites structures
tions. This approach takes use of the very nonlinear connection for clinical and biomedical applications. Analysis using confocal
among both photoluminescence intensity and excitation intensity laser scanning microscopy (CLSM) reveals that the majority of
found with PA, which reduces the cumulative distribution function nanozeolite particles cling to the surface or vicinity of the cultivated
to below 50 nm whenever the discontinuity reaches 50 [68]. In HeLa cells, but only a few adherent nanozeolites were absorbed
antiproliferative activity test for biosynthesized TiO2 and Ag NPs inside the cells. The CLSM imaging is illustrated in Fig. 12 [72].
reveals the survivability of cells through the confocal microscopy.
The biological cells treated with TiO2 nanoparticles showed a 5. Characterization techniques for nanostructures internal
consequential structural changes. Moreover, both the nucleus geometries and case studies
discrete process and condensation were observed with majority of
dead cells [69]. Wu et al. revealed the adaptable biopolymer sub- 5.1. X-ray diffraction (XRD)
strate for the advancement of nanodiamonds (NDs) as novel ma-
terials for biomedical uses. In this work, the emergence of the X-ray diffraction is currently a standard method for studying
anticancer drug doxorubicin from NDs was tracked using confocal crystalline structures and atomic spacings. It offers knowledge on
microscopy. The laser excitation of 532 nm and 560 nm filter were topologies, phases, preferred textures, and additional structural
used to acquire confocal microscopy pictures [70]. The antibacterial data, including average grain dimensions, crystallinity, strain, and
action of ZnO nanoparticles may include both formation of reactive lattice parameters. The apparatus utilized for diffraction data ex-
oxygen species (ROS) and deposit of nanoparticles in the cytoplasm periments has not evolved much since the late 1940s. Advanced
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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

Fig. 11. Illustration of confocal microscope's operating principle [65]. (Reused with Elsevier's kind consent (Lic. No. 5502880804665)).

Fig. 12. CLSM photograph of HeLa cells subjected to nanozeolite LTL. HeLa cells (A) and HeLa cells employed to 10 mg mL1 GFP adsorbed nanozeolite LTL-90 (B, C) were analysed by
CLSM (Scale bar: 50 mm) [72]. (Reused with Elsevier's kind consent (Lic. No. 5502881119100)).

equipment is distinguished by the utilization of the intermediate

computer system for control, data gathering, and data manage- nl ¼ 2dsinq (2)
ment. The patterns of X-ray diffraction are generated by the
constructive interference of a homogenous beam of X-rays reflected where l is X-rays wavelength, n is an integer value, d is the value of
at particular angles from each pair of crystal planes in a material. inter-planar spacing, and q indicates diffraction angle. The XRD
Atomic dispersion inside the crystal lattice determines the peak instrument output is commonly referred to as a diffractograms, in
intensities. X-ray diffractometers include three fundamental com- which the y-axis represents intensity and the x-axis represents
ponents: an X-ray tube, a sample stage, and an X-ray detecting scanning angle. The obtained peak forms and widths reveal crys-
sensor. Initially the electrons are ejected from cathode ray tube by tallite size, stacked faults, and antiphase limits. Along with crys-
heating filament and accelerating those ejected electrons towards talline structure and phase analysis, the X-ray diffraction technique
the sample target with the application of voltage (Fig. 13). When- is also popular in thin film observations, texture analysis, and
ever electrons have enough strength to eject inner shell electrons stress-strain observations. XRD has the potential to characterize the
from the surface, distinctive X-ray spectra are created. The corre- contents and physical structures of medicines in drug delivery
lation between incident X-rays and obtained interference satisfies applications. XRD is the most important method for solid-state
only with the Bragg's law principle (Equation (2)). This equation drug analysis, with applications in all phases of drug discovery,
establishes a relationship between the wavelength of electromag- assessment, and manufacturing. Among the most crucial pre-
netic waves, the reflection angle, and the lattice parameter of requisites for the examination of powder substance by X-ray
crystalline samples [73,74]. diffraction is appropriate sample preparation. This remark is
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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

Fig. 13. Illustrations of X-ray diffraction (XRD) [74]. (Reused with Elsevier's kind consent (Lic. No. 5502881371906)).

