Envision and Appraisal of Biomolecules and Their Interactions through Scanning Probe Microscopy

Scanning probe microscopy (SPM) has gifted a novel eye to envision nanotechnology and nanoscience. The SPM technique involves a sharp probe that moves over the sample surface and leads to produce signals, which facilitates a deep understanding of the structural, electronic, vibrational, optical, magnetic, (bio) chemical, and mechanical properties of the material. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) measure various kinds of physical properties such as electric current, force, and capacitance. Moreover, AFM shows a prominent feature over STM that it accepts insulating surfaces and can work under physiological conditions, making it feasible to study biological molecules. Herein, the SPM approach toward biomolecule imaging and appraising different physiological processes with molecular resolution is highlighted. The review raises awareness regarding current obstacles in biological sample preparation and possible ways to conquer these difficulties. Subsequently, the recent applications of STM and AFM on various biomolecules with representative examples like DNA, protein, and carbohydrates are discussed. The conductance measurement studies of the biological samples emphasize applications like drug delivery, biosensors, molecular electronics, and spintronics fields. This is followed by an outlook of future perspectives making a pavement toward the basic science and applied field of biomolecules using SPM technology.

of the tunneling effect is the emergence of the high resolution in the technique. The probe tip is a key element of STM, which is attached to a piezodrive comprised of three mutually perpendicular piezoelectric transducers (x, y, and z piezo). This can probe the local electronic properties of topographical surfaces and their functionalities and simultaneously can obtain the actual visualization of the atomic structure. [13] Moreover, the tip-sample interactions can raise the way to perform precise atomic manipulations along with surface investigation. [14] This can further elucidate various chemical reactions [15] or molecular interactions at the molecular and atomic scales.
STM is much-privileged apparatus as a broad range of nanoscience phenomena can be studied using it. Apart from surface topography, various other elemental properties like excitations, [16][17][18] magnetic, [19][20][21] and optical [22] can be appraised very proficiently. Subsequently, it allows the chemical identification of atoms; hence, today, the analysis of magnetic anisotropy energy is also an engrossing topic that is being studied with this ubiquitous machine. STM also offers electrochemical studies at the electrode/liquid interface, along with redox processes occurring at the interface, [23] metallic and semiconducting surfaces, [24][25][26] oligomers, polymer-like heptadiyne, [27,28] organic molecules or wires, [29] redox molecules, [30,31] and redox proteins. [32,33] Being a versatile machine, the use of STM in studying various biomolecules is increasing continuously. [34] Various groups have made successful efforts to study several biomolecules concerning their different properties using STM. Precise studies of bacteriophage, [35,36] metal-covered [37] and native DNA, [38] larger biomolecules like amino acids, [39][40][41] peptides, [42][43][44] proteins, [45] protein-lipid bilayer membrane, [46][47][48] and dried cells on the conductive plate [49] have been accomplished. This technique is also effective for the investigation of biomolecules conductance because the potentiostatic control of electrodes and solution provides similar conditions to the physiological environment. Different methods are being used to measure the molecules' conductance including the break-junction technique, [50] the spontaneous junction formation, [51] and scanning tunneling spectroscopy (STS). [52] In current-potential (I-V), the local conductance can be measured by modulating the probe-sample bias potential with a sinusoidal potential of sufficiently high frequency and low amplitude. This methodology can be used for several systems, that is, electrolytic solution, [53] conductors and semiconductors, [54,55] organic molecules, [56] and biomolecules [57] in air/vacuum. AFM is the second oldest tool of the SPM family, established in 1986. [58] A small cantilever is the central element of this imaging microscope. A flexible cantilever is used to place the sharp tip and used as a spring that evaluates the force between the tip and the sample surface. [59,60] Whenever the tip and the sample surface come nearby, attractive and repulsive forces get generated that deflect the cantilever. [61] This deflection leads to the bending and torsion of the cantilever. Its detection is based upon the changes in the position of reflection of the light beam. [62,63] There are different modes of operation to be followed, that is, contact mode (constant contact between the tip and the sample surface), noncontact mode (vibrating cantilever generates the image using variations of its resonance frequency), and intermittent contact (rapid movement of the cantilever with a large oscillation, between repulsive and attractive forces). [61] AFM shows a prominent feature over the STM that it can accept