processes

Review
Atomic Force Microscopy as a Tool to Study Transport
Phenomena in Biological Systems
Sneha Kandapal and Bingqian Xu *

Single Molecule Study Laboratory, College of Engineering and Nanoscale Science and Engineering Center,
University of Georgia, Athens, GA 30602, USA; [Link]@[Link]
* Correspondence: nanoxu@[Link]

Abstract: Biological interactions often involve the transport of molecules, ions, or other species
across biological membranes or between interacting proteins. The understanding of these transport
phenomena is crucial for the development of therapies for various diseases. Atomic force microscopy
is a powerful tool that has been increasingly used to study biological systems at the nano scale. The
high resolution, quantitative measurements, and the ability to probe biological interactions under
near-physiological conditions make AFM an attractive tool for investigating transport phenomena
in biological systems. In this article, we focus on the use of AFM in the study of the transport
phenomena in biological systems. We discuss the principles of AFM, its instrumentation, and its
application in the study of biomolecules and biological systems. We also provide a comprehensive
overview of recent articles that have utilized AFM in the study of biomarkers in biological systems.

Keywords: atomic force microscopy; biomarkers; transport; binding interactions; physical properties;
biological systems

1. Introduction
Atomic force microscopy (AFM) [1,2] is a technique that is used not only for high-
resolution imaging of a variety of materials, but also in the assessment of diverse physical
characteristics at the nano scale. It finds extensive applications across various disciplines,
Citation: Kandapal, S.; Xu, B. Atomic including physics, chemistry, biology, and materials science, enabling the investigation of
Force Microscopy as a Tool to Study diverse physical and chemical properties at the molecular level. AFM [3] has equipped
Transport Phenomena in Biological scientists with indispensable tools and resources to investigate the structure and properties
Systems. Processes 2023, 11, 2430. of a wide range of samples, spanning from individual atoms to complex biological systems.
[Link] These investigations hold broad applications, from fundamental research to industrial
Academic Editors: Kun Wang and endeavors. Due to its remarkable capacity to deliver high-resolution images and precise
Han Hu measurements at the nano scale, it has emerged as an ever more crucial instrument in
the examination of biosystems. This technology empowers researchers to visualize the
Received: 12 May 2023 surface of biological samples, enabling them to extract comprehensive data pertaining to
Revised: 18 July 2023
topography, structure, and arrangement. Consequently, scientists can now delve into the in-
Accepted: 20 July 2023
tricate world of diverse biological molecules, such as proteins, DNA, RNA, and lipids [4–8],
Published: 12 August 2023
unraveling their structure–function relationships and gaining valuable insights. In addition
it can serve as a valuable tool for investigating the mechanical characteristics of biological
materials [9], such as their stiffness and elasticity, and provide insights into biomolecules
Copyright: © 2023 by the authors.
and their role in biological processes such as cell adhesion [8,10], migration [11,12], and
Licensee MDPI, Basel, Switzerland. signaling [13,14]. With its remarkable ability to image and manipulate individual molecules,
This article is an open access article AFM has emerged as a potent instrument in the examination of isolated molecules, thereby
distributed under the terms and offering invaluable insights into their behavior and function. This capability has revolution-
conditions of the Creative Commons ized the study of biological molecules, including enzymes [15,16], receptors [17,18], and
Attribution (CC BY) license (https:// transporters [19,20].
[Link]/licenses/by/ AFM has proven highly valuable in the realm of investigating transport phenomena
4.0/). within biological systems. Transport phenomena involve the movement of particles or

Processes 2023, 11, 2430. [Link] [Link]

Processes 2023, 11, 2430 2 of 39

molecules across media like cell membranes or tissue barriers. In-depth exploration of
transport phenomena within biological interactions, encompassing molecular binding and
recognition processes involving proteins, nucleic acids, and lipids, sheds light on funda-
mental biological processes such as cell adhesion, protein–protein interactions, and drug
delivery. Diverse mechanisms drive these interactions, including electrostatic interactions,
hydrogen bonding, van der Waals interactions, and hydrophobic interactions. AFM finds
noteworthy applications in the study of transport phenomena, with a specific focus on
studying the blood–brain barrier (BBB) [21,22]. Acting as a complex barrier, the BBB sepa-
rates blood vessels from brain tissue and plays a vital role in regulating the transport of
nutrients, ions, and other molecules into the brain. AFM has facilitated investigations into
the mechanical properties of the BBB and the interactions between the BBB and circulating
cells and molecules. By quantifying the local mechanical properties of the BBB, AFM
offers insights into the barrier’s permeability and transport properties. Similarly, AFM
has proven to be instrumental in studying transport phenomena in ion channels [23–26].
These membrane proteins govern ion flow across cell membranes and are pivotal in various
biological processes, such as nerve impulse transmission and muscle contraction. The
exploration of biomolecular interactions through AFM has unveiled valuable insights into a
wide array of biological processes, holding potential for the development of novel therapies
and diagnostic tools. This review delves into the diverse applications of AFM in studying
transport phenomena within biological interactions.

2. Principles of AFM
AFM is a scanning probe microscopy technique that facilitates the measurement of the
interaction between a probe (Figure 1) and a specimen surface [27]. The probe is affixed to
a flexible cantilever and systematically moved across the sample surface in a controlled
manner, where it experiences various forces, such as contact, electric, magnetic, electrostatic,
and Van der Waals forces. The deflection of the cantilever is gauged using a laser beam,
which is directed onto a position-sensitive detector after being reflected off the back of the
cantilever. This deflection information is then utilized to compute the forces operating
between the probe and the sample surface. AFM can operate in multiple modes, including
contact mode, tapping mode, and non-contact mode. In contact mode, the tip continuously
maintains contact with the sample surface, and the deflection data acquired generate a
topographic image of the surface. In tapping mode, the tip oscillates near its resonance
frequency, and the resulting deflection data are utilized to create a topographic image of
the surface. In non-contact mode, the tip is brought close to the sample surface without
contacting it. The interaction between the tip and the sample surface causes a shift in
Processes 2023, 11, x FOR PEER REVIEW
the resonance frequency of the cantilever, which is measured
3 of 40
and exploited to generate a
topographic image.

Figure 1. Schematic diagram showing the major components of an atomic force microscope with an
Figure
optical 1. Schematic
beam deflection diagram
system. Reprinted showing
with permissionthe
frommajor
[27]. components of an atomic force microscope with an
optical beam deflection system. Reprinted with permission from [27].
2.1. Probe and Cantilever Design
The design of both the AFM probe and the cantilever plays a pivotal role in deter-
mining the resolution and sensitivity of the instrument. The AFM probe is composed of
silicon or silicon nitride and features a sharp tip that scans across the sample surface. To
enhance the detection of the cantilever’s motion, the tip is often coated [28,29] with a re-
flective material. The design of the tip is critical in achieving high-resolution imaging. A
sharp and well-defined tip enables the detection of small features on the specimen surface,
whereas a blunt or irregular tip can result in poor resolution and inaccurate measure-
Processes 2023, 11, 2430 3 of 39

2.1. Probe and Cantilever Design

The design of both the AFM probe and the cantilever plays a pivotal role in determin-
ing the resolution and sensitivity of the instrument. The AFM probe is composed of silicon
or silicon nitride and features a sharp tip that scans across the sample surface. To enhance
the detection of the cantilever’s motion, the tip is often coated [28,29] with a reflective
material. The design of the tip is critical in achieving high-resolution imaging. A sharp and
well-defined tip enables the detection of small features on the specimen surface, whereas a
blunt or irregular tip can result in poor resolution and inaccurate measurements. Addition-
ally, the dimensions and geometry of the tip affect the interaction forces with the sample,
thereby influencing the precision and sensitivity of the measurements. The cantilever, on
the other hand, is a thin and flexible beam that provides support to the AFM probe and
detects its deflection as it scans the sample surface. Like the AFM probe, the cantilever is
typically made of silicon or silicon nitride and is coated with a reflective material to enhance
the detection of its motion. The stiffness, length, and width of the cantilever all contribute
to its resonant frequency, which determines the speed and accuracy of the measurements. It
is crucial to match the resonant frequency of the cantilever with the frequency of the AFM
feedback loop to achieve optimal performance [30,31]. Proper tuning ensures effective
interaction between the cantilever and the sample surface, enabling precise and reliable
measurements. There are many distinct types of AFM probes and cantilevers available,
each with its own advantages and disadvantages. For example, the use of ultrathin can-
tilevers [32] can improve resolution and sensitivity but can also lead to increased noise
and decreased stability. Similarly, the use of specialized probe coatings [33] can improve
the detection of specific properties, such as magnetic or chemical properties, but can lead
to increased tip wear and reduced probe lifetime. In conclusion, the design of the AFM
probe and cantilever is critical for achieving high-resolution, high-sensitivity imaging in
AFM. The choice of probe [34,35] and cantilever design should be based on the specific
requirements of the experiment and the properties of the sample being investigated. By
meticulously considering these variables, AFM becomes an instrumental tool for exploring
the intricate properties of surfaces and materials at the nano scale.

2.2. Detection of Cantilever Deflection

The precise detection of cantilever deflection constitutes a pivotal aspect of AFM, en-
abling in-depth exploration of the nanoscale properties exhibited by surfaces and materials.
The deflection of the cantilever is directly proportional to the interaction forces occurring
between the probe and the specimen, serving as a potent tool for investigation. Among the
various methods available for detecting cantilever deflection in AFM, optical detection [36]
stands out as the most commonly utilized. In this approach, a laser beam is directed onto
the cantilever, and the reflected light is captured by a photodetector. As the cantilever
deflects, the position of the reflected beam undergoes changes, facilitating precise measure-
ment of the cantilever’s deflection. This method provides high sensitivity and excellent
temporal resolution, allowing for real-time monitoring of the cantilever’s position. Another
method for detecting cantilever deflection is piezoresistive detection [37,38]. In this method,
the cantilever is coated with a piezoresistive material that changes its electrical resistance
as the cantilever deflects. This change in resistance can be measured by a Wheatstone
bridge circuit, providing a direct measurement of the cantilever’s deflection. Piezoresistive
detection provides high sensitivity and is well suited for use in high-frequency AFM ap-
plications. Capacitive detection [39] is another method for detecting cantilever deflection
in AFM. In this approach, the cantilever is positioned between two capacitive plates, and
the alteration in capacitance resulting from the deflection of the cantilever is quantified.
This method provides excellent linearity and sensitivity but is typically more complex and
expensive than optical or piezoresistive detection. Finally, there are several specialized
detection methods that have been developed for specific AFM applications. For instance,
magnetic detection is utilized to identify the deflection of a magnetic cantilever in magnetic
force microscopy (MFM), while thermal detection is employed to measure the deflection
Processes 2023, 11, 2430 4 of 39

of a thermally sensitive cantilever in thermal AFM applications. Thus, the detection of

cantilever deflection is a critical aspect of AFM, and a variety of methods are available for
achieving high sensitivity and accuracy.

2.3. Force Measurement

Force measurement using AFM is a highly effective technique for exploring the physi-
cal properties of materials [40]. The most widely used method for force measurement in
AFM is force spectroscopy. In force spectroscopy, the cantilever is brought into contact
with the sample surface and then gradually retracted at a constant velocity, while simul-
taneously measuring the deflection of the cantilever. The resulting force–distance curve
(Figure 2) [41] provides valuable insights into the mechanical characteristics of the sample,
including its stiffness, elasticity, surface roughness, adhesion, viscoelasticity, and plastic
deformation. Force spectroscopy can be conducted with a variety of probe geometries, such
as spherical, pyramidal, and conical tips, tailored to the specific experimental requirements.
Another approach for force measurement in AFM is lateral force microscopy (LFM). In
LFM [42], the cantilever is scanned across the sample while detecting the forces between
the probe and the sample. This technique enables the investigation of frictional properties
and the mapping of lateral force distributions across the sample. MFM represents a third
method for force measurement. It employs a magnetic cantilever to measure the magnetic
forces between the probe and the sample surface. It allows for the investigation of the
magnetic properties of materials and the mapping of magnetic force distributions across
the sample. Additionally, several specialized force measurement techniques have been
developed for specific applications. These include electrostatic force microscopy (EFM),
capacitance force microscopy (CFM), and nanoindentation. EFM is employed to investigate
electrical properties, CFM is utilized to explore dielectric properties, and nanoindentation
focuses on the mechanical properties of materials. Thus, force measurement using AFM
presents a potent approach for examining the physical properties of materials and surfaces
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at the nano scale, enabling comprehensive characterization and analysis.

Figure 2. Representation of a typical force–distance curve. Reprinted with permission from [41].
Figure 2. Representation of a typical force–distance curve. Reprinted with permission from [41].

2.4. Scanning Modes

An essential aspect of AFM is its versatility in operating under different scanning
modes, as mentioned earlier, each offering unique advantages and limitations. The most
utilized scanning mode in AFM is contact mode. In contact mode, the cantilever makes
direct contact with the sample surface, and the deflection of the cantilever is monitored as
the probe scans across the surface. Contact mode excels at high-resolution imaging of sur-
faces with significant topographical contrast. However, it can potentially damage the sam-
Processes 2023, 11, 2430 5 of 39

2.4. Scanning Modes

An essential aspect of AFM is its versatility in operating under different scanning
modes, as mentioned earlier, each offering unique advantages and limitations. The most
utilized scanning mode in AFM is contact mode. In contact mode, the cantilever makes
direct contact with the sample surface, and the deflection of the cantilever is monitored
as the probe scans across the surface. Contact mode excels at high-resolution imaging of
surfaces with significant topographical contrast. However, it can potentially damage the
sample surface and has limited sensitivity to other surface properties beyond topography,
such as elastic modulus, surface roughness, and topography. Another popular scanning
mode in AFM is tapping mode, also known as intermittent contact mode. In tapping
mode, the cantilever vibrates at its resonant frequency, periodically contacting the sample
surface. This minimizes harm to the sample surface and offers excellent sensitivity to non-
topographical characteristics, such as material contrast, elasticity, and stiffness mapping,
making it suitable for imaging delicate or soft samples. Nevertheless, tapping mode may
compromise spatial resolution and can be more operationally intricate compared to contact
mode. Another scanning mode employed in AFM is non-contact mode [43]. In this mode,
the cantilever remains at a fixed distance from the sample surface, with the tip interacting
with the surface through Van der Waals forces, and exhibits high sensitivity to surface
forces, making it appropriate for imaging of fragile or easily affected samples. However, it
possesses limited lateral resolution and can be influenced by environmental factors like
temperature and humidity. The selection of a scanning mode relies on factors such as the
type of sample, resolution requirements, and desired information content.