particularly accurate for sands and sandy clay that include finely extra phases. The crystallite size of the nanoparticles falls below the
split colloids, which have been poor X-ray reflectors, in addition to 12.5 nm dimension [81]. Jahan et al. synthesized and characterized
other category including iron oxide coatings and organic com- the non-toxic natured Cu particles for antibacterial applications.
pounds that complicate XRD analysis [73,75]. First, since more Five major responses were recorded at 43.49
, 50.64
, 74.38
,
distribution and instability of the component phases are defining 90.25
, and 95.48
, which are pretty consistent with the normal
characteristics of nanomaterials, their diffractograms are accom- metallic Cu spectra. Furthermore, the interplanar distance values
panied by substantial widening of patterns up towards merging were 2.084, 1.806, 1.277, 1.090, and 1.043 Å, confirming five basic
with the background. Second, the issue of establishing quantitative reflections, which are compatible with the planes of (1 1 1), (2 0 0),
relationships among phases complicates structural analyses of (2 2 0), (3 1 1), and (2 2 2) respectively. The obtained crystallite size
nanocomposites. Third, interconnections in between component was 14.8 mm with a lattice constant value of 0.3610 [82].
nanophases of a nanocomposite structure might result in nonad-
ditive scattering. Finally, the interblock borders and high structural 5.2. Transmission electron microscopy (TEM)
defects are frequently present in nanocrystalline materials,
together with localized aberrations of the periodic pattern. The Transmission electron microscopy (TEM) has developed as a
diffraction peaks varies as a result of the factors described above foundation of characterization in all branches and sub-branches of
[76]. science its debut over a century earlier. TEM delivers extensive,
Salunkhe and co-workers reported the structural analysis of directly resolved structural data and behaviour of occurrences
PVA coated and uncoated CoFe2O4 nanoparticles for biomedical spanning atoms to microns in size, which are both basic and
applications. The presence of several, comparatively high reflection beneficial for society [83]. TEM is a useful tool for examining small
peaks in the range of 20e100 nm area of CoFe2O4 nanoparticles, features with typical diameters less than 100 nm and even atomic
which are remarkably confirms that the nanoparticles possesses scale for special cases. The TEM was founded by Max knoll (German
spinel cubic structure and fd3 m space group. Fig. 14 clearly illus- engineer) in the year 1931. The initial TEM had a resolution of just
trates the diffractograms for PVA coated and uncoated CoFe2O4 around 17X. Marton and his coworkers in Brussels developed
nanoparticles. PVA-coated particles showed no change in phase, comparable microscopes, and by 1934, they had produced the first
however it is still possible to see a little reduction of the diffraction photographs of nuclei. The construction of these ancient TEMs,
peaks. The crystalline sizes for PVA coated and uncoated nano- which used a series of horizontal lenses in a column, differed from
particles were found to be 38 and 35 nm respectively [77]. The the design of current TEMs, which use a sequence of stacked
polyethylene glycol coated nanostructures for biomedical applica- vertically lenses in a column. Due to the poor magnification, the
tions possess a diffraction peaks at 30.13
, 35.48
, 43.12
and 62.81
, TEMs with lenses in lateral series were quickly discarded. However,
which confirms the crystalline planes at (2 2 0), (3 1 1), (4 0 0) and vertically aligned lenses in columns may be able to maintain good
(4 4 0) respectively. This also exhibited the cubic spinel crystalline alignments for a longer duration of time due to increased gravity
structure. The peak intensity observations reveals the decrement in pressures corresponding towards the optic axis resulting in lesser
crystallinity with PEG modification [78]. The XRD plot for the mechanical creep. Nevertheless, with in context of a TEM, almost all
synthesized biogenic silica nanoparticles showed a peak value at of the inbound electrons are transferred via sample, assuming it is
22
matching to amorphous silica nanoparticles, with no additional sufficiently thin. Even though those electrons are not ingested, they
impurities found [79]. Ghaffari and team worked on prevention of are dispersed (deflected in their course) by the sample's atoms. To
H1N1 virus infection using PEGylated zinc oxide nanostructures comprehend the generation and understanding of TEM pictures,
and compared the diffraction patterns among them. The dif- we must look into the characteristics of the diffraction mechanism.
fractogram result reveals the no changes in relative intensity and The schematic illustration of recent TEM and its principle is illus-
atomic spacing in modified ZnO nanostructures [80]. The synthe- trated in Fig. 15. The imaging procedure involves generation of
sized magnetic nanostructures using FeCl2$4H2O and FeCl3$6H2O electron beams from the gun, followed by focus through metal
(0.1 M) gained popularity hyperthermia applications, and Múzquiz- equipment and electromagnetic lenses. The behaviour of electrons
Ramos et al. reported the characterization of those nanoparticles. in the form of negatively charged particles that are deflected by
The obtained plot follows JCPDS card 39e1346 standard with no electromagnetic fields gives rise to the process for the
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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

Fig. 14. (a) Diffractograms for coated and uncoated CoFe2O4 nanoparticles, (b) PVA coated SAED pattern [77]. (Reused with Elsevier's kind consent (Lic. No. 5502890004340)).