insulating surfaces in various conditions (e.g., buffer solution) and makes it attractive for studying biological molecules in approximate physiological conditions on a nanometer scale. [64,65] AFM is conventionally used to appraise the structural characteristics of various biological samples right from proteins [66] and nucleic acids [67,68] to living cells also. [69,70] Its acceptability in aqueous environments, as biomolecules show important activities only in aqueous conditions, facilitates it as a prime tool for real-time microbe monitoring. These physiological circumstances provided to the biological samples lower the additional sample preparation task. Remarkably, in the very early days, Hansma and his research group observed the specific behavior of biological samples in action. [71] For example, a group monitored intervals in the blood clotting process, initiated by the thrombin of fibrin molecules. [72] After this benchmark achievement, many biological processes have been explored broadly using a novel surface imaging tool, AFM. [73] Moreover, this technique can also help to analyze the interactive force between the tip and the sample molecule as a function of the tip-surface www.advancedsciencenews.com www.small-structures.com distance. This is extremely useful for the evaluation of the type and bond strength of intra-and intermolecular bonds at the single-molecule level. [74][75][76] In addition, many other topographical properties of the living cells like electromechanical [77] and cell adhesion properties [78] can be investigated using the AFM technique. The use of coated or customized tips broadens the usage of the technique for different measurements such as magnetic, elastic, binding forces, and surface potentials. [79][80][81][82][83][84] Moreover, different modes of AFM contribute vital insights into the complexity of biomolecular interactions. [85] Also, this technique facilitates the detection of biomolecules or cells, which makes it a fast biodiagnostic equipment for diseases like cancer. [86] For example, AFM-based force-clamp spectroscopy measures the dissociation and association processes of the bonds in a ligand-receptor pair by maintaining force. [87] The force-clamp AFM is also being broadly used to study the mechanical un-and refolding of proteins, [88,89] formation and breaking of disulfide bonds, [90] the mechanical strength of the typical bonds, [91] lipid bilayer disruption, [92] and the development of membrane chain. [93] Reynold et al. focused on the mechanism of amyloid crystalfibril conversion during protein folding. The morphology and the molecular organization have been studied using the AFM technique and compared with theoretical observations. [94] Adamick et al. discussed protein folding and aggregation and also studied amyloid fibrils, which play a crucial role as the ground state in the folding and aggregation landscapes of proteins. This study has demonstrated the direct experimental observation of morphology and different polymorphic transitions of amyloid fibrils at the mesoscopic level. [95,96] Moreover, the onset molecular mechanism of neurodegenerative diseases has been painted by studying AFM morphological studies of fibrils and their fibrillation time. The article has directly observed the self-aggregation of amyloid β fibril. These self-aggregates fuses further to form the final structure of fibrils. The size distribution of these aggregates decides the morphology of fibrils. Consequently, studying fibril formation clears the picture of the mechanism of neurodegenerative diseases. [97] Kelvin probe force microscopy (KPFM) has been used to study different biomolecules, typically, the surface charge distribution of different biomolecules like DNA, [98] actin filament bundle, [99] microtubules, [100] etc. Lee et al. conducted single-molecule-based imaging through KPFM, which typically enables measuring the surface charge distribution of a single amyloid fibril, which is defined by structural confirmations of fibrils. This group has precisely observed that electrostatic properties of fibrils are pH dependent and provided detailed insights into the amyloid formation mechanism including its interaction between different proteins, which can be a prime cause for the amyloid-driven dysfunctioning of cellular mechanism. [101] Similarly, using KPFM for single-molecule studies, one can screen the efficacy of the drug. This kind of AFM study can be more thoughtful over drug design and nanomedicines. Park et al. studied the interaction between small molecules and tyrosine kinase using KPFM; this study can explain cellular signaling and its malfunctioning. [102] Choi et al. studied the nanomechanical properties of amyloids through single-molecule experimental and computational simulation techniques; this study helps to get more details about the biological functions of fibrils and typically and focuses on the mechanism of the protein interaction and their properties. [103] Lee et al.'s studied the conformational heterogeneity of amyloid fibril through AFM and KPFM studies, focusing precisely on microwave-assisted chemistry in the formation of amyloid fibrils and demonstrating that this chemistry is useful for controlling conformation and the population of the small aggregates in the structural formation of fibrils. By