2.5. Functionalization of AFM Tips

In the realm of life sciences or biomedical research, AFM tips are often tailored with
specific chemical groups or molecules to enable the measurement of weak forces, including
magnetic, adhesive, Van der Waals, electrostatic, and hydration forces, between the tip and
the surface molecules. This is achieved through tip functionalization [44], which plays a
critical role in enhancing the capabilities of AFM. A frequently used approach for func-
tionalizing AFM tips [45] is through the nonspecific adsorption of probe molecules. This
method is effective and uncomplicated; however, it is crucial to consider that the bonding
strength of adsorption may not always exceed the specific interaction between the ligand
attached to the tip and the target molecules on the surface. Moreover, directly immobi-
lizing biomolecules onto an inorganic support can result in denaturation, degradation,
and compression during the scanning process. To overcome these challenges, covalent
bonding is employed to attach the probe to the tip, for which linear poly (ethylene glycol)
(PEG) chains are often used. PEG provides benefits such as chemical and physical stability,
enabling the probe molecule to reorient itself quickly and freely when the AFM tip interacts
with the surface. This ensures that a single molecule on the tip can effectively identify its
corresponding counterpart on the target surface. Various functionalization techniques can
be employed by taking into account the specific molecules involved and the experimental
requirements [46]. Dazaet al. [47] explored the use of the activated vapor silanization (AVS)
process to functionalize AFM cantilevers and tips. Thin films with specific desired func-
tionalities were deposited onto silicon nitride chips, and their existence and performance
were confirmed by assessing changes in the resonance frequency of the cantilever. To gain
further insights into these films, fluorescein-derived molecules were covalently tethered
to the amine-reactive groups present on the surface. As a result, the functionalized tips
displayed resilient capabilities, withstanding repeated interactions with a model graphite
substrate even under demanding conditions, while exhibiting no noticeable adverse effects.
Additionally, the covalent attachment of a molecule to the tip permits the manipulation of
the substrate–tip interaction. Another study by Girish et al. [48] showcased the successful
functionalization of AFM tips using folic acid (FA) for the imaging of folate receptors
(FRs) in oral cancer cell lines. This functionalization technique enables well-resolved phase
images of FRs (Figure 3), offering insights into their distribution and characteristics.
 graphite substrate even under demanding conditions, while exhibiting no noticeable ad-
verse effects. Additionally, the covalent attachment of a molecule to the tip permits the
manipulation of the substrate–tip interaction. Another study by Girish et al. [48] show-
cased the successful functionalization of AFM tips using folic acid (FA) for the imaging of
folate receptors (FRs) in oral cancer cell lines. This functionalization technique enables
Processes 2023, 11, 2430 6 of 39
well-resolved phase images of FRs (Figure 3), offering insights into their distribution and
characteristics.

[Link]
Figure Topographicimage
image
andand phase
phase image
image of of L929
L929 cells,
cells, respectively,
respectively, using
using an FA-functional-
an FA-functionalized
ized
tip. tip. Density
Density of receptors.
of receptors. Applied
Applied Physics
Physics Letters,
Letters, 2009,2009, 95(22):
95(22): p. 223703.
p. 223703. Reprinted
Reprinted withwith permis-
permission
sion from [48].
from [48].

2.6. Investigating Mechanical Properties of Biological Materials

AFM has emerged as a powerful and versatile tool for investigating the mechanical
properties of biological materials at the nano scale. It allows one to probe and manipulate
biological samples, providing valuable insights into their stiffness, elasticity, adhesion,
and topography. Before determining mechanical properties, high-resolution imaging and
surface topography of the biological sample should be obtained using AFM in contact mode
or tapping mode. This step allows the visualization and selection of appropriate regions
of interest for subsequent mechanical measurements, ensuring that the chosen areas are
representative of the sample’s properties. The force–distance curve is the primary technique
used to determine mechanical properties using AFM. In this technique, the AFM tip is
brought into contact with the sample surface, and a small indentation force is applied. The
deflection of the cantilever is measured as a function of the piezo displacement, resulting
in a force–distance curve. This curve provides information about the sample’s response to
applied forces and allows for the calculation of various mechanical properties.

2.6.1. Elasticity and Young’s Modulus

From the force–distance curve [49], one can determine the elasticity or Young’s modu-
lus of the biological sample. Young’s modulus is a key parameter for characterizing the
mechanical properties of samples. It represents the stiffness, rigidity, or compliance of the
material and can provide insights into intermolecular interactions. AFM nanoindentation
is one of the most powerful techniques for the determination of Young’s modulus. The
main idea is to indent the sample surface with the AFM tip and to measure the applied
load at each indentation depth. The cantilever base or the sample is moved vertically by a
piezoelectric actuator (Z-axis), causing the tip–sample gap to decrease until contact occurs,
and the cantilever tip experiences a repulsive force, deflecting while indenting the sample
 Processes 2023, 11, 2430 7 of 39

of interest. Force–distance curves are typically composed of a baseline, contact between the
tip and the surface, loading of the tip and indentation into the sample, the jump to contact
point where the direction of the Z motion is reversed, adhesion where the tip is bound to
the sample surface beyond the point of contact as the tip–sample contact is being separated,
and finally, the return to zero interaction force, as shown in Figures 2 and 4. During the
analysis of the force–indentation curves, the fitted function is assumed to take the form
of the power law y = a · xb , where the value of b depends on the assumed shape of the
intended AFM tip. The final Young’s modulus is calculated considering all values obtained
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from a whole set of force–indentation curves. The resulting distribution [50] is fitted with8 of 40
the Gaussian function. In force–distance curves, a steeper slope indicates a higher Young’s
modulus, suggesting a more rigid or resistant region.

Figure
Figure (a)The
4.4.(a) The force–displacement
force–displacement curves
curvesmeasured
measured for glass and a cell.
for glass and Reprinted with permission
a cell. Reprinted with permis-
from [49]. (b) The force–indentation curve fitted with the Hertz model. (c) Determination
sion from [49]. (b) The force–indentation curve fitted with the Hertz model. (c) Determination of Young’s of
modulus
Young’s from thefrom
modulus Gaussian function fit.
the Gaussian Reprinted
function fit. with permission
Reprinted withfrom [50].
permission from [50].
2.6.2. Adhesion Force
2.6.2. Adhesion Force
AFM force–distance curves provide valuable information about the adhesion proper-
tiesAFM force–distance
between curves
the AFM tip and providesurface.
the sample valuable information
By analyzing about
these theasadhesion
curves, shown in prop-
Figure 2, one can gain insights into the interactions and forces involved
erties between the AFM tip and the sample surface. By analyzing these curves, as shownat the nanoscale
in level.
Figure The adhesion
2, one property
can gain is typically
insights into thecharacterized
interactionsby theforces
and adhesion force, at
involved which rep-
the nanoscale
resents
level. Thethe force required
adhesion to separate
property the AFM
is typically tip from the
characterized bysample surface after
the adhesion contact.
force, which rep-
It is measured as a negative force value on the force–distance curve when the tip is being
resents the force required to separate the AFM tip from the sample surface after contact.
retracted. The magnitude of the adhesion force indicates the strength of the interaction
It is measured as a negative force value on the force–distance curve when the tip is being
retracted. The magnitude of the adhesion force indicates the strength of the interaction
between the tip and the sample. In addition to the adhesion force, other parameters can
be derived from the force–distance curve to assess the adhesion properties. These include
the pull-off distance, which is the distance at which the tip completely detaches from the
Processes 2023, 11, 2430 8 of 39

between the tip and the sample. In addition to the adhesion force, other parameters can
be derived from the force–distance curve to assess the adhesion properties. These include
the pull-off distance, which is the distance at which the tip completely detaches from
the surface, and the adhesion energy, which represents the energy required to break the
adhesive bonds between the tip and the surface. The shape and characteristics of the
force–distance curve can provide further information about the adhesion properties. For
example, a steep and abrupt increase in force during the approach phase, followed by a
sudden drop during retraction, indicates strong adhesion between the tip and the sample.
On the other hand, a gradual increase and decrease in force suggests weaker adhesion. By
analyzing the adhesion properties obtained from AFM force–distance curves, one can gain
insights into various phenomena and processes. This includes studying the adhesion be-
tween biomolecules, assessing surface properties and coatings, investigating the strength of
molecular interactions, and understanding the adhesion mechanisms in biological systems.
Thus, AFM has revolutionized the characterization of mechanical properties of biolog-
ical materials at the nano scale. Advancements in AFM technology continue to enhance our
understanding of the mechanical properties of biological materials, contributing to various
fields, such as biomaterials, tissue engineering, and biomechanics.

2.7. Recent Advancement in AFM

Significant variations in the measured properties of the same sample are often ob-
served, which can be attributed to two primary sources: inherent biological diversity,
and experimental inaccuracies. Experimental inaccuracies can stem from deviations in
instrument performance and discrepancies in data gathering and analysis protocols. To
counteract and diminish these errors, it is essential to undertake a comprehensive normal-
ization endeavor spanning across multiple laboratories, instruments, and operators. This
collaborative approach is vital for overcoming the inherent limitations associated with
single-laboratory studies. Schillers et al. [51] introduced SNAP, a standardized nanome-
chanical AFM procedure that enhances the accuracy and reproducibility of mechanical
measurements. It reduces variability in elastic modulus measurements for soft samples and
living cells caused by technical factors and ensures precise calibration of the AFM optical
lever system, independent of the instrument, laboratory, or operator. It addresses errors by
accurately calculating deflection sensitivity (Figure 4) based on spring constants determined
using a vibrometer. Wang [52] developed a practical transition model to explain complex
ricin–aptamer interactions. It combines the Bell–Evans model and Markov-type transition
matrices, providing detailed information about molecular structures, unbinding forces, and
activation energies. This approach can be applied to study other single-molecule interac-
tions with multiple reaction pathways. In addition, the authors proposed a method [53]
that enables the investigation of binding conformations and affinities of biomolecules. The
combination of AFM techniques and molecular simulations enables a novel approach for
detecting and studying the mechanisms of aptamers. Adams et al. [54] achieved high-speed
AFM imaging (HS-AFM) [55] in air by using SU-8 polymer cantilevers. These polymer-
based cantilevers mimic the damping of liquid environments and provide an imaging-in-air
detection bandwidth 19 times faster than conventional cantilevers with similar properties.
Puppulin et al. [56] conducted a study for recognizing specific target molecules on mica sub-
strates and lipid membranes. They focused on the MET receptor and used the macrocyclic
peptide aMD4 to functionalize the HS-AFM tip. The imaging results revealed the selective
interaction between the aMD4-conjugated tip and the hMET receptors, leading to enhanced
phase delay in the cantilever’s oscillation. By using aMD4, they were able to discriminate
between human MET and the murine homologue. Yang et al. [57] introduced an advanced
platform for nanoscale analysis called spectral-spatially encoded array AFM (SEA-AFM)
(Figure 5). Unlike conventional methods, it utilizes alternative readout techniques, such
as piezoelectric sensors and high-resolution optical detection, to enable simultaneous and
independent addressing of multiple cantilevers. Thus, this method successfully tackles
interferences while preserving remarkable sensitivity and expandability, thereby present-
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ing a formidable instrument for thorough nanoscale analysis with enhanced efficacy and
versatility.

Figure 5. Elastic modulus of gels from different labs measured using (A) a conventional procedure
and (B) using SNAP. Reprinted with permission from [51].

Park et al. [58] developed a novel technique called DP-pAFM (Figures 6 and 7) for
high-resolution morphological and optical analysis of samples, which offers improved
Figure contrast
5. Elastic and Figure
modulus Elastic
[Link] modulus
from of gels labs
different from different
measured labs measured
using (A)using (A) a conventional
a conventional procedure
procedure
image sensitivity while avoiding excessive power,
and (B) using SNAP. Reprinted with permission from [51].
thereby minimizing the
and (B) using SNAP. Reprinted with permission from [51].
risk of damage to the sample or the cantilever tip. Two identical lasers generate separate
Park et al. [58] developed a novel technique called DP-pAFM (Figures 6 and 7) for
beams with a controlled time delay, which areand combined using mirrors which
and a beam split-
Park et al. [58] developed a morphological
high-resolution novel technique optical
calledanalysis of samples,
DP-pAFM (Figuresoffers6 andimproved
7) for
ter, coupled into a single-mode
image contrast fiber, and obliquely
and sensitivity illuminated
while avoiding onto thereby
excessive power, the target sample
minimizing the
high-resolution morphological and optical analysis of samples, which offers improved
through a focusing system. The first
risk of damage laser
to the sampleheats
or thethe area under
cantilever tip. Twothe cantilever
identical tip, followed
lasers generate separate
image contrast and beamssensitivity while avoiding
time delay, excessive power,usingthereby
mirrorsminimizing the
by the second laser after a with
short a controlled
delay. This which are combined
induces unique cantilever and a beam splitter,
oscillations, allow-
coupled into a single-mode fiber, and obliquely illuminated
risk of damage to the sample or the cantilever tip. Two identical lasers generate separate onto the target sample through
ing for the mapping of opticalsystem.
a focusing structures inlaser
The first small-molecule semiconductor
heats the area under films.
the cantilever tip, followed by the
beams with a controlled time delay, which are combined using mirrors and a beam split-
second laser after a short delay. This induces unique cantilever oscillations, allowing for
ter, coupled into a single-mode fiber, structures
the mapping of optical and obliquely illuminated
in small-molecule onto the
semiconductor target sample
films.
through a focusing system. The first laser heats the area under the cantilever tip, followed
by the second laser after a short delay. This induces unique cantilever oscillations, allow-
ing for the mapping of optical structures in small-molecule semiconductor films.