concentrating phenomena of electrons. This process produced a own, including or excluding very little manual interaction, as big
beam of electrons with a clearly defined energy spectrum since data evaluation and machine learning advance [88].
only electrons inside a narrow energy range allowed to pass across. Kumar and coworkers reported the synthesis and analysis of
This transmitted electron beam is subjected through the sample zirconia nanostructures for oral cancer detection application. The
and phosphorescent plate glow will be observed after hitting the nanostructures shape and size were analysed using TEM, which
electrons that were passed through sample [84e86]. According to reveals the sheet structure and hexagonal shape of the particles.
the recent publication [87], single proteins may be seen using an The separate ZrO2 nanoparticle was subjected to HR-TEM obser-
electron microscope based on a multi-pass measuring methodol- vations, and it reveals the polycrystalline nature of the nano-
ogy without aggregating geometries across multiple pictures. This structures. It was clearly observed the lattice spacing of 0.318 nm
method has a wide variety of applications and is anticipated to corresponds to (1 1 1) plane [89]. The nanocomposites made of
enhance accuracy and resolution for a number of electron micro- hydroxyapatite and graphene oxide exhibited an excellent
scopy imaging modalities, such as, for instance, scanning and biocompatibility suitable for drug delivery and orthopaedic pur-
spectroscopy methods. As of now, TEM data collecting mainly de- poses. The TEM morphology confirms the absence of agglomera-
pends on the expertise and skill of the TEM specialist. It is antici- tions inside the nanocomposites and estimated the HAp nanorods
pated that computer would obtain high-quality TEM data on their length range between 60 and 85 nm [90]. Yuvakkumar et al. utilized

Fig. 15. Schematic illustration of transmission electron microscope [84]. (Reused with Elsevier's kind consent (Lic. No. 5502890215498)).

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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

TEM instrument for characterizing the surface adhesion by the tip [93]. The MRFM technique utilizes cantilever as like AFM in-
biosynthesized extract and for the analysis of ZnO bioinspired strument to achieve resonance spectrum. AFM and NMR are
morphology (Fig. 16). It is obvious that the biosynthesized ZnO had effectively combined in MRFM. As per Fig. 17 illustration, the whole
to have a sphere and hexagonal shape, an estimated size from 50 to setup mainly divided into northwest and southeast parts, where
100 nm, and also have some agglomerations in the nanostructures. those are corresponds to AFM and NMR respectively. If the elec-
The variety of shapes and sizes were observed with an increase of tromagnetic wave frequency created by a coil meets the resonance
incubation time. After 4 days of incubation, the multi-sized chain criterion at a particular location, the magnetization of sample
structures were observed and the sphere shaped particles inter-
twined themselves [91]. Rasheed et al. [92] worked on character-
ization of biosynthesized Ag nanoparticles with the help of
Artemisisa vulgaris leaves extract for human cancer cell treatment.
Numerous TEM images used to investigate the morphology of AV-
AGNPS revealed that the silver nanoparticles were of various
sizes and shapes, including a median particle size of about 25 nm.
Even though the majority of the particles did not coalesce, some
nanoparticles appeared to be covered in irregularly shaped
materials.

5.3. Magnetic resonance force microscopy (MRFM)

For fundamental research in biology and medical field, magnetic

resonance force microscopy (MRFM) offers a paradigm-shifting
potential by sensing MRI signals from nano-scaled samples.
Nanoscale magnetic resonance imaging aims to visualize nuclear
spin concentrations inside the materials in three dimensions with
near-atomic magnification and on length scales as small as some
100 nm. MRFM is the high calibre candidate for magnetic resonance
imaging. The technique makes use of a very sensitive nano-
mechanical transducers to identify magnetic force interactions
among nuclear movement inside the material and a tiny magnetic
Fig. 17. Illustration of the MRFM configuration [94]. (Reused with Elsevier's kind
consent (Lic. No. 5502891137231)).

Fig. 16. TEM images: (a to c) 1, (d to f) 4, (g to I reaction result from 0.1 M Zn(NO3)26H2O and 10 mL extract after 7 days [91]. (Reused with Elsevier's kind consent (Lic. No.
5502890388902)).