measuring the surface charge potential, it has been concluded that electrostatic interaction plays an important role in the radial growth of the fibrils. Hence, the study concluded clearly about heterogeneity in the confirmations of fibrils, which are typically induced by alteration in the thermodynamics of protein aggregation due to microwaveassisted chemistry. Understanding the heterogeneity further helps to gain insights into amyloid-driven pathogenesis and also can be useful for designing biomimetic materials. [104] Another research group has used multifrequency atomic force microscopy (MF-AFM) to study the fundamental mechanical properties of the membrane protein aquaporin-0 (AQP0). This study has typically studied hydrophilic and hydrophobic properties of the molecules which clearly defines water affinity of the molecules. [105] Typically, hydrophobic forces play important role in biological systems; however, quantitative measurement of such forces on complex biosurfaces is quite challenging. Chemical force microscopy (CFM) equipped with hydrophobic tips was implemented to quantify the local hydrophobic forces on organic surfaces and live bacteria. It has been observed that CFM plays a better role in measuring wettability, demonstrating hydrophobicity, which subsequently provides a detailed insight into the nature of hydrophobic forces. The hydrophobic forces present on the surface of mycobacteria were measured, which symbolize an important permeation barrier for the drugs. [106] In another study, a novel AFM mode (i.e., peak force tapping with chemically functionalized tips) was used to measure and image the hydrophobic forces on organic surfaces and microbial pathogens. The adhesion images obtained through this method specifically provide quantitative information regarding the distribution and strength of hydrophobic forces of single proteins. [107] Force-distance (FD) curve-based AFM can be used to study the biophysical properties of biomolecules. The research group used advanced FD-based technology in combination with biochemically modified tips to image filamentous bacteriophages extruding from living bacteria. The quantitative imaging of nanobiomaterials method helps in mapping the physical properties and their molecular interactions from viruses to tissues. [108] Similarly, FD-based AFM has also been applied to study different mechanical properties of cells, [108] viruses, [109] protein membranes, [110] lipid membranes, [111] proteins, [112] and fibrils. [113] Recently, the biophysical properties of the SARS-CoV-2, typically, S-glycoprotein binding to ACE2 receptors, were studied using the FD curve-based AFM. The study demonstrated a specific binding mechanism between the Sl subunit and the ACE2 receptor. The study has preferentially highlighted that SARS-CoV-2 binding to ACE2 is dominated by the RBD/ACE2 interface. [114] Biomolecules are the basic building blocks of living entities. An appropriate availability of biomolecules is much important for the structural build up and fundamental functioning of living cells. Any inappropriate changes in the concentration of biomolecules may lead to the dysfunction of the cells and organisms.
Biomolecules include carbohydrates, proteins, lipids, etc., which are employed in forming structural integrity and are typically responsible for specific functions like reproducibility, sustainability, and cell cycle. Especially, the carbohydrates such as monosaccharides, oligosaccharides, polysaccharides, and macromolecules such as amino acids, peptides, proteins, nucleobases, nucleotides, oligonucleotides, nucleic acids (DNA/RNA), and lipids are the basic units of the living cells. [115,116] It takes billions of years for biomolecules to modify into a distinct cellular structure to get specific molecular recognition. These molecular recognition properties play a vital role in maintaining the structural and functional actions of the living cell cycle. Biomolecules and their interactions may produce typical biological systems or materials. [117,118] Studying explicitly the biomolecules, their properties, and interactions has become a source of inspiration to material chemists and biologists to develop new biomaterial frameworks as a mimic of biomolecules for various applications like drug delivery, green electronics, etc. [119][120][121] SPM could be one of the assured sources for the thorough appraisal of biomolecules and their interactions.
Here, in this review, we have focused on the SPM technique and its significant use for biomolecules imaging, including studying different physiological processes and interactions of the biomolecules at the molecular resolution. The review has covered the current hurdles in biological sample preparation and possible ways to conquer these difficulties. Subsequently, specific attention has been given to the current approaches of STM and AFM on various biomolecules with representative examples like DNA, protein, and carbohydrates. The specific electrical conductance measurement studies of the biological samples can be applied to drug delivery, biosensors, molecular electronics, and spintronics fields ( Figure 2). This is followed by an outlook of future perspectives, making a pavement for researchers toward the basic science and applied field of biomolecules using SPM technology.