Figure 6. (a) SEA-AFM system. (b) Calibration grid of 3 µm. (c) AFM image of fixed neural progenitor
Figure 6. (a) SEA-AFM cells.
system. (b) Calibration
Reprinted grid
with permission fromof[57].
3 µm. (c) AFM image of fixed neural progen-
itor cells. Reprinted with permission from [57].

Figure 6. (a) SEA-AFM system. (b) Calibration grid of 3 µm. (c) AFM image of fixed neural progen-
OR PEER REVIEW 11 of 40

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(a)

(b) (c)
Figure 7. (a) Schematic diagram
Figure of DP-pAFM.
7. (a) Schematic diagram of(b,c) Topographic
DP-pAFM. imagesimages
(b,c) Topographic of SP-ofand DP-pAFM,
SP- and DP-pAFM, re-
spectively. Figure (a,b,c) reprinted
respectively. Figure with
(a,b,c) permission
reprinted with from [58].from [58].
permission

Cheng et al. [59] introduced an algorithm called network-based automatic clustering

Cheng et al. [59] introduced
algorithm (NASA) toan algorithm
address called
the challenge network-based
of automatically automatic
specifying unbindingclustering
forces to
binding sites in single-molecule data. It extracts unbinding
algorithm (NASA) to address the challenge of automatically specifying unbinding forces forces, constructs a network
representing their relationships, and detects community structures corresponding to bind-
to binding sites in ing
single-molecule data. It extracts unbinding forces, constructs a network
sites. They applied NASA to decode the interaction between heparan sulfate (HS) and
representing theirantithrombin
relationships,(AT) onand detects
different community
endothelial structures
cell surfaces corresponding
as a model system, successfully to
binding sites. They applied NASA to decode the interaction between heparan sulfate
detecting peaks, calculating unbinding forces, and identifying three force clusters (HS)
that corre-
sponded to different binding sites. NASA shows potential for analyzing other AFM-based
and antithrombin measurements,
(AT) on different endothelial cell surfaces as a model system, success-
such as antibody–antigen and DNA–protein interactions, characterized by
fully detecting peaks, calculating
“saw-tooth” unbinding
force–distance forces,
curves with and identifying
force-induced three force clusters
unbinding events.
that corresponded to different binding sites. NASA shows potential for analyzing other
AFM-based measurements, such as antibody–antigen and DNA–protein interactions,
characterized by “saw-tooth” force–distance curves with force-induced unbinding events.

3. Applications of AFM in the Study of Biomolecules

Processes 2023, 11, 2430 11 of 39

3. Applications of AFM in the Study of Biomolecules

AFM enables the visualization of biomolecules in three dimensions, providing valuable
insights into their shape and structure at the nano scale. It operates in diverse environments,
making it suitable for studying biological systems. It plays a crucial role in investigating
biomolecular interactions, including protein–protein, lipid–protein, and DNA–protein
interactions. It allows direct, label-free measurements of molecular forces and interaction
energies, facilitating the study of structural and mechanical properties. In this section, we
explore the application of AFM as a tool for studying transport phenomena in biological
interactions.

3.1. Protein
Proteins play a vital role in biological systems, participating in essential cellular
processes such as protein synthesis, signal transduction, and enzymatic reactions. They
are also implicated in the development of various diseases, including neurodegenerative
disorders like Parkinson’s and Alzheimer’s, as well as localized conditions such as type
2 diabetes and cataracts. Understanding protein aggregation is crucial in clinical settings for
comprehending disease pathology and developing diagnostics and therapies. One example
of protein aggregation is pseudoexfoliation syndrome (PEX), a disorder characterized by
extracellular matrix protein aggregation that obstructs the eye’s aqueous outflow, posing a
significant risk of glaucoma. Creasey et al. [60] utilized AFM-based antibody recognition
imaging (Figure 8a) to investigate the molecular nature of PEX aggregates on human lens
capsules. Their findings revealed an association between lysyl oxidase-like 1 (LOXL1)
and an increased susceptibility to the syndrome. Another notable example is Alzheimer’s
disease, which is characterized by the self-assembly and accumulation of fibrillar amyloid-
β (Aβ) peptides in the brain. Biophysical studies, including AFM, have significantly
advanced our understanding of the mechanisms underlying amyloid formation and its role
in the pathogenesis of Alzheimer’s.
AFM plays a crucial role in studying protein folding and unfolding processes, which
are fundamental to cellular function. Proteins transition from an unfolded state to well-
defined three-dimensional structures, unique to each protein, for their biological activity.
AFM enables direct visualization of the topographic structures of individual proteins
and provides mechanical insights into their unfolding dynamics. Protein misfolding can
lead to the formation of aggregated protofibrils, contributing to various diseases, such as
Alzheimer’s, prion diseases, cystic fibrosis, and amyotrophic lateral sclerosis. AFM studies
on protein folding have focused on proteins with tandem repeats of similar modules, such
as tenascin, spectrin, and titin, which exhibit mechanical strength that is essential for their
physiological functions. Best et al. [61] aimed to investigate the resistance of proteins to
force and discovered that proteins that are not specifically designed for tensile strength
may not exhibit the same resistance to force as those that are. They also observed that
proteins with similar unfolding rates in solution may exhibit different unfolding properties
under force, emphasizing the importance of considering protein-specific characteristics
when studying their response to mechanical forces using AFM. Kawakami and Smith [62]
introduced a novel force-ramp modification that enables precise control of multiple unfold-
ing events in a multi-modular protein. This advancement utilizes software-based digital
force feedback control to maintain a constant force-loading rate, regardless of the length of
the soft elastic linkage or the number of unfolded polypeptide domains. By applying this
technique, one can observe distinct pathways of unfolding in a controlled manner. Peng
and Li. [63] conducted an experimental study that provided empirical evidence for the
kinetic partitioning mechanism involved in the mechanical unfolding of T4 lysozyme—a
compact protein composed of two subdomains. By subjecting T4 lysozyme to applied
stretching forces from its N and C termini, they observed multiple distinct pathways of
unfolding (Figure 8b,c). The majority of T4 lysozyme molecules exhibited an all-or-none
unfolding behavior, surpassing a dominant kinetic barrier. However, a minority fraction of
T4 lysozyme molecules followed a three-state unfolding pathway involving intermediate
 Processes 2023, 11, 2430 12 of 39

states. Notably, these three-state unfolding pathways displayed variability and diversity,
deviating from well-defined routes. These findings provide direct evidence for the presence
of kinetic partitioning in the mechanical unfolding pathways of T4 lysozyme. Moreover,
the complex unfolding behaviors observed highlight the stochastic nature of kinetic barrier
R PEER REVIEW 13 of 40
rupture during mechanical unfolding processes. In a study by Mahmood, Moheimani, and
Bhikkaji [64], the effectiveness of integrating genetic manipulation with the AFM technique
was investigated as a robust method to study the responses of proteins to forces within liv-
ing cells. The findings underscore the potential of this approach for unraveling the intricate
of proteins’ aggregation, folding,
mechanisms and unfolding
underlying processes.
protein mechanics They context.
in a cellular shed light
Thus,onAFMthestudies
struc-have
tural and mechanical properties
significantly of proteins,
contributed to our offering insights
understanding into their
of proteins’ biological
aggregation, func-
folding, and un-
folding processes. They
tions and their roles in disease development. shed light on the structural and mechanical properties of proteins,
offering insights into their biological functions and their roles in disease development.

(a)

(b) (c)
Figure 8. (a) AFM images of8.a lens
Figure capsule
(a) AFM imagesfrom a PEX
of a lens patient
capsule fromacquired using
a PEX patient an anti-LOXL1-anti-
acquired using an anti-LOXL1-
body-functionalized tip, from an area near
antibody-functionalized tip,the
fromcenter
an area of the
near thecapsule. Reprinted
center of the with permission
capsule. Reprinted with permission
from [60]. (b) Typical force–extension curves of cysteine-free pseudo wild-type T4 lysozyme (T4L*)
showing three-state unfolding behaviors. (#-second unfolding force peaks) (c) A series of force–ex-
tension curves of the same T4L* measured during repeated stretching–relaxation experiments. Re-
printed with permission from [63].
Processes 2023, 11, 2430 13 of 39

from [60]. (b) Typical force–extension curves of cysteine-free pseudo wild-type T4 lysozyme (T4L*)
showing three-state unfolding behaviors. (#-second unfolding force peaks) (c) A series of force–
extension curves of the same T4L* measured during repeated stretching–relaxation experiments.
Reprinted with permission from [63].

3.2. Nucleic Acids

The direct imaging of nucleic acids [65] in aqueous solutions has long been a chal-
lenging task, primarily because of the relatively weak interactions between nucleic acids
and substrates. However, advancements in substrate modifications and detection methods
have improved the quality and resolution of nucleic acid imaging. Zhu et al. [66] pro-
vided a method for studying DNA methylation that cannot be detected using conventional
DNA probes. They used an antibody-tethered AFM cantilever to measure the distances
between 5-methylcytidine bases in DNA strands, with a resolution of 4 Å. By binding to
two 5-methylcytidine bases, the cantilever retraction produced a unique rupture signature
reflecting the spacing between the tagged bases. Shim et al. [67] developed a force mapping
method to directly quantify low-abundance DNA containing methyl-CpG at a specific site.
Their approach utilized methyl-CpG-binding (MBD) proteins to distinguish methylation
states. The MBD protein was positioned on the probe opposite the target DNA site of
interest. If the target site was methylated, the MBD protein would identify the methylated
CpG, while it would disregard the site if it was not methylated. This method allowed
for the detection of specific methylation patterns without the need for chemical treatment
or amplification of the target DNA. Strobl et al. [68] investigated the factors influencing
nucleic acid condensation and release in individual parvovirus particles. A single virus
particle was attached to a cantilever and exposed to various physicochemical conditions,
where the pH and salt concentration were manipulated to induce the condensation or
release of nucleic acids. The study revealed that parvovirus particles are highly responsive
to changes in physicochemical conditions for nucleic acid condensation and release.

3.3. Polysaccharides
The application of AFM in the study of polysaccharides [69] is not as extensive as it
is for other biomolecules, despite the crucial biological roles that they play. Nevertheless,
there have been several AFM investigations that have aimed to unravel the structural
characteristics of polysaccharides found in bacteria, plants, and fungi, as well as densely
glycosylated peptides and proteoglycans. Furthermore, AFM proves valuable in explor-
ing the molecular interactions between polysaccharides and other biomolecules, such as
proteins and lipids. In addition to these efforts, recent advancements in AFM techniques
offer opportunities to delve deeper into the characterization and functional analysis of
polysaccharides, providing a promising avenue for future research in this field. An example
of interest in this field is the investigation of heparan sulfate (HS), a linear polysaccharide
found in all animal cell plasma membranes. Guo et al. [70] investigated the interaction
between heparan sulfate (HS) and antithrombin (AT) on the surface of a single endothelial
cell under near-physiological conditions to understand the role of critical sulfates responsi-
ble for AT binding. The specific interaction between HS and a protein ligand is primarily
determined by the sulfation patterns on the HS chain. The research revealed that AT
interacts with endothelial HS through multiple binding sites, and the presence of N-, 2-O-,
and/or 6-O-sulfates on HS is essential for this interaction.
Processes 2023, 11, 2430 14 of 39

3.4. Peptide
Peptides are short chains of amino acids that can have a wide range of functions in
biological systems, including signaling molecules, neurotransmitters, cell regulation and
homeostasis, etc. Li et al. [71] conducted a study focusing on peptide-assembled hydro-
gels, investigating the morphology and mechanical properties of individual nanofibrils
during the gelation and degradation processes of these hydrogels. With the help of topo-
graphic imaging, they were able to observe distinct assembly behaviors of peptide-formed
nanofibrils throughout the gelation and degradation stages, revealing a correlation between
these behaviors and changes in nanofibrillar mechanics. Gaspar et al. [72] conducted a
study investigating the impact of a peptide called APN-1 on the mechanical character-
istics of cancer cells. Their findings revealed that APN-1 can induce alterations in the
mechanical properties of cancer cells, resulting in cell death and suppressed tumor growth.
Specifically, APN-1 was observed to enhance the stiffness of cancer cells and disrupt their
cytoskeleton—a network of protein fibers responsible for cellular structural support. These
changes in mechanical properties were associated with increased cell death and diminished
tumor growth. These findings suggest the potential of APN-1 as an antitumor agent, with
its effects on cellular biomechanics playing a crucial role in its therapeutic activity.