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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

location will change. Consequently, the force felt by the test spec- towards high magnification spectroscopy, which was formerly
imen on the cantilever also varies. This level of deflection in the called ESCA (electron spectroscopy for chemical analysis) [100]. The
cantilever is recorded using interferometer. This is due to magne- XPS approach highly dependent on chemical formulations of an
tization force observed by the MFRM and the potentiality of the atom and chemical shift phenomenon. The binding energy be-
gradient decides the image and force resolutions [94]. In the im- tween core electrons is a key parameter in analysing the bonding
aging apparatus, a nanometer-scale magnet creates a strong structures through reflected electrons. The whole instrument
gradient field. The resonance segment, whereby magnetic reso- works on the principle of photoelectric effect that was invented by
nance takes place, is narrow in comparison to specimen thickness. Hertz. Fig. 18 depicts the conceptual setup for XPS studies. Upon
The resultant signal is composed of the specimen picture and the analysis, x-rays light of defined energy are used to incinerate the
inherent spectrum. The main principle behind the MFRM is the sample, resulting in the photoelectric effect. After incidence of
detection of magnetization force among electron spinning in the electrons, some are reflected back from the sample surface and are
sample and ferromagnetic tip of the equipment. In order to expand received at spectrometer analyser. The amount of electrons pro-
the MRFM approach to nuclear atoms spins, which is essential for duced per unit time is correlated with their energy. This plot of
imaging organic substances, the saturation magnetization sensi- energy v/s intensity gives XPS spectrum. In the advanced XPS
tivity must be increased by at least a factor of 1000, as nuclear spins apparatus, mainly two approaches are preferred for sample radi-
interact approximately 600 times less strongly than electronic ance using monochromatized source. In the first scenario, the X-ray
spins. Recent applications of extremely sensitive MFRM to mag- beam is concentrated together into small point (probe) with a
netic resonance imaging have enabled 3D imaging with a diameter of hardly few micrometers, allowing for spatially-resolved
geometrical resolution of 4 nm in a biological material [95]. studies, or it is rastered more than the sample area to be studied if
Another important factor that defines the spatial resolution is this is not necessary. Nevertheless, the need to concentrate x-rays
deconvolution processing. Concerning the deconvolution phase, an to a tiny point necessitates a smaller radius, resulting in an adverse
exact force reaction must be calculated employing precise readings effect on the energy precision and X-ray propagation. In the alter-
of the magnetic field distribution of the nanoparticles. Restoration nate method, the region to be studied is determined by the entry
of an MRFM picture is difficult since resonance happens in a shell slit in the analyser apertures and the material is effectively inun-
with a significant curvature which goes further than the scanning dated with a rather broad x-ray beam of 1e2 mm [101,102]. Modern
limit [96]. XPS devices are trustworthy and consistently deliver the same re-
Magnetic nanoparticles contain exceptional magnetic charac- sults from the same samples, usually with a fraction of fluctuation.
teristics, such as superparamagnetic activity, and range in size be- Care should be exercised when directly comparing data collected
tween 1 and 100 nm. Consequently, they are of tremendous value in using various XPS instrument settings. For instance, high pass en-
biological applications, particularly in catalysis and cancer-target ergy is ideal for carrying out a quantitative examination of atomic
treatment. They remind readers that the typical chemotherapy compositions since the counts are larger and phenomena such
medications that are injected into the blood circulatory system spectrometer scattering have less of an impact. A low-pass energy,
have poor control over drug delivery, and as a result, they highly magnified spectrum is necessary to differentiate an ele-
frequently cause severe harm to both cancer cells and undamaged ment's several simultaneous states since this is possible. The pro-
tissues. Targeted medication delivery, on the other hand, reduces portion of components at every state may be determined using the
toxicity, and significantly improves the efficacy of pharmaceuticals. lower power spectrum, and this knowledge paired with the high
As prospective magnetically directed drug solid lipid nanoparticles, pass energy quantitative analysis [103]. The binding energy that
several types of magnetic nanoparticles have been created and resides between photon electrons and the kinetic energy of the
explored for these objectives. Characterization of these magnetic electrons were used to analyse the quantitative analysis, which
nanoparticles requires a special magnetic resonance imaging includes component identity and chemical condition. Different XPS
technique for clear analysis. In these nanoparticles list, the most image kinds are employed in some applications to ascertain the
preferred one is iron-oxide and its different forms [97,98]. Sievers
et al. established a magnetic moment measuring resolution of
1018 A m2 for nanoparticles. For their measurement equipment
employing a commercialized apparatus operating in self-excitation
mode, the resolution of their apparatus limits the magnetic
moment value that may be successfully determined [99].

6. Characterization techniques for nanostructures

composition analysis and case studies

6.1. X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS) has emerged as one of

the most widely adopted approaches for surface investigation, and
XPS apparatus has grown more operator-friendly, making the
technique accessible to a vast number of scholars. This technique
involves bombarding the sample surface with x-rays and
measuring the reflected electrons. It will be considered for kinetic
energy measurements. This approach is highly effective due to its
surface sensitivities and capability to provide chemical data frames
from the study sample. This technique is feasible for almost all
types of materials and elements except helium and hydrogen. The
first XPS was invented by Kai Siegbahn in the 1960s. In the year Fig. 18. Conceptual illustration of XPS setup [101]. (Reused with Elsevier's kind con-
1981, he was awarded the Nobel Prize for his developments sent (Lic. No. 5502891349326)).