Sample Preparation
To date, sample preparation is one of the major limitations to studying biomolecules for SPM applications. In the case of SPM, most of the time, single or very few sightings have been observed in many attempts. This obstacle is mainly due to the fact that biomolecules are thick and insulators in nature; hence, the current could not pass subsequently, and the system fails to get a proper image. [122] On the other hand, the prime aspect of the substrate is related to the flatness and stiffness that should allow the mobility of biological molecules in an aqueous medium to preserve biological samples. [123] For imaging, a smooth and flat surface is required, as the rough surface can suppress the presence of sample particles. In the case of the sample, small molecules show more stability in the liquid environment due to their viscosity at the liquid-solid interface, enabling high-resolution imaging. However, a large biological molecule (e.g., 30 nm) at the interface can be very soft and deformable. During scanning, the SPM tip must scan over the 30 nm range, and the fast-scanning rate can damage soft biomolecules. However, the slow scan led to the formation of a blurred image due to the Brownian motion of the sample. This can be observed more evidently in the liquid environment. [124] To date, no universal solution is found for this issue, but making specific changes in developing each type of the sample is the most necessary thing to proceed with good-resolution images and minimum sample damage. For biological samples, binding between the sample and the substrate should be firm enough to avoid dragging the sample due to tip movement. Usually, this can be done with the help of an atomically flat substrate, which generally shows better binding with the sample to be examined. To get the better binding or adsorption of sample with the substrate, modifications in pH or solution composition/concentration play an important role as it affects the typical binding forces like electrostatic or Van der Waal's forces. [125] In STM, a gold (Au) (111) substrate can be the right choice as a substrate, that shows extremely flat facets. [126] These are smooth enough that even a single protein can be fixed on them and will be visible. [127] Moreover, this substrate also shows typical properties, such that it can be prepared easily, is highly conductive, and adsorptive. Previously, Lindsay and Barris used Au substrate for STM studies of DNA under water. [128] Moreover, Au substrate can be facilitated with immobilizing anchors to achieve immobilization of the biological objects. For instance, the thiol, organic group -SH, can form a strong S─Au bond on gold surfaces. Also, other groups such as -OH, -CH 3 , -CHO can be the immobilizing anchors to the molecules of interest. Molecules containing carboxyl (-COOH) and amine (-NH 2 ) functional groups are also of interest for biomolecule immobilization. [129,130] Clean copper (Cu) (111) is another surface that has been used for DNA imaging, where DNA was deposited onto the substrate using the oblique pulse injection method. [131] Chaplygin and group used pyrophyte as a substrate for STM studies, as it allows to obtain a lateral resolution and an atomically resolved image. [132] Guchenberger et al reported that numerous biomaterials can be imaged in humid air as the monolayer of water on the substrate can be enough for investigating insulator samples in STM imaging. [34] The various biological samples like Cytochrome c, [133] HIV-1 regulatory protein, [134] flagellin, [135]   wheat gluten protein, [135] miscellaneous proteins, [136,137] and DNA [37,138] have shown clear imaging in the presence of air or under ultrahigh-vacuum (UHV) conditions too. An appropriate visualization of molecules in AFM needs the dynamic interactions between the tip and the sample surface via weak bonds. Hence, the sample preparation method must be addressed to fulfill such a challenging requirement. During the scanning, avoiding displacement of the sample due to AFM probe movement and adsorption of biological species onto their substrate is not an easy task. Different ways can be followed to attach the sample to the substrate like substrate modification to enhance the sample adhesion probability. In addition, the porous substrates can be used to achieve the biological species immobilization for the timescale measurement. The immobilization also can be achieved through covalent linkages or weaker noncovalent interactions, suspending a sample in such matrices and forcing the sample to move in a particular orientation with the suppressed motion in a repeated manner, leading to the study of the exact dynamic biological activities. Tight binding between the substrate and the sample is the prime need during scanning. In contrast, while working on a real-time biological activity, sometimes too-tight adherence may disturb its structure and activity. Choosing a reliable substrate for imaging for topography and surface chemistry analysis is also a prime piece of experimentation, for example, Mica and highly ordered pyrolytic graphite (HOPG). Mica is a natural muscovite or synthetic fluorophlogopite, that can be conventionally used as its surface is atomically flat with a large area, making it a promising substrate for SPM imaging. Due to the presence of a net negative charge on its surface, it can adsorb biomolecules and various proteins readily on its surface. [129,139,140] Adsorption strength can be controlled by changing the pH/addition of divalent cations such as Mg 2þ and Ni 2þ . [141,142] A mica surface can be modified with the deposition of liposomes, which will be useful for the immobilization of specific proteins like biotinylated proteins and His-tagconjugated proteins and lipids with biotin. [143] In addition, protein molecules show a specific affinity toward DNA origami structures and can be bound to them very easily. [144] This has been observed by anchoring the typical functional group to the DNA structure. [145,146] Yuki Yamamoto's group immobilized streptavidin (SA) protein by fixing it on DNA origami. The designed DNA origami facilitates two different binding sites for two different SA molecules, that is, in-SA and on-SA molecules. The SA protein is shown in Figure 3a, which has the capability of specific binding with vitamin B7. In the 'in-SA condition', a biotin-functionalized staple single-stranded DNA (ssDNA) plays a vital role in binding between DNA origami and a single SA molecule. While in the 'on-SA' condition, the direct binding between SA molecule and DNA has been observed. Figure 3b shows a topographic image obtained from frequency modulation (FM-AFM), where the bright spots in the image depict SA molecules in both the conditions and have been differentiated with their height difference of 0.9 nm. The dark region on the right of the in-SA molecule indicated by the arrow is the exposed mica area in the window, which is slightly lower than that of the DNA origami surface. The study reported here that the in-SA condition exhibited a stronger binding than that of the on-SA condition. Other substrates, that is, glass can be easily used for imaging, as its transparency is useful in optical or fluorescence images. For instance, glass coverslips have been used as a substrate for imaging cells. [147] 3. Biomedical Applications 3.1. STM Studies of Biomolecules

Deoxyribonucleic Acid (DNA)
It is a large molecule, essential for life, as it plays a prime role in inheritance and genetic expression. DNA shows a doublestranded (ds) helical structure, which is composed of two polynucleotides (ssDNA). The typical chemical composition of the whole structure consists of four nucleobases, namely, adenine (A), thymine (T), guanine (G), and cytosine (C). Phosphate sugar is the backbone of the whole structure, in which a specific chemical structure of nucleobases, 'A' can pair up with 'T' and 'G' can bind with 'C', are connected with hydrogen bonding. The doublehelical structure of DNA is primarily the reflection of this base sequencing. After the discovery of the double-helix structure of DNA by Watson and Crick in 1953, [148] the innovation of STM has attracted many scientists' curious attention to the direct visualization of the DNA structure, leading to many reports aiming to observe DNA structure. [149,150] In 1989, STM images of DNA structures were reported in which DNA was deposited onto pyrolytic graphite substrate by dropping DNA solution in air. [151,152] Later reports have recognized that these reported "DNA images"  [147] Copyrights 2020, IOP publishing Ltd.