3.5. Enzymes
Enzymes help in speeding up chemical reactions in physiological processes such as di-
gestion, metabolism, and DNA replication. Enzymes have been extensively studied due to
their significance and diverse applications. Zhang et al. [73] investigated the enzymatic hy-
drolysis of pretreated biomass and observed real-time changes in the cellulose structure of
plant cell walls during enzymatic hydrolysis (Figure 9a,b). The enzymes’ action depended
on the size and width of the cracks in the cellulose. Smaller cracks led to progressive degra-
dation, while larger cracks caused the peeling of glycan chains. The combination of CBH I
and β-G enzymes effectively hydrolyzed the biomass, emphasizing the role of crack size in
the depolymerization and peeling of cellulose microfibrils. Cellulolytic enzymes, such as
CBM3a, play a crucial role in binding specifically to crystalline cellulose. Zhang et al. [74]
utilized CBM3a-functionalized gold nanoparticles (GNPs) to monitor the binding activity of
CBM3a to poplar cell-wall cellulose. The GNPs-CBM3a complexes showed specific binding
to the cellulose surface, aligning with the cellulose fibril axis. This work advances the
comprehension of biomass–enzyme interactions and facilitates the development of efficient
cellulolytic enzymes for biofuel production. In their research, Zhang and Wang et al. [75]
investigated the affinity interactions between the carbohydrate-binding module (CBM) and
plant cell-wall cellulose at the single-molecule level. They used AFM tips functionalized
with CBM3a and CBM2a to determine the binding efficiencies of CBMs to cellulose. Recog-
nition imaging (Figure 9c) revealed that CBM3a exhibited slightly higher binding efficiency
and affinity than CBM2a on both natural and extracted cellulose surfaces. Moreover, both
CBMs showed higher affinities towards natural cell-wall cellulose microfibrils compared
to extracted single cellulose microfibrils. This study provides valuable insights into the
binding properties of CBMs to cellulose, highlighting the differences between CBM3a and
CBM2a and their interactions with cellulose surfaces. They [76] also further examined the
CBM–cellulose binding process on extracted crystalline cellulose. CBM3a molecules were
utilized for both functionalizing the AFM tip and binding as free CBM molecules. The in
situ AFM imaging revealed the efficient and regular binding of CBM molecules to cellulose,
particularly within the initial 60–120 min. This research significantly contributes to in-depth
understanding of the binding mechanism between CBM and crystalline cellulose at the
single-molecule level.
Processes 2023, 11, x FOR PEER REVIEW 16 of 40
Processes 2023, 11, 2430 15 of 39

Figure 9. (a,b)
(a,b) Topography, recognition images, and cross-sectional analysis
analysis of pretreated poplar
cellulose incubated with EG at 0 and 70 min, respectively. Reprinted with
cellulose incubated with EG at 0 and 70 min, respectively. Reprinted with permission from
permission [73].[73].
from (c)
Extracted single cellulose microfibrils. Reprinted with permission from [75].
(c) Extracted single cellulose microfibrils. Reprinted with permission from [75].

AFM for
4. AFM for Studying
Studying Transport
Transport Phenomena in Biological Interactions
Recent advancements
Recent advancements in in scanning
scanning probe microscopy techniques have expanded the
application of force microscopy
of force microscopy in in
biology, providing
biology, simultaneous
providing simultaneousand specific information
and specific infor-
about biological interactions. One area of focus is the study of transport
mation about biological interactions. One area of focus is the study of transport phenom- phenomena in
biological interactions, which can be investigated by assessing molecule interaction
ena in biological interactions, which can be investigated by assessing molecule interaction dynam-
ics. AFM isAFM
dynamics. particularly useful in
is particularly studying
useful protein–ligand
in studying interactions,
protein–ligand as it can as
interactions, measure
it can
bond rupture
measure bondforces,
rupturerevealing information
forces, revealing about affinity
information and
about kinetics.
affinity andFurthermore, AFM
kinetics. Further-
enables the visualization of cell surfaces and their interactions with substrates,
more, AFM enables the visualization of cell surfaces and their interactions with substrates, offering
insights into
offering important
insights cellular processes
into important such as adhesion,
cellular processes migration,migration,
such as adhesion, and differentiation.
and dif-
ferentiation. Endothelial cells, which act as a barrier between blood and tissues,
Endothelial cells, which act as a barrier between blood and underlying underlyingare par-
tis-
ticularly intriguing for AFM investigations. Kolodziejczyk et al.
sues, are particularly intriguing for AFM investigations. Kolodziejczyk et al. [77] [77] conducted a study
con-
on endothelial
ducted a study cells (EA.hy926)cells
on endothelial exposed to silver
(EA.hy926) nanoparticles.
exposed to silverThey discovered
nanoparticles. thatdis-
They the
elasticitythat
covered of the
thecells decreased
elasticity of the with
cells higher concentrations
decreased with higher of silver nanoparticles,
concentrations of silver poten-
nano-
tially due to cytoskeleton reorganization and reinforcement of
particles, potentially due to cytoskeleton reorganization and reinforcement ofthe cell cortex caused bycor-
the cell the
presence of nanoparticles. Additionally, agglomerates of silver nanoparticles
tex caused by the presence of nanoparticles. Additionally, agglomerates of silver nanopar- were observed
on the cell membrane and inside the cells. These findings suggest that force spectroscopy
ticles were observed on the cell membrane and inside the cells. These findings suggest
can serve as a bio-indicator of the physiological state of endothelial cells when exposed to
that force spectroscopy can serve as a bio-indicator of the physiological state of endothelial
nanoparticles. In another study, Dong and Sahin [78] investigated bond lifetimes, observ-
cells when exposed to nanoparticles. In another study, Dong and Sahin [78] investigated
ing notable increases in molecular interaction forces with lifetimes of approximately 5 µs.
bond lifetimes, observing notable increases in molecular interaction forces with lifetimes
This nanomechanical interface provides opportunities for studying shortlived molecular
of approximately 5 µs. This nanomechanical interface provides opportunities for studying
processes and advancing biological imaging, single-molecule manipulation, and assembly
shortlived molecular processes and advancing biological imaging, single-molecule ma-
technologies. By utilizing AFM, one can gain valuable insights into the dynamic nature
nipulation, and assembly technologies. By utilizing AFM, one can gain valuable insights
Processes 2023, 11, 2430 16 of 39

of biological interactions and contribute to the development of various fields within the
biological sciences.

4.1. Unraveling the Mechanics of Antigen–Antibody Interactions

Antigen–antibody interactions [79,80], which involve the binding of an antibody
molecule to a specific antigen molecule (typically a protein or carbohydrate on the surface
of a pathogen or target molecule), can be studied using AFM. This technique allows for the
measurement of interaction strengths and bond breaking, providing valuable information
about the strength and specificity of the interaction. Hinterdorfer et al. [81] investigated
recognition events between antibodies and antigens, as well as the localization of antigenic
sites. They observed unique force signals with an unbinding force of 244 ± 22 pN for
single antibody–antigen recognition events. The unbinding forces were found to result
from the dissociation of individual Fab fragments from a human serum albumin molecule.
The study revealed that the two Fab fragments of the antibody demonstrated independent
and equiprobable binding, facilitated by a flexible linkage that allowed for a 6 nm range
of binding and a high binding probability of 0.5 within encounter times of 60 ms. In a
study by Berquand et al. [82], the forces involved in antigen binding were examined by
studying individual Fv fragments of anti-lysozyme antibodies. The force–distance curves
exhibited the rupture of a single antibody–antigen pair. The injection of free lysozyme led
to a significant decrease in adhesion probability, confirming the specificity of the antibody–
antigen interaction. By recording force–distance curves at different loading rates, the study
revealed two distinct linear regimes with increasing slopes, indicating the presence of
multiple energy barriers in the interaction dynamics. Kienberger et al. [83] focused on
investigating the strength of molecular binding between antibodies and their corresponding
antigenic sites. They examined the binding of anti-Sendai antibodies to Sendai epitopes
fused into bacteriorhodopsin molecules. By studying bacteriorhodopsin’s unfolding, the
study observed distinct intramolecular force patterns, allowing for clear differentiation
between wild-type and Sendai bacteriorhodopsin molecules based on their length distri-
butions. The unbinding force between the antibody and antigen was significantly lower
than the force required to extract and unfold the epitope-containing helix pair from the
membrane, indicating that the intermolecular unbinding forces were weaker than the in-
tramolecular unfolding forces responsible for maintaining the native protein conformation.
Chen et al. [84] conducted a study on the interactions between the HIV-1 envelope protein
gp120 and its receptors, sCD4 and CD4. They examined the binding and dissociation
forces involved in the gp120–sCD4 and gp120–CD4 antigen–antibody interactions. The
results showed that the binding forces of gp120–sCD4 were stronger compared to those of
gp120–CD4 antigen–antibody interactions. Additionally, they investigated the impact of
glycosylation on the gp120–sCD4 interaction and found that the presence of carbohydrates
weakened the interaction. Thus, AFM proves to be a powerful tool for studying antigen–
antibody interactions, providing insights into their structure, binding mechanisms, and
specificity. This technique enables the investigation of bond rupture forces, localization of
antigenic sites, and characterization of molecular binding strengths, contributing to a better
understanding of these interactions in various biological contexts.

4.2. Aptamers: Versatile Tools for Molecular Probing

Aptamers [85], as small and chemically stable biomolecules, offer high affinity and
specificity in binding to targets, serving as versatile alternatives to antibodies for molecular
recognition. Wang and Yadavalli [86] demonstrated the versatility of oligonucleotide ap-
tamers as probes for imaging and spatially locating targets on surfaces. By coupling DNA
aptamers with force recognition mapping, they successfully localized specific proteins at
the single-molecule level and detected changes in binding due to environmental variations.
Topographic images revealed distinct protein patterns on the surface, and the measured
unbinding forces aligned with individual interactions between the DNA aptamer and
protein. In a significant development related to aptamers, Junior et al. [87] devised a rapid
Processes 2023, 11, 2430 17 of 39

NS1 detection platform using a specific DNA aptamer and electrochemical techniques. The
non-structural protein NS1 encoded by the dengue virus exhibits continuous secretion into
the bloodstream by infected host cells. The aptamer-based sensor showed exceptional sen-
sitivity across a wide dynamic range in both buffer and undiluted human serum, offering
rapid response time, cost-effectiveness, and high selectivity against other dengue proteins.
It effectively detected serotypes 1 and 4 within clinically relevant ranges for primary and
secondary infections, paving the way for potential application in miniaturized devices and
point-of-care settings. Leitner et al. [88] conducted an insightful investigation into the inter-
action between the DNA aptamer sgc8c, immobilized on an AFM tip, and its corresponding
receptor, protein tyrosine kinase-7 (PTK7), present in the membrane of acute lymphoblastic
leukemia cells (Jurkat T cells). The aptamer exhibited remarkable affinity and specificity for
binding to PTK7. Through concurrent topography and recognition imaging experiments
(TREC) employing AFM tips functionalized with sgc8c aptamers on immobilized Jurkat
cells, they achieved label-free determination of PTK7 distribution under near-physiological
conditions. In a related study by Poturnayová et al. [89], interactions between the DNA
aptamer sgc8c and protein tyrosine kinase (PTK7) were investigated in the membranes of
leukemia lymphoblastic (MOLT-4) and lymphocyte cell lines, as well as PTK7-negative
U266 myeloid leukemia cells. They utilized the thickness shear-mode acoustics method,
developing an extremely sensitive, label-free biosensor for detecting leukemia cells. The
biosensor demonstrated an impressive limit of detection of 195 ± 20 cells/mL.
These pioneering investigations shed light on the fascinating interplay between DNA
aptamers, their target receptors, and their applications in label-free imaging and biosensing
for leukemia cells. The use of aptamers in conjunction with advanced techniques and
technologies holds promise for advancements in molecular recognition, diagnostics, and
therapeutic applications.

4.3. Understanding Interactions in Polymer Systems

Understanding the interactions in polymer systems is crucial for advancing various
fields, including biofuel production. Lignocellulosic biomass, a promising renewable re-
source for biofuel production, consists of cellulose, hemicellulose, and lignin. However,
the presence of lignin poses a significant challenge, as it inhibits the enzymatic conversion
of cellulose into glucose. Specifically, lignin causes cellulase enzymes, responsible for
breaking down cellulose, to bind irreversibly to it, deactivating the enzymes and reduc-
ing their overall activity. To gain a better understanding of the non-productive binding
phenomenon [90] between lignin and cellulase enzymes, which hinders the enzymatic
conversion of cellulose into glucose in lignocellulosic biomass used for biofuel production,
Qin, Clarke, and Li [91] embarked on a study to investigate the interactive forces involved
in this interaction. Their objective was to identify the underlying mechanisms and factors
contributing to the reduced activity of cellulase in the presence of lignin. To conduct their
investigation, they utilized specialized tips with specific moiety groups attached to them.
These modified tips allowed them to examine the interaction forces between immobilized
cellulase and different surface chemistries. Specifically, they compared hydrophobic tips
with COOH- and OH-coated tips. The results of their study were intriguing. They found
that the interaction forces between the hydrophobic tips and cellulase exhibited the highest
adhesion, surpassing the adhesion forces from COOH- and OH-coated tips by 13% and
43%, respectively. These findings indicated that hydrophobic–hydrophobic interactions
played a significant role in the non-productive binding of cellulase to lignin in this simpli-
fied enzymatic hydrolysis system. The implications of these findings are substantial. By
uncovering the importance of hydrophobic interactions in the non-productive binding of
cellulase to lignin, one can devise strategies to mitigate this interaction and improve the
efficiency of enzymatic biofuel production processes. Developing methods to minimize
the binding of cellulase to lignin will enhance the overall activity and effectiveness of
the enzyme, leading to increased glucose yield and improved biofuel production. Under-
standing the intricate interactions within polymer systems, such as the interaction between
Processes 2023, 11, 2430 18 of 39

lignin and cellulase, opens up avenues for targeted modifications and advancements in
biofuel production. By gaining insights into these fundamental processes, one can optimize
enzymatic hydrolysis and overcome the challenges posed by lignin, bringing us closer to
sustainable and economically viable biofuel production.