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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

distributions of constituents and chemical states as a function of 6.2. Energy Dispersive X-ray spectroscopy (EDX)
surface position. The energy scale of the XPS device must be
accurately calibrated, and the recharging of non-conductors during The discipline has advanced quickly since the initial demon-
XPS measurements must be correctly controlled or corrected [104]. strations of atomic-resolution electron energy-loss spectroscopy in
Isacfranklin et al. performed XPS analysis for single phase Cr2O3 scanning transmission electron microscopy (STEM). Utilizing
nanostructures that are well suited for cancer treatment applica- energy-dispersive x-ray spectroscopy (EDX), a specific atom spe-
tions. The main peak in the XPS spectra was correlated to Cr(III), cies' locations in nanostructures have been mapped using in STEM
accompanying to 576.86 eVe576.96 eV and Cr2p1/2 exist in the fields. This technique involves raster scanning a coherent focused
range from 593.8 eV to 594.01 eV. The other peaks at probe all over the object while recording the resulting x-ray
530.5 eVe530.64 eV and 530e531 eV corresponds to the CreO and emission spectra at each probe point. An elemental mapping is then
hydroxyl groups respectively [105]. Jiang and team worked on created using this spectrum [110]. In many biological fields, scien-
bimetallic XPS analysis on AueAg nanomaterials for clinical appli- tists and medical professionals use EDX elemental research. Semi-
cations. The combined spectra between Au0 and Ag0, which was qualitative and quantitative information are revealed in the bands
specifically illustrated for Au and Ag, respectively, in Fig. 19A and B, produced by EDX research. The EDX technique is useful in phar-
is explained by the peaks at 83.87 and 88.27 eV (Au0) and 373.37 macological research because it can be used to pinpoint nano-
and 367.57 eV (Ag0). The peaks at 533.8 eV and 167.2 eV, which particles, such as in the examination of drug dispersal. EDX is also
indicate the participation of the O 1s as well as the S 2p, respec- used to analyse environmental contaminations and describe min-
tively, and illustrate the metal-S binding's capacity to build Au0 and eral bioaccumulation in tissues. Despite being a potent instrument,
Ag0 there at S-containing core and outside of the protein scaffold EDX has some drawbacks, such as the failure to differentiate be-
[106]. In the literature, an interesting work was reported by doping tween ionic and non-ionic elements and the need for an air-free
cerium (IV) into hydroxyapatite and characterized the same for vacuum area. Inter-element interference, also referred to as peak
clinical applications due to its atoxic potentiality. The XPS spectrum overlap in X-ray spectrometry, typically has a major detrimental
analysis performed over nanostructure revealed that the Ca2 p has effect on X-ray detection, and presents significant difficulties for
two unique peaks at 347.5 and 350.4 eV, which are Ca2 p3/2 and Ca2 elemental analysis. As a result, elements with atomic values higher
p1/2, accordingly. The existence of the phosphate group (P2p) in than 10 are common [111]. The majority of peaks in the EDX
HAP constrained the distinctive peak at 133 eV. An oxygenated sp2 spectrum are generated by X-rays that are created by distinctive
carbon, CeO, is supported by a peak at 286.29 eV, while there are fluorescence light. Every element with in periodic table has such a
two anoxygenic aromatic sp2 carbon rings (C1s), CeC and 284.5 eV, specific atomic arrangement that results in a specific pattern of
respectively [107]. The calcium phosphate nanostructures doped by peaks on an X-ray wavelength range, which is significant to this
selenium and silver particle incorporation showed a diverse po- evaluation approach. The sample generates recognizable X-ray
tentiality in biomedical field. All the anticipated elements, such as emission whenever a high-energy potential emitted particles, such
Ca, O, C, P, and Se, were detected in the XPS spectra of selenium as electrons, protons, or an X-ray beam, is impacted on it. In energy-
based nanostructures during the production procedure. The peak at dispersive evaluation, the emerged particles energy is sorted to
58 eV, which was SeO32- [Se (IV)] correlated, suggested that SeO32- look into a certain fluorescence radiation. The amount of individual
group had been integrated in BCP-NPs structure. Additionally, the element contained in the sample determines the intensity of each
absence of Se (0)'s peak at 55 eV demonstrated that the redox re- radiation. An EDX unit comprises of various components, and
action took place while doing sample preparation [108]. Khan and Fig. 19 illustrates the operational procedures. A solid-state sensor is
group worked on biosynthesis and characterization of chromium a feature of all energy-dispersive spectroscopy [112,113]. The Si(Li)
oxide nanoparticles from Abutilon Indicum extract for advanced detector, which can be chilled to cryogenic levels using liquid ni-
biomedical applications. Five peaks were found in the XPS spectra, trogen, is currently the most used detector. Nevertheless, silicon
with binding energies of 284.5, 400.9, 530.9, 576.9, and 586.8 eV. drift detector with Peltier cooling systems are frequently used in
These peaks stand for C1s, N1s, O1s, Cr2p3/2, and Cr2p1/2, corre- modern models [114].
spondingly. Except chromium and hydrogen, the nitrogen and Singh and coworkers conducted an eco-friendly biosynthesis of
carbon peaks owing to phytomolecules adsorption [109]. Au and Ag nanoparticles and characterized the same for biomedical
applications. AgNPs and AuNPs both had their largest peak in the
EDX spectra at 3 keV and 2.15 keV respectively. Moreover, the metal
ions from copper and carbon were also observed in the normal
peaks [115]. The synthesized biogenic nanostructures from rice
husk possesses excellent properties for tissue engineering appli-
cations, and the EDX spectra for these shows an existence of the
elements like silica, carbon and copper. Both copper and carbon
components matched the carbon-coated copper grid system uti-
lized to mount the samples, in accordance with the EDX data. The
outcomes supported the fact that the manufactured samples were
silica nanoparticles that have been extremely pure [116]. An inno-
vative multifunctional bioactive glass nanostructures incorporated
with Mn has higher potential for bone tissue applications. The EDX
results revealed the incorporated Mn and P elements in the
nanostructures, and EDX mapping confirms the uniform distribu-
tion of Si, Ca and Mn elements. Nanostructures' P and Ca peak in-
tensities are rising while their Si peak intensities are falling,
according to EDX spectra. The calcium phosphate deposition was
confirmed in nanostructures after immersion in simulated body
Fig. 19. A description of the principle behind energy-dispersive X-ray spectroscopy fluid [117]. Sood et al. reported a synthesis of gold coated Fe2O3
and elements [112]. (Reused with Elsevier's kind consent (Lic. No. 5502900226567)). nanoparticles and discussed the significance in drug delivery and
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P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