are nothing but the grain boundaries of the used graphite substrate. [153] Unfortunately, thereafter, a lack of high-resolution microscopic images of the DNA structure in scientific journals has occurred due to inappropriate sample preparations to deposit DNA onto the proper substrate without it getting contaminated.
Humid air plays an important role in STM experiments that have been observed previously as the probability of an adsorbed monolayer of water onto the substrate is taken into consideration for observed conductivity. [154,155] Following that, Guckenberger and group demonstrated the STM imaging of plasmid DNA on mica in humid air. Figure 4a,b shows STM images of the plasmid (pUC18) DNA deposited on the mica substrate. The height difference on the right side of Figure 4a exhibited the height of one mica layer, that is, 1 nm. This occurred due to the conductivity difference between the substrate and the plasmid loop. Figure 4b shows lateral high resolution of the DNA structure obtained in early times. The inset has confirmed the width of DNA as 3.5 nm, closer to the actual diameter of %2.5 nm. [156] Moreover, the theoretical studies have observed structural modifications in the DNA structures as cross-and T-shaped structures. [156] To clarify these modifications, STM and AFM are used, as these techniques can produce an image of a single molecule with an appropriate resolution. In early times, these techniques exhibited linear and circular structures of DNAs, under ambient, vacuum, and liquid medium. [157][158][159] However, intact DNA structures have been reported in most of the cases, where the branched structure of DNA has been observed by AFM under vacuum. [160] Recently, Terasaki et al. observed DNA images under UHV, deposited onto Au (111) using the electrospray method. [161] Figure 4c shows an STM image of linear DNA, a bright string of DNA observed with 190 nm length and 6 nm width. Although the width of the DNA is known to be %2 nm, [162] however, the 6 nm width observed here in Figure 4c is wider than the reported one. Such a wider width has been observed previously by SPM probably due to noncontact interaction between the SPM tip and the sample. [158,159] The inset of Figure 4c shows the high-resolution STM image of DNA, composed of periodic bright protrusions of a double-helix structure with a periodicity of 4.5 AE 0.2 nm. The previously reported periodicity is %3 nm [162] and obtained through SPM with 2.6-3.8 nm. [158] In the study mentioned here, about 4.5 nm has been observed, which may be due to structural relaxation occurring through molecule-surface interactions. In Figure 4d, branched DNA has been shown. The green and blue lines of widths are about 6 and 3 nm, indicating dsDNA and two ss DNA respectively. In Figure 4e, it looks like the dsDNA opened up into two ssDNA. Along with extended DNA structures, the folded structures also have been observed in Figure 4f. Narrow strings of the folded structure may be of  [156] Copyrights 1994, American Association for the Advancement of Science. d) Branched DNA on Au(lll) (300 nm Â 300 nm) exhibiting three branch points. e) The same image as (d) shows the marking of dsDNA (green) and two ssDNA (blue) f ) Folded (or looped) DNA on Au(lll). (50 nm Â 75 nm). (d-f ) Reproduced with permission. [157] Copyrights 2019, American Chemical Society.