4.4. Investigating Protein Interactions through AFM Manipulation

Protein–protein interactions [92,93] are fundamental to the normal functioning of
biological systems, playing critical roles in a wide range of biological processes, including
signaling, transport, and regulation. Assessing these interactions using AFM involves the
manipulation of individual proteins or protein complexes to measure the forces required
for separation. The microvascular endothelium, which forms a monolayer of cells along the
inner surface of blood vessels, serves as a vital structural barrier between the bloodstream
and surrounding tissues. Due to its significant role in tissue and organ homeostasis, the
microvascular endothelium holds substantial diagnostic and therapeutic potential. En-
dothelial cells exhibit complex morphology and functionality depending on their location
within the body, actively contributing to maintaining homeostasis. Liver sinusoidal en-
dothelial cells (LSECs), characterized by fenestrae and sieve plates, represent the most
permeable type of endothelium, providing insights into their physiological state. Recent
advancements in AFM imaging techniques have facilitated successful visualization of
both fixed and live LSECs [94]. In a notable study conducted by Bonanni et al. [95], the
molecular interaction between the protein C551 and gold-immobilized AZ was metic-
ulously examined using AFM. The proteins were strategically positioned on both the
substrate and the AFM tip to facilitate their mutual interaction. AZ was immobilized on
gold via specific residues, orienting the protein configuration in a manner that ensured
that its hydrophobic patch, responsible for interaction with C551, faced away from the
electrode surface and towards the AFM tip. This study highlighted the significance of
protein–protein interactions in biological systems and underscored the potential of AFM
in investigating these interactions. The regulation of gene expression heavily relies on
DNA-binding proteins, which play critical roles across various organisms, from bacteria
to mammalian cells. These proteins interact with DNA, and their significance extends to
numerous essential biological processes, such as DNA replication, packaging, and repair.
Interference in DNA–protein interactions is implicated in various diseases. Manipulating
these interactions through chemical means enables control over gene expression, offering
potential therapeutic interventions for numerous ailments. A study conducted by Sanchez,
Humberto, et al. [96] focused on the interaction between human RAD54w protein and
DNA. They investigated the dynamic interaction of RAD54 with the sample surface and its
influence on protein mobility. They observed that the presence of ATP and AMP-PNP in
the imaging buffer affected the protein’s diffusion constant. Anomalous diffusion behavior
was observed when the protein interacted with surrounding molecules, such as DNA.
The study further revealed that RAD54 moved along DNA through diffusion, and protein
translocation in a DNA-crowded environment increased the likelihood of hopping and facil-
itated transfer to different DNA locations. Despite the development of various techniques,
the understanding of DNA–protein interactions remains incomplete. The initial step in
such studies involves identifying and isolating DNA-interacting proteins. Advancements
in AFM and related techniques continue to contribute valuable insights into the intricate
mechanisms and dynamics of these interactions, leading to a deeper understanding of
biological processes and potential therapeutic strategies.

4.5. Carbohydrate Recognition in Biological Systems: Exploring Receptor Specificity

Carbohydrates play a pivotal role in the composition of the cell membrane and actively
participate in diverse biological processes, including energy production, cellular adhesion,
and glycosylation. Lectins, which are proteins that bind to specific sugars, play a critical
role in recognizing glycoconjugates and exhibit a distinct affinity for their corresponding
sugar components. Gaining insights into the molecular interactions between lectins and
Processes 2023, 11, 2430 19 of 39

carbohydrates is fundamental to understanding cellular interactions and exploring innova-

tive bioanalytical applications. In a study conducted by Touhami et al. [97], the interaction
between the lectin concanavalin A (Con A) and oligoglucose saccharides was investigated.
Gold-coated substrates functionalized with Con A and hex saccharide molecules terminated
with thiols were utilized. Analysis of force–retraction curves revealed that approximately
half of the curves displayed unbinding forces of 96 ± 55 pN, accompanied by elongation
forces and rupture lengths ranging from 0 to 200 nm. These characteristics were absent in
measurements conducted with mannose or a hydroxyl-terminated probe. The obtained
results confirm that the measured unbinding forces arise from specific interactions between
the lectin and carbohydrates. In another study by Zhang et al. [98], AFM recognition imag-
ing was employed to examine structural changes in crystalline cellulose on the cell-wall
surfaces of poplar, switchgrass, and corn stover. The effects of natural dilute sulfuric acid
pretreatment and delignification were investigated. The results indicated that the coverage
of crystalline cellulose on the cell-wall surface increased from 17–20% to 18–40% after dilute
acid pretreatment at 135 ◦ C, with variations based on the acid concentration (Figure 10a–c).
After delignification, the coverage reached 40–70%. These findings highlight the efficacy
of dilute acid pretreatment in enhancing the accessibility of cellulose on plants’ cell-wall
surfaces. Through these studies, a deeper understanding of the role of carbohydrates in
biological processes and their interactions with proteins can be obtained, paving the way
for advancements in bioanalytical techniques and applications. The molecular insights
provided by AFM investigations contribute to the development of innovative approaches
for studying and manipulating carbohydrate–protein interactions, which have significant
implications in various fields, including drug discovery, biomaterial engineering, and
diagnostics.

Figure 10. (a) Area type distributions of natural and dilute-acid-pretreated cell-wall surfaces of corn
stover. (b) Area type distributions of delignified cell-wall surfaces of corn stover. (c) Recognition area
percentage (RAP) summary of natural and pretreated poplar, switchgrass, and corn stover cell-wall
surfaces. Reprinted with permission from [98].
Processes 2023, 11, 2430 20 of 39

5. Unveiling the Mechanical Properties of Cells

Cells are complex systems composed of interconnected molecules and structures,
both on the surface and within the cell. These molecular components play vital roles
in various physiological processes, including cell adhesion, tissue function, and gene
expression regulation through DNA methylation. Understanding how biological systems
adapt their properties to fulfill their functions is a significant challenge in biology. At the
nanoscale level, the structural, biophysical, and chemical properties of biological systems
determine their functionality in response to the surrounding environment. Cells experience
mechanical loads and deformations in their natural environment, triggering biochemical
changes and interactions with neighboring cells and the extracellular matrix. The process
by which cells sense mechanical stimuli and convert them into biochemical signals, known
as mechanotransduction, is crucial for normal functioning and development. It involves the
transmission of external forces to the cytoskeleton, influencing transcriptional processes and
gene expression. AFM offers a powerful tool for quantifying the mechanical properties of
cells, including their stiffness and adhesion characteristics. AFM-based force spectroscopy
allows for the indentation of cell surfaces and generates force–distance curves that can
be analyzed. Alterations in the mechanical properties of cells have been associated with
various diseases, such as muscular dystrophies and cancers. AFM has been employed to
differentiate between cancerous cells and non-malignant cells based on their mechanical
properties, holding great promise for identifying pathological changes in cells and tissues
associated with diseases like cancer, arthritis, and cardiovascular disorders. Various studies
have compared the mechanical properties of aggressive cancer cells with non-malignant
cells using AFM, providing insights into metastasis mechanisms and disease progression.
For example, Faria et al. [99] investigated the elastic properties (Figure 11c) of prostate
cells at different stages of disease progression and successfully discriminated between
benign prostate hyperplasia (BPH), non-invasive prostate cancer cells, and metastatic
prostate cancer cells based on their Young’s moduli. Li et al. [100] compared the mechanical
properties of red blood cells (RBCs) with three aggressive cancer cell types, providing
insights into metastasis mechanisms. The results (Figure 11a) showed that RBCs had
smaller diameters and lower Young’s moduli compared to the cancer cells. Additionally,
aggressive cancer cells exhibited lower Young’s moduli than indolent cancer cells, providing
further insights into metastasis mechanisms. Other studies have explored the mechanical
properties of tissues and spheroids associated with pathological conditions, such as ovarian
tissues (Figure 11b) [101] and tumor spheroids (Figure 11d) [102], providing insights into
physiological and pathological mechanisms.
Lekka et al. [103] measured the stiffness of erythrocytes under physiological conditions
using the Hertz model. They observed variations in the Young’s modulus distributions
among donors, which correlated with factors such as disease type, sex, age, and cigarette
smoking. Li et al. [104] examined the elastic properties of individual lymphoma cells
and measured the CD20–rituximab binding force on the surface of B-cell lymphoma cells.
Rituximab, an anticancer drug used for the treatment of B-cell lymphoma, was linked to
the AFM tip to assess the CD20–rituximab binding force on the lymphoma cell surface.
The force curves exhibited a clear, sudden peak during retraction [105]. Wang et al. [106]
examined the elastic properties of ricin and its interactions with anti-ricin aptamers, re-
vealing distinct binding conformations with unique elastic properties. They developed
a method to differentiate specific unbinding pathways by analyzing individual force–
extension curves in a multi-pathway system. Another study by Li et al. [107] investigated
the impact of methotrexate on the viscoelastic properties of different cell types. AFM in-
denting with conical and spherical tips was employed to quantitatively measure changes in
cellular viscoelastic properties, including Young’s modulus and relaxation time. The results
demonstrated that methotrexate stimulation significantly reduced both the cellular Young’s
modulus and the relaxation times (Figure 12a–d). AFM imaging was utilized to visualize
the morphological changes induced by methotrexate. Furthermore, AFM has been used
to investigate the elastic properties of individual lymphoma cells. In a study conducted
 Processes 2023, 11, 2430 21 of 39

by Wei et al. [108], inverse finite element simulation was used to extract the viscoelastic
parameters of living cells based on experimental stress–relaxation curves. This approach
allowed for the quantification of the cells’ viscoelastic properties. These investigations high-
light the potential of AFM in studying the mechanical properties of cancer cells and tissues,
providing valuable information for cancer diagnosis, understanding disease progression,
and developing new therapeutic approaches, as discussed in the following sections. The
Processes 2023, 11, x FOR PEER REVIEW 22 of 40
combination of AFM with other techniques offers a multidimensional understanding of
cancer at the cellular and tissue levels, paving the way for improved cancer detection and
treatment strategies.

Figure
Figure11.
11.(a)(a)
Histogram
Histogram of of
Young’s modulus
Young’s modulus forfor
RBCs,
RBCs, Raji, Hut,
Raji, and
Hut, K562
and cells.
K562 Reprinted
cells. Reprintedwith
permission from [100]. (b) Stiffness versus degree of coalignment of F-actin. Reprinted
with permission from [100]. (b) Stiffness versus degree of coalignment of F-actin. Reprinted with with permis-
sion from [101].
permission from(c) [101].
Apparent Young’s moduli
(c) Apparent Young’sfor LNCaP,
moduli for PC-3,
LNCaP, BPH, andBPH,
PC-3, PNT2-C2 cells. Reprinted
and PNT2-C2 cells.
with permission from [99]. (d) Moduli G’ (f) and G” (f) as a function of frequency f (Hz)
Reprinted with permission from [99]. (d) Moduli G’ (f) and G” (f) as a function of frequency f (Hz)
for different
spheroids of various sizes. Reprinted with permission from [102].
for different spheroids of various sizes. Reprinted with permission from [102].

Lekka et al. [103] measured the stiffness of erythrocytes under physiological condi-
tions using the Hertz model. They observed variations in the Young’s modulus distribu-
tions among donors, which correlated with factors such as disease type, sex, age, and cig-
arette smoking. Li et al. [104] examined the elastic properties of individual lymphoma cells
and measured the CD20–rituximab binding force on the surface of B-cell lymphoma cells.
Rituximab, an anticancer drug used for the treatment of B-cell lymphoma, was linked to
the AFM tip to assess the CD20–rituximab binding force on the lymphoma cell surface.
The force curves exhibited a clear, sudden peak during retraction [105]. Wang et al. [106]
examined the elastic properties of ricin and its interactions with anti-ricin aptamers, re-
vealing distinct binding conformations with unique elastic properties. They developed a
method to differentiate specific unbinding pathways by analyzing individual force–exten-
sion curves in a multi-pathway system. Another study by Li et al. [107] investigated the
impact of methotrexate on the viscoelastic properties of different cell types. AFM indent-
 ing sections. The combination of AFM with other techniques offers a multidimension
understanding of cancer at the cellular and tissue levels, paving the way for improve
cancer detection and treatment strategies.
Processes 2023, 11, 2430 22 of 39

Figure 12. (a–d) Young’s modulus of C2C12, L929, A549, and HEK 293 cells, respectively, after 24 h
Figureof 12.
stimulation with methotrexate.
(a–d) Young’s modulus of (* pC2C12,
< 0.05; ** p < 0.01;
L929, A549,*** pand
< 0.001)
HEKReprinted
293 cells,with permission after 24
respectively,
from [107]. with methotrexate. (* p < 0.05; ** p < 0.01; *** p < 0.001; ) Reprinted with permissio
of stimulation
from 6.
[107].
Biomarkers
Diseases can give rise to structural and compositional changes in the body, which can
6. Biomarkers
serve as early indicators for disease detection. It has been demonstrated that micro- and
nano-stiffness
Diseases canalterations
give risecan be observedand
to structural evencompositional
before visible morphological
changes inchangesthe body,occur.
which ca
Understanding these changes is crucial for unraveling the underlying physiological mecha-
serve as early indicators for disease detection. It has been demonstrated that micro- an
nisms leading to disease symptoms. Such knowledge not only enables improved symptom
nano-stiffness
managementalterations can bethe
but also establishes observed
groundworkeven forbefore
effectivevisible
diseasemorphological
prevention [Link] o
cur. Understanding
Therefore, AFM can these
servechanges
as a valuableis crucial for unraveling
tool in studying thewhich
biomarkers, underlying physiologic
are molecules
mechanisms leadingoftospecific
that are indicative disease symptoms.
diseases Such knowledge
or physiological not only encompass
conditions. Biomarkers enables improve
proteins, nucleic acids, or other detectable molecules present
symptom management but also establishes the groundwork for effective disease in body fluids, tissues, or cells. preven
Using AFM, one can analyze the topography and mechanical properties of biomolecules.
tion strategies. Therefore, AFM can serve as a valuable tool in studying biomarkers, whic
For instance, AFM can measure the stiffness of a protein, providing insights into its folding
are molecules
and [Link]
This isare indicative
significant of specific diseases
for understanding or physiological
how environmental conditions. B
changes or interac-
omarkers encompass
tions with proteins,
other molecules affect anucleic acids,
particular or other
biomarker. detectablethemolecules
By immobilizing biomarker on present
bodyafluids,
surface tissues, or [Link]
and quantifying Using
force AFM,
requiredone can analyze
to detach thedetermine
it, one can topography and mechanic
the strength
of the interaction and investigate its modulation by numerous
properties of biomolecules. For instance, AFM can measure the stiffness of a protei factors. High-resolution
imaging enables the identification of the spatial distribution and concentration of specific
providing insights into its folding and stability. This is significant for understanding ho
biomarkers, thereby exploring their presence in different cellular compartments or tissues.
environmental
AFM transport changes or interactions
is particularly valuable for with otherbiomarkers,
studying moleculessince affect a particular
it enables biomarke
the direct
By immobilizing
measurement of the biomarker
physical on aand
properties surface and quantifying
interactions at the individualthebiomolecular
force required level,to detac
it, one can determine the strength of the interaction and investigate its modulation
which helps in the identification and characterization of biomarkers and can be utilized for by nu
merousdiagnostic or prognostic
factors. purposes. imaging enables the identification of the spati
High-resolution
6.1. Cancer Biomarkers
The role of AFM in characterizing cancer-related biomarkers involves studying bioma-
rker interactions in cancer progression, with cell stiffness serving as a crucial biomarker
Processes 2023, 11, 2430 23 of 39