contrast agent applications. The EDX analysis revealed the peaks for resolving power of a substance of the examined solid, Fig. 20 (a)
Au, Fe, and O elements, which validates the existence of Fe2O3 and demonstrates that the incoming electron beam impacting the ob-
Au nanoparticles inside the sample. The spectroscopy also showed ject permeates to an in-depth of 1e3 mm in such an enlarged solid.
a peak for sulphur, indicating that the altered SA was successfully Due to the extremely short inelastic mean free paths, which are
attached to the iron oxide and gold core nanostructures. Carbon depicted by the grey squared box shown in Fig. 20 (b), the majority
contained in the sample container may cause a peak to emerge that of the Auger electrons created with in activation volume inside the
corresponds to carbon [118]. The potentiality of silver oxide nano- case of such a stretched solid, though, cannot move near to the
particles has been applied as anticancer agents, and their EDX surface. Only those produced inside the particular data depth of a
spectrum showed the formation of oxides after the chemical re- few nanometres that defines the limited AES analysis volume that
actions. As a consequence, an investigation of the findings obtained will get to the analyser. The AES illumination volume may indeed
has demonstrated that the target product is free of any additional bi be limited to that same volume of the nanoparticle in the instance
products, such as nitrates or other metal oxide ions. The spectrum of nanostructures analysis. The application of purely elements
reveals that the measured silver oxide atoms proportion is quite sensitivities factor derived with semi-infinite, pure materials will
comparable to the provided data values [119]. The cobalt doped result in significant measurement errors in quantitative in-
Fe2O3 nanostructures EDX spectra presence of O, Fe, and Co ele- vestigations of nanomaterials [124e126].
ments with their atomic percentages of 54.51, 30.7, and 14.79 The charging impact on non-conducting objects is among the
respectively. This study also verified the precise CoFe2O4 stoichi- most frequent constraints in AES. Coatings used to lessen specimen
ometry (1:2:4) [120]. charging for those other electron-beam-based methods are often
not utilized in AES owing to its large surface responsiveness since
6.3. Auger electron spectroscopy (AES) they may consume the full sampling depth and prevent the sam-
ples from being examined. However, due to significant charging
The elemental formation of surfaces, thin films, and interfaces problems with these extremely insulated biological materials, it
can be determined semi-quantitatively using the noninvasive core- was required to sputtering coat the latter with AuPd [127]. Rades
level electron spectroscopy technique known as Auger electron et al. characterized the silica nanoparticles with the help of Auger
spectroscopy (AES). AES has recently made advancements that have electron spectroscopy and discussed the methodological aspects.
broadened its application further than just analyzing the elemental Analysis of dielectrics, such as silica, can reveal charge phenomena
formation of the substrate. For instance, magnetic solid surfaces that impact Auger electrons and subsequently Auger spectra.
may be studied using the spin polarization of Auger electrons. Charged particles may be identified by their brighter edges and
Additionally, the outcomes of investigations using resonant Auger darker inside. On the spectrum, meanwhile, only minor peak en-
electron spectroscopy offer data pertinent to femtosecond charge ergy changes can be seen. In regard to variables that are well-
carrier kinetics. Surface morphology can also be detected by auger established for SiO2, the silicone peaks' energies move towards
electron diffraction [121]. The Auger impact, a series of radiation- lower energies while the oxygen peak's does so into higher energies
free transformations that occur when one of an atom's internal [128]. The existence of Ag, Ti, and O elements in the composite layer