www.advancedsciencenews.com www.small-structures.com ssDNA. Usually, the ss DNA tends to fold back and form loop-like structures. In the studies performed through the AFM technique, the folded ss DNAs have been observed. [163,164] In addition, some DNA coil structures have been analyzed in poor solvents. [165] Real-time imaging of molecules on the atomic scale is possible with STM. In this viewpoint, various research groups have attempted sequencing a single DNA molecule. [166,167] The detection of tunneling current plays a crucial role in sequencer technology. In the very first study, Tanaka et al. [57] conducted the partial sequencing of single-stranded monomer DNA by STM in a different way than previously studied reports. [168] STM and spectroscopic studies of ss DNA (M13mp18) and PNA (peptide nucleic acid) have confirmed the existence of guanine. Figure 3a-d) shows typical STM images of single-stranded monomers of fluorescein isothiocyanate (FITC)-modified PNA (FITC-TTGACC) and DNA (FITC-TTGGCC) along with the spectroscopic data of these samples. It is quite difficult to identify and assign a typical base sequence of oligomers, only by observing the STM images. However, the comparison between two types of oligomers helps to find the existence of guanine. Figure 5a of PNA (FITC-TTGACC) shows only one base pair shining in the middle of the base sequence. while in Figure 5c of DNA (FITC-TTGGCC two base molecules that are seen to be shining in the large-area image are shown. Furthermore, the tunneling current measurement was performed by lock-in detection. Guanine molecule can be detected easily as it has the lowest potential [169] among four base molecules. It shows a characteristic peak at the applied voltage of À1.6 V (Figure 5b,d). The typical electronic state of the Guanine base can easily distinguish the molecule from the other nucleic acid bases.
In a recent report, the visualization and recognition of adenine in ssDNA have been demonstrated using STM. [131] In this study, ssDNA (TET-AAAAA, TET-A5 for short), tethered to 5A-tetrachloro-fluorescein phosphoramidite (TET), was used as a fluorophore. Figure 5e shows that DNA is bound with TET. In the enlarged image, Figure 5f shows the linear structure of nucleotides, showing intervals of about 0.65 nm, elucidated in the previous reports. [57,170] Figure 5g shows the STM image of the TET-A5 oligomer and confirmed the presence of all five nucleotides. However, only three of them appeared to be in the linear conformation. Figure 5h shows a graph measuring the spectral data and density of states (DOS) peaks in the bias voltage from À1.2 to À1.6 V. Since no peak has been observed at point 5, the characteristic peak of guanine appears at À1.6 V. The DOS peak of adenine shows a similar peak to that of guanine, with a bit weaker intensity. These results logically state that the full sequencing of dsDNA can be obtained by studying all purine bases in every single strand. In this way, base discrimination can be possible. Adenine and guanine can be differentiated from each other.
Moreover, the structural characteristics of the DNA molecule make it ideal for electronics applications such as molecular electronics and spintronics. [171,172] Its specific properties like conductance and charge transport have been employed in the applications like lab on chip. [173,174] Typically, DNA exhibits the nucleobase sequence-dependent conductance property.  [57] Copyrights 2009, Springer Nature. e) Wide-and narrow-range STM images of TET-A5 deposited onto a Cu (111) surface. f ) Linear structure of nucleotides with the white arrows indicating individual nucleotides. g) Magnified STM image of TET-A5. h) dI = dV spectra measured at points 1-5, shown in (a). (e-h) Reproduced with permission. [131] Copyrights 2017, IOP publishing ltd. Extensive research is being carried out to understand the conduction mechanism along with double-helical DNA molecules. Specifically, it occurs through π-orbitals overlapping in the sequenced nucleobases. [175][176][177] Short-range charge transportation occurs through a nontunneling resonant regime, [178] while long-range charge transportation is attributed to the π-electron hopping between the nucleobases. [175,179] Typically, among the four nucleobases, guanine takes part in electron hopping, as it has the highest occupied molecular orbital (HOMO) level closer to the electrode Fermi level. [180] Xiang et al. [181] reported conductance measurement of a DNA molecule substituting a base in double-helical DNA with a redox group using an STM break junction technique. [182,183] The study demonstrated that one of the DNA bases has been replaced with anthraquinone (Aq), and a redox group shows reversible oxidation and reduction ( Figure 6b). It allows a continuous π-π stacking pathway and results in efficient charge transportation. The conductance has been compared between redox-modified DNA, referred to as Aq-DNA, and unmodified DNA as u-DNA. In this specific technique, Au tip coated with wax has been used to reduce ionic conduction on the Au substrate ( Figure 6a). The monitored current between the tip and substrates shows the plateau in the current traces and pulling forces, exhibiting the formation of a single junction at Au-DNA-Au molecular junctions. The studies revealed the conductance for Aq-DNA and u-DNA as 4.0 AE 0.2 Â 10 À4 G0 and 14 AE 1 Â 10 À4 G0, respectively, where G0 = 7.748 Â 10 À5 S, the conductance quantum ( Figure 6c). This conductance difference is attributed to the intercalation of the redox group into the base pairs in DNA, as shown in the literature [184] (Figure 6b). This can introduce an active control on DNA conductance and can switch it reversibly between oxidizing or reducing groups. This optimistic strategy can be implemented in developing active device building blocks.