for metastatic potential. Li et al. [109] conducted research to explore the connections
between cell structure, mechanical properties, and breast cancer. They compared non-
malignant (MCF-10A) and malignant (MCF-7) human breast epithelial cell lines. The results
(Figure 13a) showed that higher loading rates led to increased stiffness in both cell types,
which indicates that cells appear stiffer when probed at higher loading rates, primarily due
to the contribution of cell viscosity. At the physiological temperature of 37 ◦ C, the apparent
Young’s modulus of MCF-10A cells was significantly higher than that of MCF-7 cells at the
same loading rate. This implies that non-malignant cells possess greater stiffness compared
to malignant cells. The observed difference in cell elasticity was attributed to variations in
the organization of the sub-membrane actin structures between the two cell types. MCF-
10A cells exhibit well-aligned filamentous structures below the membrane, referred to as
stress fibers, which contribute to their higher stiffness. In contrast, MCF-7 cells display
less-defined and disorganized filamentous structures, resulting in reduced cell stiffness.
These findings suggest a correlation between cell structure, mechanical properties, and
breast cancer. In study by Xu et al. [110], non-malignant ovarian cells (IOSE) were compared
to ovarian cancer cells (HEY). They found that IOSE cells exhibited higher stiffness than
various ovarian cancer cells (HEY) (Figure 13b). This was determined by analyzing the
force–indentation curves and calculating the Young’s modulus of individual cells. They
also examined the relationship between cell stiffness and metastatic potential. HEY A8 cells,
which were derived from the same tumor specimen as HEY cells, were found to be more
compliant (i.e., less stiff) than HEY cells. This finding is significant, because HEY A8 cells
also exhibited higher tumorigenicity and increased invasiveness and migratory activity.
This suggests an inverse correlation between cell stiffness and metastatic potential. To
understand the molecular basis of the observed differences in cell stiffness, gene expression
analysis was performed on HEY and HEY A8 cells. The analysis identified numerous
differentially expressed genes between the two cell lines, particularly genes related to
cytoskeletal remodeling pathways. These findings support the hypothesis that changes
in actin-mediated cytoskeletal remodeling contribute to the differences in cell stiffness.
Microscopic examination of the cells confirmed that non-malignant cells had denser and
well-aligned F-actin, while ovarian cancer cells displayed less organized and randomly
oriented F-actin. The degree of coalignment of F-actin fibers was correlated with cell
stiffness, further supporting the relationship between cytoskeletal remodeling and cell
stiffness. Li et al. [111] utilized AFM peak force tapping imaging mode to visualize and map
CD20 molecules on human lymphoma cells using rituximab-tethered tips. Recognition
spots, denoted by green arrows (Figure 13c,d), were observed in the adhesion image,
indicating the presence of CD20 molecules on the cell surface. In contrast, normal blood
cells from healthy volunteers did not exhibit recognition spots, as they did not express
CD20. Quantitative analysis of the cluster sizes of CD20 molecules on the surface of Raji
cells was performed using recognition images. The recognition spots were quantitatively
analyzed, and the cluster size histogram revealed that CD20 organizations were mainly
distributed in the range of 100–4000 nm2 . Furthermore, they applied PFT imaging to
visualize CD20 molecules on cancer cells obtained from a clinical lymphoma patient.
Recognition spots were observed in the adhesion image, and the cluster size histogram
showed a distribution similar to that observed on Raji cells. AFM imaging allowed for the
localization and quantification of CD20 molecules, providing valuable insights into their
spatial organization and nanoscale behavior. In another study by Paul et al. [112], AFM-
based high-resolution imaging was used to differentiate extracellular vesicles (EVs) derived
from colon cancer cells (HCT 116) and healthy colon cells (CCD 18CO). They determined
the morphology and ultrastructural characteristics of the EVs, with HCT-116-derived EVs
having at least two times higher density compared to CCD-18CO-derived EVs. The CD9-
antibody-functionalized AFM tips showed higher rupture forces and frequencies for HCT
116 EVs, indicating a higher density of CD9 molecules on their surface. Spectroscopic
techniques confirmed the presence of hyaluronic acid (HA) in HCT 116 EVs, but not in
CCD-18CO EVs. The hydrodynamic diameter of the EVs was measured to be around
 derived EVs having at least two times higher density compared to CCD-18CO-derived
EVs. The CD9-antibody-functionalized AFM tips showed higher rupture forces and fre-
quencies for HCT 116 EVs, indicating a higher density of CD9 molecules on their surface.
Spectroscopic techniques confirmed the presence of hyaluronic acid (HA) in HCT 116 EVs,
Processes 2023, 11, 2430 but not in CCD-18CO EVs. The hydrodynamic diameter of the EVs was measured 24 of 39 to be

around 100 ± 20 nm by dynamic light scattering. The study suggested that HA-enriched
EVs could serve as potential biomarkers for colon cancer. Overall, AFM plays a crucial
100 ±
role in20 nm by dynamic
characterizing light scattering.
cancer-related The study
biomarkers bysuggested that HA-enriched
investigating EVs
biomarker interactions
could serve as potential biomarkers for colon cancer. Overall, AFM plays a
in cancer progression. It enables the study of cell stiffness as a valuable biomarkercrucial role in for
characterizing cancer-related biomarkers by investigating biomarker interactions in cancer
metastatic potential and provides insights into the mechanical properties, structural or-
progression. It enables the study of cell stiffness as a valuable biomarker for metastatic
ganization, and spatial distribution of molecules in cancer cells and extracellular vesicles.
potential and provides insights into the mechanical properties, structural organization, and
These findings contribute
spatial distribution to a better
of molecules understanding
in cancer of cancer’s
cells and extracellular mechanisms
vesicles. and the po-
These findings
tential development
contribute of novel diagnostic
to a better understanding andmechanisms
of cancer’s therapeuticand
approaches.
the potential development
of novel diagnostic and therapeutic approaches.

Figure 13. (a) Apparent elastic modulus against loading rate for MCF-10A and MCF-7 cells at 37 ◦ C.
Reprinted with permission from [109]. (b) Young’s modulus of different cells. Reprinted with
Figure 13. (a)
permission Apparent
from elastic
[110]. (c,d) modulus
Overlay against image
of recognition loading rate
and thefor MCF-10A and
corresponding MCF-7 cells
topographic imageat 37 °C.
Reprinted with permission
and the distribution from [109].
of recognition (b) Young’s
spot size modulus of
of CD20 molecules ondifferent
lymphoma cells.
RajiReprinted with per-
cell surfaces,
mission fromwith
without and [110]. (c,d) Overlay of
rituximab-tethered recognition
tips, image
respectively. and the
Reprinted withcorresponding
permission fromtopographic
[111]. image
and the distribution of recognition spot size of CD20 molecules on lymphoma Raji cell surfaces,
6.2. Neurodegenerative
without Disease Biomarkers
and with rituximab-tethered tips, respectively. Reprinted with permission from [111].
AFM has made significant contributions to the understanding of neurodegenerative
disease markers. One area of investigation is the mechanical properties of amyloid fibrils
and aggregates. Alzheimer’s disease (AD) poses challenges for early detection and accurate
diagnosis, leading to extensive research on identifying biomarkers. Understanding the
mechanics of brain tissues has emerged as a crucial aspect in diagnosing brain diseases.
Park et al. [113] conducted a study using nanoindentation to investigate the viscoelastic
properties of human autopsy brain tissues as a potential biomarker for AD. The stress–strain
 the mechanics of brain tissues has emerged as a crucial aspect in diagnosing brain dis-
eases. Park et al. [113] conducted a study using nanoindentation to investigate the viscoe-
lastic properties of human autopsy brain tissues as a potential biomarker for AD. The
stress–strain curves revealed two common factors: a linear relation between stress and
Processes 2023, 11, 2430
strain during the pre-loading phase, and increased stiffness with higher loading frequen-
25 of 39
cies, consistent with soft tissue characteristics. However, brain tissues affected by AD
showed moderate slopes during the pre-loading phase, along with greater hysteresis, in-
dicating
curveshigher
revealedenergy dissipation.
two common Comparing
factors: the Young’s
a linear relation modulus
between values
stress and between
strain during nor-
maltheand AD-affected brain tissues (Figure 14a) showed higher values
pre-loading phase, and increased stiffness with higher loading frequencies, consistent in normal tissues—
by with
23.5% fortissue
soft graycharacteristics.
matter and 27.9% However,for brain
whitetissues
matter. Similar
affected trends
by AD weremoderate
showed observed in
storage
slopesmodulus,
during the loss modulus,
pre-loading phase, and loss
along withfactor
greatermeasurements. Storage
hysteresis, indicating moduli
higher energywere
dissipation. Comparing the Young’s modulus values between normal
higher in normal brain tissues, while loss moduli exhibited more overlap. The loss factor and AD-affected
brain tissues
indicated a more(Figure
viscous 14a) showedinhigher
response values brain
AD-affected in normal tissues—by
tissues, especially23.5% for gray
at lower loading
matter and 27.9% for white matter. Similar trends were observed in storage modulus, loss
frequencies, suggesting changes in the viscosity and stiffness of extracellular matrix com-
modulus, and loss factor measurements. Storage moduli were higher in normal brain
ponents during AD’s progression. Another study by Nirmalraj et al. [114] demonstrated
tissues, while loss moduli exhibited more overlap. The loss factor indicated a more viscous
the response
applicability of AFM brain
in AD-affected for analyzing proteinataggregates
tissues, especially lower loading on frequencies,
RBCs fromsuggesting
patients with
neurocognitive
changes in the viscosity and stiffness of extracellular matrix components during aggregate
disorders. The findings highlighted the variations in protein AD’s
morphology
[Link] assembly
Another studypatterns,
by Nirmalrajproviding insights
et al. [114] into the
demonstrated thepathophysiological
applicability of AFM pro-
cesses
for associated with AD.
analyzing protein They analyzed
aggregates on RBCsthe fromsize, shape,with
patients morphology, assembly
neurocognitive patterns,
disorders.
andThe findings highlighted
prevalence of protein the variationsreferred
aggregates, in protein toaggregate
as physicalmorphology and assembly
biomarkers, on red blood
patterns,
cells (RBCs)providing insights
from patients withinto the pathophysiological
neurocognitive processes associated
disorders—particularly with AD. dis-
Alzheimer’s
ease (AD)—and revealed variations in height across RBCs from both patients protein
They analyzed the size, shape, morphology, assembly patterns, and prevalence of with disor-
aggregates, referred to as physical biomarkers, on red blood cells (RBCs) from patients with
ders and healthy controls. They found that the size, shape, morphology, assembly pat-
neurocognitive disorders—particularly Alzheimer’s disease (AD)—and revealed variations
terns, and prevalence
in height across RBCsof protein
from aggregates
both patients withon RBCs and
disorders varied depending
healthy controls. on
Theythe age and
found
severity
that theof size,
the neurocognitive
shape, morphology, disorder.
assembly They also and
patterns, correlated
prevalencetheofAFM measurements
protein aggregates of
fibrillar
on RBCsaggregates with the Aβ42/40
varied depending on the ageratio
and in cerebrospinal
severity fluid (CSF),disorder.
of the neurocognitive which could
They be a
potential biomarker
also correlated the for
AFMADmeasurements
pathology. The analysis
of fibrillar revealedwith
aggregates the presence
the Aβ42/40of spherical/an-
ratio in
cerebrospinal
nular oligomers,fluid (CSF), which
protofibrils, and could
fibrilsbe
onaRBCs,
potential
with biomarker for AD of
the prevalence pathology. The
fibrils increasing
analysis revealed the presence of spherical/annular
with the severity of the neurocognitive disorder. oligomers, protofibrils, and fibrils on
RBCs, with the prevalence of fibrils increasing with the severity of the neurocognitive disorder.

(a) (b)
Figure 14. (a) Young’s modulus (E) of normal and AD-affected human autopsy brain tissues.
Reprinted with permission from [113]. (b) Young’s modulus of different cells. Reprinted with
permission from [115].