level gets ionized, serves as the foundation for Auger electron as-obtained was verified by the AES analyses of Ag sputter-
spectroscopy (AES). An Auger electron, a kind of electron, is ejected deposited TiO2 nanotubes for antibacterial coating application.
at the conclusion of such a procedure. In the past, the French sci- Domestically obtained Auger survey spectrum at the Ag/TiO2 NT
entist Pierre Auger and the Austrian scientist L. Meitner developed surface are shown in Fig. 21(a). Upon that surface of an Ag-
independently the Auger effect in 1922 and 1923, respectively. The incorporated TiO2 nanotube, the usual AES spectra of silver was
majority of contemporary, commercialized Auger electron spec- observed with a resolution of 0.2 eV in Fig. 21(c), alongside the
trometers offer extremely sensitive, non-destructive chemical corresponding silver reference spectrum. The Zn, O, and Ti ele-
investigation of a solid substrate. Because surfaces are the primary ments existence in the as-obtained composite as illustrated in
objects of concern in several scientific and technical domains, this Fig. 21(b) was validated by the AES analyses of ZnO electro-
method is extensively used in those sectors. Additionally, studies of deposited TiO2 nanotubes. With the proper zinc referenced spec-
open surface and grain-boundary stratifications in polycrystalline trum, Fig. 21(d) displays a standard AES spectrum of zinc recorded
materials constitute a significant field of applications for AES [122]. at a range of 0.2 eV on a ZnO-loaded TiO2 nanotubes substrate
AES needs an ultrahigh vacuum (UHV) environment to maintain a [129]. Roguska and team studied the antibacterial behaviour of Ag
clean surface while collecting data since it is a surface-sensitive loaded TiO2 nanotubes and characterized the same using AES in-
technology. Around 106 torr, one single layer of external addi- strument. Prior to and after deposition of Ag, the standard Auger
tives will typically be accumulated in 1 s assuming every contam- electron spectroscopy (AES) inspection scans were taken on the
ination atom in the vacuum system that touches the sample surface TiO2-NT/Ti surface. The indications from Ti and O can be clearly
clings to it. Auger examination of accumulated surface really distinguished from both scenarios. The TiO2-NT/Ti surface also
wouldn't disclose the original surface components, only the shows contaminants with C and a helpful integration of F, which
contaminated layer. Base pressures in the limit of 1010 are presumably occurs from the procedures. The existence of Ag inside
frequently necessary to reduce the aggregation during analysis the composite layer as-obtained was verified by AES analyses of
[123]. The cylinder chamber of the analyser are positioned TiO2 NTs with sputtering of Ag [130].
concentrically in AES equipment, with the inner cylinder being held
at earth potential and the outermost cylinder being maintained at a 6.4. Secondary Ion Mass Spectroscopy (SIMS)
negative potential. Through an opening, electrons from the mate-
rial under analysis reach the area among the cylinders where they Secondary Ion Mass Spectrometry (SIMS) is an established
are refracted by that of the negative potential on the outermost method of surface analysis with several uses in materials research.
cylinder. Auger electrons with a range of energies could flow via the SIMS is a surface examination method with trace element sensi-
analyser by changing the potentiality on the outside cylinder. The tivities, sub-nanometer depth accuracy, and a horizontal resolution
main elements attached to the concentric cylinder analyser are of 5 nm. It is possible to create a 3D SIMS chemical mapping of a
electron gun, optical tools, deflection plate, and detector. According substance by combining SIMS in-depth profiling with imaging
to its first kinetic energy and inelastic backscattering linked to the [131]. In a typical SIMS experimental procedure, an intense primary
137
P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