Proteins
Proteins play a vital role not only in the formation of the cytoskeleton but also in various important cell life activities. [185] At the molecular level, proteins behave similarly to machines, taking part in cellular life cycle processes including DNA replication, catalytic metabolism, cell proliferation, etc. [186,187] Proteins can be visualized at atomic resolution through SPM. [188] STM shows favorable horizontal and vertical resolutions of 0.1 and 0.01 nm, respectively, which are the most desirable ranges for biological sample imaging. After DNA imaging, different proteins also were studied using STM. Albreacht et al. observed STM images of heme protein MOP-C in solution. [188] In another study, metalloproteins were also imaged in solution by Friis et al. [189] . Flagellin protein was studied in STM by adsorbing it on a flat Au (111) substrate. [135] The morphological studies of viral protein R (V pr ) fragment, V pr 13-33, were performed on graphite surface using STM and b-sheet-like structures of V pr 13-33(HIV) were observed. [134] Amyloid protein (A) plays a vital role in Alzheimer's disease, for the detailed STM study. Amyloid protein (A) was adsorbed onto a flat Au substrate, where the small globular structures were observed. [136] Arakawa et al. studied STM imaging of turtle alpha microglobulin protein using STM. [190] The blue copper protein azurin [191,192] was immobilized on the Au and imaged at the resting state subsequently, by applying a potential across the tip and the sample. STM imaging of K27C azurin was done following the potential applied across the tip and the sample, which induced dissociation of the dimers pair. This study will be useful in observing protein distribution on the biocompatible surface and can be applied to devices like a single-molecular array, biosensors, etc. [137] Wang et al. studied submolecular images of large biomolecules using liquid-phase STM (L-STM). [193] They further reported imaging of a single streptavidin protein in solution under physiologically favorable conditions. Figure 7a shows an STM image of streptavidin that resembles the crystal structure. The difference between the structure and image observed is probably due to the images being acquired in solution; hence, these molecules can orient in different shapes. The marked region in the enlarged image of the protein in Figure 7b,c presented various features consistent with the crystal structure model in Figure 7c. In this study, the detailed surface analysis of the streptavidin in solution has been done in submolecular resolution.
Following the globular structures of proteins, the unfolded and folded morphologies also have been imaged. Cytochrome Figure 6. a) Illustration of the experiment. STM tip and substrate are the source and drain electrodes, respectively, and EC gate is a silver electrode inserted in the solution. DNA molecule bridged between two electrodes via thiol linker groups, and charge transport indicated in red arrows via overlapping n-orbitals. b) From left to right: redox-modified DNA (Aq-DNA), where a base was replaced with an anthraquinone (Aq) moiety (highlighted in blue), intercalated in between the two guanine bases, acts as a hopping site (red arrows). Aq moiety is shown in blue. DNA without the Aq moiety (u-DNA), as control. c) Conductance histograms of Aq-DNA (in blue) and u-DNA (in red) show the difference in the conductance peaks with Gaussian distribution fitting. (a-c) Reproduced with permission. [181] Copyrights 2017, Springer Nature. C (CytC), a protein central, plays an important role in various mechanisms including electron transfer (ET) in mitochondria and apoptosis. Deng and his co-workers demonstrated imaging of folded and unfolded CytC protein ions layered on a flat substrate in UHV (10 À10 mbar) using STM. [133] For implementing sample molecules on crystalline surfaces in UHV, electrospray ion-beam deposition (ES-IBD) method was followed. [194,195] Experimentally, CytC, folded proteins, were adsorbed onto the metal surfaces in vacuum, despite their large structure and insulating nature; these molecules have been imaged by STM successfully and show globular morphology. These molecules were studied by depositing on three surfaces: Cu (001), the highly interacting surface, where molecules can be easily immobilized; Au (111), a comparatively less-interactive surface but shows pinning centers, helpful for immobilization of the molecule; [196] and the third substrate was the monatomic boron-nitride nanomesh (BN) layer on Rh (111), weakly interacting with the sample but facilitated with the modulated surface; this offers a template for the patterned organization of molecules. [197] The deposition of the molecule on three different surfaces has identified two types of structures.  Figure 7g). The study vouches for an ES-IBD technique to form high-quality surfaces coated with selective molecules. Samples of fragile and complex molecules like proteins can be easily prepared using this method.