Additionally, Strijkova-Kenderova et al. [115] investigated the ultrastructural charac-

teristics of RBCs as a peripheral cell model for specific signatures of neurodegenerative
pathologies such as Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and AD.
They found significant differences in the shape distribution, surface roughness, volume,
and Young’s modulus between RBCs from patients with neurodegenerative disorders and
healthy cells. The percentage of biconcave cells decreased in neurodegenerative disor-
Processes 2023, 11, 2430 26 of 39

ders, with PD cells exhibiting the lowest proportion of biconcave shape. Differentiated
morphology and aging-induced shape transformations distinguished neurodegenerative
disorder RBCs from healthy cells. The volume of different cell shapes also differed, allow-
ing for differentiation between AD and other disorders. Surface roughness was lower in
neurodegenerative disorder cells but similar between PD, ALS, and AD cells. Increased
membrane stiffness, reflected in higher Young’s modulus values (Figure 14b), indicated
decreased deformability in neurodegenerative disorder RBCs. These findings align with
previous studies reporting higher Young’s modulus values in PD and AD patients. The
study concluded that neurodegenerative disorders lead to distinct alterations in RBCs’ sur-
face nanostructure, morphometry, and nanomechanical properties, potentially serving as
markers for these disorders. Accumulated misfolded proteins/aggregates and their impact
on RBCs’ cytoskeleton and lipid bilayer may contribute to the observed morphological
transformations.

6.3. Infectious Disease Biomarkers

Utilizing AFM to study biomarkers in infectious diseases allows for the investigation
of pathogen–host interactions and their impact on biomarker behavior. One such disease is
dengue, a mosquito-borne illness caused by dengue virus (DENV) that affects a large num-
ber of people worldwide. Unfortunately, the lack of specific treatment options for dengue
stems from an incomplete understanding of how viral components interact with cellular
structures in the host. To address this knowledge gap, researchers such as Patil et al. [116]
have visualized the intramolecular structure of antibodies, while Gilbert et al. [117] demon-
strated the capability of AFM to assess the forces and dynamics of molecular interactions in
living bacteria. They showed the capability of AFM using vancomycin tips to quantitatively
assess the forces and dynamics of the vancomycin/D-Ala-D-Ala interaction (Figure 15a).
They also successfully imaged individual D-Ala-D-Ala sites on the division septum of
living Lactococcus lactis bacteria. Advancements have also been made in studying viral
infections using AFM. Rankl et al. [118] investigated the attachment and internalization of
human rhinoviruses (HRVs), providing detailed information on virus–receptor interactions
at the single-molecule level. Their findings revealed a time-dependent transition from
single- to multiple-receptor binding (Figure 15b). The unbinding forces required to detach
the virus from the cell membrane increased over several hundred milliseconds. Further-
more, Fantner et al. [119] utilized HS-AFM to enable real-time imaging of live bacterial
cells, revealing nanometer-scale resolution and high temporal resolution. They focused on
the interaction between antimicrobial peptides and Escherichia coli cells, uncovering the
multistep process of bacterial cell death (Figure 15c). Investigating the interaction between
dengue virus (DENV) capsid protein (C) and low- and very-low-density lipoproteins (LDL
and VLDL, respectively) using various techniques, Faustino et al. [120] discovered a specific
interaction between DENV C and VLDL, while no interaction was observed with LDL. The
results revealed stronger binding forces between DENV C and VLDL (40 pN) compared
to LDL (20 pN). This binding was dependent on potassium and involved the N-terminal
region of DENV C (Figure 15d,e). They successfully inhibited DENV C–VLDL binding
using a peptide drug lead. In addition to their work on DENV, they also investigated the in-
teractions between Staphylococcus epidermidis and host proteins absorbed onto biomaterials
during biofilm formation. In their research, Herman-Bausier and Dufrêne [121] investi-
gated the binding mechanism of the S. epidermidis CWA protein serine-aspartate repeat
protein F (SdrF) to type I collagen. They observed that SdrF enables bacterial adhesion to
collagen-coated surfaces through both weak and strong bonds (Figure 15f,g). It was also
revealed that these bonds involve the A and B regions of SdrF, indicating the protein’s
dual ligand-binding capability. Notably, the weak and strong bonds exhibited high dis-
sociation rates, suggesting a less stable nature compared to the conventional “dock, lock,
and latch” mechanism. These findings shed light on novel ligand-binding mechanisms
employed by CWA proteins. Bacterial and viral infections have significant implications
for human health, making it essential to study these phenomena at the molecular and
Processes 2023, 11, 2430 27 of 39

cellular levels using AFM. AFM has been successfully employed in numerous studies to
investigate bacterial and viral interactions, cell death processes, intramolecular structures,
and the attachment of viruses to host cells. These advancements have greatly contributed
to our understanding of infection processes and hold potential for the development of
new strategies for diagnosis, treatment, and prevention. For example, Newton et al. [122]
introduced a novel AFM technique that quantifies binding events between enveloped
viruses and surface receptors on live animal cells, offering insights into early virus–cell
interactions. Kiss et al. [123] conducted a study on the topographical and nanomechanical
properties of SARS-CoV-2—the virus responsible for the COVID-19 pandemic—revealing
its dynamic nature and unique mechanical characteristics (Figure 15h,i). The dynamics of
the surface spikes were identified as potentially contributing to the virus’s unusually high
infectivity, while its mechanical and self-healing properties may facilitate adaptation to
diverse environmental conditions. By leveraging the capabilities of AFM, these studies
have expanded our knowledge of bacterial and viral infections, shedding light on their
intricate mechanisms and interactions at the molecular level. This information can aid in
the development of novel therapeutic approaches, the design of antiviral agents, and the
understanding of viral pathogenesis. Ultimately, studying bacterial and viral infections
using AFM offers valuable insights that contribute to our ability to combat these infectious
diseases effectively.

Figure 15. (a) Force histogram of adhesion. Reprinted with permission from [117]. (b) Surface
variation as a function of time. Reprinted with permission from [118]. (c) Force spectroscopy of the
interaction between HRV2 and MBP-V1-8. Reprinted with permission from [119]. (d) Force histogram
of the interaction between DENV C and VLDL (e) Force histogram when K+ is replaced by the same
concentration of Na+. Reprinted with permission from [120]. (f,g) SdrF–collagen interactions of weak
and strong Cn-binding forces, respectively. Reprinted with permission from [121]. (h) Force–distance
curves of a single SARS-CoV-2 virion. (i) force–distance curves obtained during retraction. Reprinted
with permission from [123].
Processes 2023, 11, 2430 28 of 39

6.4. Hematological Disorders

Hematological disorders are medical conditions characterized by a propensity for
excessive bleeding. The formation of blood clots through fibrinogen polymerization is an es-
sential process to prevent bleeding. However, elevated levels of fibrinogen and erythrocyte
aggregation can increase the risk of cardiovascular and cerebrovascular diseases. Previous
research has indicated that fibrinogen-induced erythrocyte hyperaggregation is mainly
attributed to nonspecific binding. Studies on erythrocyte aggregation in hypertension have
suggested the involvement of additional mechanisms. Although AFM combined with force
spectroscopy has been used to investigate related systems, the interaction between the en-
tire fibrinogen molecule and its receptors on blood cells has not been specifically examined.
Xing et al. [124] conducted a study focusing on a patient with elliptocytosis complicating
idiopathic thrombocytopenic purpura (ITP) to investigate the morphological properties of
erythrocytes at the nanometer scale. They observed the characteristic biconcave shape and
morphological properties of healthy erythrocytes using AFM imaging. In contrast, erythro-
cytes from the patient group exhibited deformities and surface architecture deformation.
Carvalho et al. [125] investigated the binding between fibrinogen and an unidentified re-
ceptor on human erythrocyte membranes. They found that fibrinogen-induced erythrocyte
aggregation resulted from nonspecific binding to erythrocyte membranes, while platelets
possessed a specific fibrinogen integrin receptor. A study by Liu et al. focused on ultra-
structural changes in erythrocytes in Waldenström macroglobulinemia (WM), a rare form
of lymphoma. They observed significant deformations in the shape and surface membrane
of erythrocytes in WM patients compared to healthy controls and patients with multiple
myeloma (MM). The WM erythrocytes exhibited ravines, protrusions, and larger particle
aggregation on the surface. In contrast, the MM erythrocytes showed smaller deformations
and smaller particle aggregation. Liu et al. [126] aimed to investigate the ultrastructural
changes in erythrocytes at a nanometer scale in Waldenström macroglobulinemia (WM),
a rare form of lymphoma. The results of the study showed significant deformations in
the overall shape and surface membrane of erythrocytes in WM patients when compared
to healthy controls and MM patients. The healthy erythrocytes exhibited a characteristic
biconcave shape, and the ultrastructure showed a regular nanoscale network of membrane
proteins. The particles on the surface of healthy erythrocytes were uniform, with a diameter
of 10 nm. For the WM patients, the erythrocytes showed dramatic deformations in shape
and surface membrane. The cells did not exhibit their regular biconcave shape, and the
center of the cell surface was swollen. Ultrastructural examination revealed large ravines
and protrusions on the cell surface. Larger particles (greater than 40 nm) were observed on
the surface, indicating significant aggregation of membrane proteins. The membrane pro-
teins were reorganized into stripe patterns in one direction. The statistical analysis showed
that the length, width, peak, valley, peak-to-valley value, and average surface fluctuation
of the WM erythrocytes were significantly different from those of healthy erythrocytes,
with increased roughness and peak-to-valley values. The MM patients also exhibited
deformations in erythrocyte shape and surface membrane. The cells did not exhibit their
regular biconcave shape, and small holes were observed on the cell surface. The particles
on the surface were smaller than those observed in WM erythrocytes, and their distribution
was heterogeneous. Li et al. [127] used AFM combined with magnetic bead cell isolation to
measure the viscoelastic properties of human primary B lymphocytes. They isolated B lym-
phocytes from healthy volunteers and quantitatively measured the instantaneous modulus
and relaxation time of living B lymphocytes. The results showed that the instantaneous
modulus of normal human B lymphocytes was 2–3 kilopascals (kPa), and the relaxation
times ranged from 0.03 to 0.06 s and from 0.35 to 0.55 s. Jembrek et al. [128] investigated
the cellular and molecular consequences of oxidative injury induced by hydrogen peroxide
(H2 O2 ) in P19 neurons to understand the neuroprotective mechanisms of quercetin beyond
its antioxidant activity. It was observed that the viability of P19 neurons decreased in a
concentration-dependent manner when exposed to H2 O2 , while quercetin treatment did
not decrease cell survival even at high concentrations. Quercetin treatment prevented the
Processes 2023, 11, 2430 29 of 39

downregulation of Bcl-2 and the upregulation of p53. Additionally, they also analyzed the
expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a metabolic enzyme
with non-glycolytic functions that is involved in oxidative stress sensing and cell death
induction. While H2 O2 did not affect GAPDH expression, quercetin treatment led to pro-
nounced upregulation of GAPDH. The neurons exposed to H2 O2 exhibited an irregular
circular shape and degenerated cell bodies, while those treated with quercetin displayed
a more regular morphology resembling that of control neurons. Feng et al. [129] investi-
gated the nanomechanical properties of extracellular vesicles (EVs) from the fluid biopsies
of clinical hematological cancer patients to understand the role of EV mechanics in the
development of hematological cancer. The results showed significant differences in EV me-
chanics between multiple myeloma patients, lymphoma patients, and healthy volunteers.
The mechanical properties of EVs were found to be associated with their geometric features,
such as height, radius, and deformation degree. Furthermore, the study demonstrated
dynamic changes in the mechanical properties of EVs during the development of hemato-
logical cancer. Multiple myeloma patients showed smaller Young’s moduli and viscous
coefficients of EVs in the bone marrow compared to the blood, while lymphoma patients
showed the opposite trend. The mechanical and geometric alterations of EVs were highly
heterogeneous for different types of fluid biopsies and hematological cancers. By utilizing
AFM in these studies, the researchers gained insights into the morphological, mechanical,
and ultrastructural properties of erythrocytes, the binding mechanisms between fibrinogen
and erythrocytes, the viscoelastic properties of B lymphocytes, the effects of quercetin on
neuronal morphology, and the mechanical characteristics of EVs in hematological cancer
patients. These findings contribute to our understanding of bleeding disorders, cellular
behaviors, and the pathogenesis of hematological cancers, facilitating the development of
potential treatments and diagnostic strategies.

7. The Multifaceted Applications of AFM in Drug Therapy

Understanding the interactions between drugs and their target molecules is crucial
for optimizing drug molecule design and enhancing their effectiveness in the field of
drug discovery and development. The efficacy of a drug is closely linked to its ability to
interact with specific target molecules. Traditionally, techniques such as binding assays
and spectroscopic methods have been employed to investigate these interactions, but
they often provide limited insights into the mechanical properties and forces involved.
To overcome these limitations, AFM—a high-resolution imaging and force measurement
technique—offers a unique opportunity to probe drug–target interactions at the single-
molecule level. It provides valuable information on the strength of these interactions
and their contributing factors. By studying the interactions between drugs and their
target molecules, one can assess their strength and analyze various influencing factors,
contributing to the optimization of drug molecule design and enhancing their effectiveness.
Furthermore, AFM plays a crucial role in investigating drug delivery to cells or tissues.
By performing high-resolution imaging, AFM enables the tracking of drug molecules’
location and distribution within the sample, shedding light on their uptake by cells or
tissues. This knowledge aids in the development of improved drug-delivery strategies and
enhances targeted delivery to specific cells or tissues, potentially improving therapeutic
outcomes. Additionally, AFM can be used to monitor the efficiency of drug therapy by
measuring the mechanical properties of cells or tissues before and after drug treatment,
providing more effective treatment approaches. In the context of chemotherapy, the targeted
release of anticancer drugs at tumor sites to induce cancer cell death is vital. However, the
impact of drugs on the mechanical properties of cancer cells is not yet fully understood.
Moreover, the stiffness of tumor tissue is significantly influenced by factors such as tumor
stage, invasiveness, and location within the tumor, which are related to the deposition
of extracellular matrix and affect cellular behavior at the single-cell level. Consequently,
studying the impact of hypoxia on cellular structure and its relationship with drug effects
provides essential insights into the tumor microenvironment’s dynamics and its influence
Processes 2023, 11, 2430 30 of 39

on treatment outcomes. In this section, we explore the multifaceted applications of AFM

in drug therapy. This encompasses the study of drug–target interactions, drug delivery,
monitoring treatment efficiency, and investigating binding mechanisms. AFM offers a
powerful tool to unravel the intricacies of drug interactions and optimize therapeutic
strategies in the field of drug discovery and development.