Fig. 20. (a) For Auger electrons, the fundamental excitation volume and escape depth, (b) The reported kinetic electron energy ranges for AES and XPS (box) are given [124]. (Reused
with Elsevier's kind consent (Lic. No. 5502900593110)).

Fig. 21. Regional survey AES spectra were collected at the following surfaces: (a) Ag/TiO2 NT surface; (b) ZnO/TiO2 NT surface; (c) high resolution Ag MNN spectrum; and (d) high
resolution Zn LMM spectrum [129]. (Reused with Elsevier's kind consent (Lic. No. 5502900872105)).

ion beams such as one of Gaþ, Csþ, or Arþ is directed onto the sensitivity and prevents compositional depth characterization
surface of a substrate under extremely high vacuum. Subordinate [132,133]. By giving actual compositional data at the 50 nm to many
ions are released from the specimen surface due to the primary ion micron length scale, the SIMS method to biological applications has
beam's contact with it, and they are then collected and injected into the capability to connect the gap among atomic-range structures
a mass spectrometer. Insufficient secondary ion concentrations and and traditional light microscopy. Depending on the equipment ar-
beam-induced damage impacts resulting from the employment of chitecture, there are two basically distinct methods for SIMS anal-
atomic primary ion beams have historically prevented the broad ysis of biological specimens: ToF-SIMS and dynamic SIMS. In ToF-
application of SIMS for the investigation of organic structures. SIMS type, the principal ion beam is flashed, and a ToF mass
Organic materials often necessitate “static SIMS” analysis under spectrometer finds the resultant secondary ions. With this method,
circumstances in which the principal ion fluency is regulated below the entire mass spectra may be viewed while the assessment is
a certain dosage to preserve the surface's integrity, which decreases being done. In comparison, dynamic SIMS uses a continuous

138
P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

principal ion beam and a magnetic segment mass spectrometer to

identify a predetermined set of ions [134,135]. Fig. 22 displays a
diagram of the SIMS fundamental concept. In a nutshell, SIMS
equipment employ an ion gun to create a finely concentrated,
powerful primary ion beam that assails the sample surface. This
beam generally has between 25 and 30 keV of kinetic energy. A
colliding cascade is created during the main ion incident on the
higher surface layers, and as a result of this energy loss, bindings
are disrupted and secondary particles are released. Most of the
nanoparticles that are released are neutral, but a tiny fraction (less
than 1%) possesses either polarity and, depending on the electric
field that is placed between the extractor and the specimen, are
amenable to examination by a mass spectrometer. The main ion is
transported towards the surface at a relatively high energy, which
causes significant atom breakup and reorganization as a conse-
quence of the process. By using deflecting plates to scan the
concentrated primary ion beam across a specific region of interest
(ROI) on the material, images with the help of ion intensity maps
may be generated (Fig. 23). Since only sputtering materials can be
studied, the SIMS approach is fundamentally destructive [136,137].
The investigation of chemical compounds localization in sub-
cellular components with cluster ToF-SIMS imaging - the only
label-free technology capable of tracking both endogenous and
external small molecules in complicated biological structures with
high spatial magnification and high sensitivities. Apart from using
ToF-SIMS photography on mammalian cells, using ToF-SIMS to find
chemicals in bacteria about the size of mitochondria is a substantial Fig. 23. The positive TOF-SIMS images of cells treated with nanoparticles with
technical development. The potential for this technology to become different concentrations [139]. (Reused with Elsevier's kind consent (Lic. No.
5502901303937)).
an essential analysis instrument for biological and pharmaceutical
sciences might be increased with the creation of low-damage gas
clustered ion beams as well as further improvement in ionization
capabilities [138]. The potential toxicity of ZnO nanoparticles on ZnO NPs increases. The findings indicate that TOF-SIMS was a good
human skin cells was examined by Lee and his team. The imaging approach for determining cell response to NPs in a timely manner
method TOF-SIMS was used to assess the cytotoxicity of the [139]. A new objective for SIMS imaging is the development of
nanomaterials to the selected cells. A TOF-SIMS apparatus was focused imaging approaches, particularly for big macromolecules
operated using a Gaþ main ion gun with a pulse on time of 1 pA and such as proteins. In even more newly invented equipment, such as
25 keV. Fig. 23 depicts an example picture of ZnO NPs functioning the J105 and nano-SIMS, a single beam may be used to actively
as a tracer with HaCaT cells. The positive ion TOF-SIMS pictures erode the sample while acquiring 2D pictures throughout the
reveal that Zn migrates toward the core as that of the proportion of sample's depth [140].

Fig. 22. Diagram of typical SIMS equipment configurations [136]. (Reused with Elsevier's kind consent (Lic. No. 5502901127669)).

139
P. Jagadeesh, S.M. Rangappa and S. Siengchin Advanced Industrial and Engineering Polymer Research 7 (2024) 122e143

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