7.1. Investigating Drug Delivery

AFM plays a crucial role in investigating drug delivery to cells or tissues. By perform-
ing high-resolution imaging, AFM enables the tracking of drug molecules’ location and
distribution within the sample, shedding light on their uptake by cells or tissues. This
knowledge aids in the development of improved drug-delivery strategies and enhances
targeted delivery to specific cells or tissues, potentially improving therapeutic outcomes.
For example, Zhang et al. [130] conducted a study on the binding mechanism of diosmetin
to human serum albumin (HSA). The topographic image of HSA molecules absorbed
onto mica was observed. By averaging the width of 50 single HSA molecules, the mean
width and height of individual HSA molecules was determined to be 25 ± 4.5 nm and
4.1 ± 2.6 nm, respectively. Upon interaction with diosmetin, the HSA molecule appeared to
be more swollen on the mica substrate, and flocculation or aggregation of HSA molecules
could be observed. The mean width and height of the molecules increased to 102 ± 9.1 nm
and 12.5 ± 3.0 nm, respectively—significantly larger than the original HSA dimensions.
This change in size suggested that there was an interaction between HSA and diosmetin.
The interaction with diosmetin altered the microenvironment around the HSA molecule,
exposing it to a more hydrophobic environment. To minimize the unfavorable factors and
stabilize the structure, the HSA molecule reduced its surface area in contact with water by
undergoing molecular aggregation. This resulted in the observation of larger molecules
on the mica substrate. The AFM analysis thus confirmed the presence of hydrophobic
interactions between HSA and diosmetin. Similarly, Domingues et al. [131] investigated
the interaction between rBPI21—an antimicrobial peptide—and two types of bacteria:
Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). They found
that rBPI21 caused changes in the bacterial membranes. In E. coli, the peptide led to mem-
brane vesiculation and flattening at low concentrations, progressing to complete membrane
disruption and bacterial lysis at higher concentrations. In S. aureus, rBPI21 increased the
surface roughness, disrupted the bacterial structure, and caused lysis. They also studied
the influence of soluble LPS on the interaction between rBPI21 and the bacteria, observing
that LPS reduced the binding frequency of rBPI21 to both E. coli and S. aureus. In the
case of S. aureus, the binding forces were shifted to lower values in the presence of LPS,
indicating the involvement of another ligand—potentially lipoteichoic acid or wall teichoic
acid. In another study by Rajendran et al. [132], new insights into the binding of drug–
quadruplex intermediates was presented. They proposed molecular mechanisms involved
in drug-induced quadruplex folding. The ligand used in the study was bisquinolinium
pyridine dicarboxamide bearing a biotin linker (PDC-biotin). DNA origami frames were
employed to control the DNA sequences and investigate tetramolecular antiparallel and
(3 + 1)-type structures. They observed small-molecule-induced conformational switching
using AFM and confirmed the presence of the ligand in the intermediate structures through
streptavidin localization. The study also examined the formation of G-hairpin and G-triplex
intermediates induced by the ligand. These findings provide valuable insights into drug–
intermediate binding and have implications for the development of novel anticancer drugs
targeting G-rich regions. Thus, AFM serves as a valuable tool in investigating drug delivery,
molecular interactions, and conformational changes. It enables high-resolution imaging,
facilitating the tracking of drug molecules and providing insights into their uptake by cells
or tissues.
Processes 2023, 11, 2430 31 of 39

7.2. Monitoring Treatment Efficiency

AFM has the potential to monitor the efficiency of drug therapy by assessing the
mechanical properties of cells or tissues before and after treatment. The impact of drugs on
the mechanical properties of cancer cells, in particular, is not yet fully understood. Factors
such as tumor stage, invasiveness, and location within the tumor significantly influence
the stiffness of tumor tissue, which is related to the deposition of extracellular matrix and
affects cellular behavior at the single-cell level. Understanding the effect of hypoxia on
cellular structure and its relationship with drug efficacy provides crucial insights into
tumor microenvironment dynamics and treatment outcomes. In a study by Li et al. [133],
the effects of different drugs on the mechanical properties of lymphoma (Figure 16e,f)
were investigated, and it was revealed that rituximab significantly enhanced the efficacy
of chemotherapy drugs in inducing cell death. Similarly, Ren et al. [134] examined the
effects of eight anticancer drugs on the mechanical properties of human prostate cancer
cells (PC-3) using a control-based nanoindentation measurement protocol on an AFM. By
measuring the Young’s modulus of PC-3 cells treated with the drugs, it was observed that
the drugs substantially increased the Young’s modulus, with a more pronounced effect
at higher force loading rates. Two distinct patterns were observed: one group of drugs
(disulfiram, paclitaxel, and MK-2206) showed a relatively unchanged exponent coefficient
of the frequency–modulus function, while the other group (Celebrex, BAY, tomatine, TPA,
and valproic acid) significantly increased the exponential rate. The study suggested that
the changes in mechanical properties may be correlated with the ability of cancer cells to
metastasize, highlighting the potential of physical properties as targets for the development
of anticancer drugs. In another study by Alhalhooly et al. [135], the impact of the drugs
gemcitabine, doxorubicin, vincristine, and mitoxantrone on the mechanical properties of
four cancer cell lines (pancreas, breast, glioblastoma, and prostate) was investigated, and it
was found that brain, breast, and pancreatic cancer cells exhibited a decrease in stiffness,
while prostate cancer cells showed increased stiffness (Figure 16a,b). This was attributed
to drug-induced disruption of the cytoskeletal structure. Also, it was noted that the rate
of stiffness change was reduced by up to twofold in hypoxia, suggesting a relationship
between cellular stiffness and drug resistance in the hypoxic tumor microenvironment.
Overall, the assessment of cellular mechanical properties through AFM provides valuable
insights into the efficacy of drug therapy. Studies have demonstrated that certain drugs
can alter the mechanical properties of cancer cells, potentially influencing their behavior
and response to treatment. Understanding these mechanical changes can contribute to the
development of novel therapeutic strategies and, ultimately, improve treatment outcomes.

7.3. Investigating Binding Mechanisms

AFM plays a significant role in investigating the binding mechanisms between drugs
and their target molecules. By studying nanoscale interactions, AFM can provide in-
sights into specific binding mechanisms and alterations in the geometric characteristics of
molecules upon drug stimulation. These findings have implications for the development
of more precise and efficient drugs. For instance, Taranta, Bizzarri, and Cannistraro [136]
conducted research on the impact of a P53 drug on azurin, revealing unbinding forces of
approximately 70 pN (Figure 16c,d). In another study by Zhang et al. [137], the ability of
the drug LHRH-PE40 to recognize LHRH-Rs on the surface of living cells was compared to
that of LHRH and LHRH-R using dynamic force spectroscopy, revealing that the LHRH
moiety retained its specific recognition capability for LHRH-R, suggesting that the recom-
binant protein LHRH-PE40 holds promise as a targeted drug. This indicates that the LHRH
analogue exhibits a high affinity for its target receptor, making it a potential candidate for
effective carcinoma treatment. The study also provided insights into the specific binding
mechanisms between the analogue and the receptor, which could guide the development of
more precise and efficient drugs in the future. Furthermore, Xiao et al. [138] studied the in-
teraction of metformin with transforming growth factor-β1 (TGF-β1), a molecule involved
in the development of various diseases. Through AFM, they demonstrated that metformin
Processes 2023, 11, 2430 32 of 39

inhibits the binding of TGF-β1 to its receptor, TβRII, by conducting TGF-β1-TβRII binding
force measurements on live cells. The force distribution histogram obtained from the
experiments exhibited a single peak, indicating the measurement of single-molecule forces.
In cells treated with metformin at a concentration of 50 µM, the binding forces between
TGF-β1 and TβRII on the cell surface were similar to those in the control group However,
metformin significantly reduced the binding probabilities, from 21.7 ± 3.5% to 9.9 ± 1.2%.
This reduction in binding probability was comparable to the results obtained with the
TGF-β1 antibody treatment (6.4 ± 1.9%). Additionally, Li et al. [139] explored the nanoscale
interactions between plasmid DNAs and drugs (methotrexate and cisplatin) and demon-
strated significant alterations in the geometric characteristics of the plasmid DNAs upon
drug stimulation. Moreover, Yun et al. [140] investigated the biomechanical properties of
HeLa cells before and after treatment with docetaxel. The AFM images revealed differences
in cell morphology between control and docetaxel-treated cells. The untreated cells exhib-
ited lamellipodia, while the treated cells showed a significant reduction in lamellipodia and
an increased apparent height. Measurements of cell height indicated that docetaxel-treated
cells had a significantly higher average height (3.73 ± 0.53 µm) compared to untreated cells
(2.43 ± 0.59 µm). This suggests that docetaxel treatment led to changes in cell structure.
The surface brush length, viscosity factor, and adhesion work were significantly reduced
in docetaxel-treated cells compared to untreated cells. The brush length decreased by
approximately 250 nm. The reduced brush length and adhesion work suggest alterations
in cell surface properties. Lastly, Song et al. [141] studied the interaction between the type
2 diabetes drug pioglitazone and the outer mitochondrial membrane protein mitoNEET
(mNT) using AFM. They examined the unfolding pathways and kinetics of mNT in the
presence and absence of pioglitazone. Without pioglitazone, mNT unfolded in a one-step
process, with a contour length change of 12.7 ± 1.2 nm. Interestingly, multiple peaks of
18 nm were observed from a different region of the protein. On the other hand, when piogli-
tazone was present in excess, a smaller force peak of 13 nm was observed at 106 ± 59 pN.
The unfolding force of mNT was found to be dependent on the loading rate, and a linear
relationship was observed. The off rate (koff) for mNT was measured to be 1.4 s−1 , while for
the mNT–pioglitazone complex it was 4.0 s−1 . Stepwise unfolding events of mNT were also
observed, including a two-step unfolding event with peaks of 8 nm and 5 nm. Pioglitazone
binding had significant effects on the kinetic stability of the protein. It decreased the off
rate of the 5 nm peak from the metal cluster by 10-fold and the off rate of the 8 nm peak
from the protein structure outside the cluster by 3-fold. Furthermore, the rupture of the
labile Fe-N bond in the metal cluster resulted in stepwise force peaks, and pioglitazone
had a lesser effect on the kinetic stability of the partially ruptured cluster. Pioglitazone was
found to increase the kinetic stability of the metal cluster by stabilizing the Fe(III)-N(His87)
bond, inhibiting its release/transfer. These findings highlight the influence of pioglitazone
on the unfolding pathways, kinetics, and kinetic stability of mNT, particularly targeting the
metal cluster. In conclusion, AFM-based investigations play a pivotal role in unraveling
the binding mechanisms between drugs and their target molecules. These studies provide
crucial insights into nanoscale interactions and the consequent alterations in molecular
characteristics induced by drug stimulation. Such knowledge holds immense promise for
the development of more precise and efficient drugs in the future.
 ronment. Overall, the assessment of cellular mechanical properties through AFM provides
valuable insights into the efficacy of drug therapy. Studies have demonstrated that certain
drugs can alter the mechanical properties of cancer cells, potentially influencing their be-
havior and response to treatment. Understanding these mechanical changes can contrib-
Processes 2023, 11, 2430 ute to the development of novel therapeutic strategies and, ultimately, improve treatment
33 of 39
outcomes.

Figure16.
Figure (a)Young’s
16.(a) Young’smodulus
modulus(E)
(E) compared
compared for
for four
four different
different cancer
cancer cell
cell lines (****pp << 0.0001).
lines (**** 0.0001). (b)
(b) Time
Time tracetrace of Young’s
of Young’s modulus.
modulus. (a,b)
(a,b) Reprintedwith
Reprinted withpermission
permissionfrom
from [135].
[135]. (c) Force
Force diagram
diagram of
of unbinding events. (d) Unbinding force distribution comparison of before and after blocking the
p53 monolayer. (c,d) Reprinted with permission from [136]. (e,f) Histogram of Young’s moduli of
cisplatin (e) and cisplatin and Rtx (f). Reprinted with permission from [133].

8. Conclusions and Perspective

The field of biology has undergone a remarkable transformation with the advent of
AFM, which allows for the characterization and manipulation of various interfaces at the
molecular level. It not only enables high-resolution imaging of biological surfaces, but also
facilitates the measurement of biomolecular interactions. The use of force–distance curves
generated by AFM holds tremendous potential for unraveling both inter- and intramolecu-
lar interactions under physiological conditions and investigating mechanical properties.
By modifying the AFM tip, one can explore a wide range of interactions, from molecules
to cells. The force spectroscopic modes of AFM provide a valuable tool for characterizing
real-time molecular interaction dynamics. However, despite these advancements, there are
still several technical challenges when it comes to investigating living cells at the single-
molecule level. One of the key challenges is distinguishing between specific and nonspecific
Processes 2023, 11, 2430 34 of 39

binding within the complex cellular environment. This poses difficulties when attempting
to measure specific biomolecular interaction forces accurately. In summary, AFM-based
measurements will continue to play a crucial role in future biological research, significantly
enhancing our understanding of the structures and properties of various cellular molecular
mechanisms at the single-molecule level. The integration of AFM with other techniques
holds great promise for providing further insights into transport phenomena in biologi-
cal systems and may lead to the development of novel therapeutic strategies for various
diseases. Future research in this field is expected to focus on the development of new AFM-
based techniques for studying molecular interactions in biological systems and integrating
AFM with other analytical techniques to gain a more comprehensive understanding of
these interactions.

Funding: This research was partially funded by the US National Science Foundation (ECCS 2010875).
Data Availability Statement: Data sharing is not applicable here, as we did not generate any data.
Conflicts of Interest: The authors affirm that they do not possess any conflict of interest.

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