ET occurs in proteins while taking part in different physiological processes. Several techniques are being used to study ET in the proteins including electrochemical STM (ECSTM). [198,199] STM break junction (STM-BJ) approach [182] through the redox gate effect produces protein tunneling conductance. [200,201] The report explored here has demonstrated the transistor-like behavior of proteins using STM-BJs for the first time. [202] An Azurin is a redox protein model, [203] consisting of a central copper ion coordinated by protein residues. This structure facilitates a switch for its redox state that enables protein for accepting and transporting electrons in the respiratory process of bacteria. Experimentally, azurin was deposited onto Au <111> substrate. [201] The movement of the ECSTM probe was used to produce a tunneling current (Figure 8a). The obtained I(z) plots show the current steps (Figure 8b), which (a-c) Reproduced with permission. [193] Copyrights 2016, Springer Nature. d) STM topography of folded proteins on Cu(001); inset: high-resolution individual globular resolution. Boron nitride nanomesh e) on Au (111) and f ) on Rh (111). g) Line profiles showing completely and partially folded proteins as high (1.0-1.8 nm), globular features and unfolded proteins as features of low height (0.2-0.3 nm). (d-g) Reproduced with permission. [133] Copyrights 2012, American Chemical Society. are absent for plots measured on a clean gold (inset of Figure 8b). Thus the molecular junction formation and rupture with azurin bridging the two electrodes (the Au substrate and ECSTM probe) have been observed. Such "wired" molecular junctions are important for studying tunneling current in the ECSTM. [204] In the conductance histogram, (Figure 8c), azurin (red plot) shows a Gaussian distribution peak fitting yielding an average conductance of (7.3 AE 3.5 Â 10 À6 G0 (where G0 = 2e 2 h À1 % 77.4 μS). The black histogram represents conductance for a clean Au sample that decays monotonically, used as a control. This result interpretation exhibited a general agreement with conductance measurements using c-AFM in the air. [205] The obtained conductance and on/off redox gating ratios are well compared with the previous reports. [206] This STM-BJ approach demonstrates that biomolecular transistors can be developed with single metalloprotein junctions. Therefore, it can act as a better asset in biomolecular electronics and [207] biosensing [208,209] applications; where the electronic performance of novel devices based on single proteins can be easily appraised.

Carbohydrates
Carbohydrates (saccharides or glycans) are the key components of cell structures, [210,211] recognition, [212] and chemical energy of living cells. [213] In addition, abundantly available molecules, that is, carbohydrates, are important constituents of plants' cell structure. [214] Monosaccharides are the basic building blocks of saccharides that develop different structural configurations with linkages like αor β-forming complex and irregularly branched patterns with highly flexible backbones and different probabilities of stereochemistry. This is pretty challenging to appraise it structurally. SPM is the most promising technique to study these structural isomers with their different confirmations as it allows the direct visualization of monosaccharide blocks.
That further needed to be correlated with the polysaccharide sequence to its basic molecular structure and flexibility, which are important factors in studying cellular functions and saccharides properties. [215] Despite their highly flexible nature, these molecules can lead to the formation of stable crystals through hydrogen bonding between the peripheral OH-groups. These crystalline structures can be assessed by diffraction techniques. [216] In the very early STM studies, cyclodextrins, the structures of carbohydrates, were imaged by default. [217] The images of glycogen showed ellipsoidal forms with laminar structures on the surface ascribed to the short amylase-like helical branches. [217] The researchers have studied l-and x-carrageenan, [218] different cellulose derivatives, [219] and polysaccharide xanthan. [198] Abb et al. studied the sucrose molecule (a-d-glucopyranosyl-(1!2)-b-dfructofuranoside), an abundant disaccharide that forms an anhydrous monoclinic 3D structure. [220] Sucrose is a nonreducing molecule that does not form isomers in an aqueous solution. Hence, the soft-landing ES-IBD [221] technique was used to deposit deprotonated sucrose molecules on Cu(100) surface. The figure shows a 2D island arrangement of almost all the molecules in a periodic porous network (Figure 9a). The elongated double lobes have been observed forming a node as the prime building block of the network. The observed dimensions of each lobe are 1.0 AE 0.2 nm in length and 0.5 nm in width, which exactly fit the expected size of the sucrose molecule. Hence, each (double) lobe has been assigned as a single molecule. The magnified image has been shown in Figure 9b, representing a molecule (i.e., each double lobe) distinguished as two well-defined, circular features, which vary in apparent height by 0.3 Å. On the right side of the image, a pictorial  presentation of the molecule has been shown. A green oval shape is sketched with two distinct round structures of different colors, attributed to the variation of the intensities observed by STM. In the perfect assembly of a 2D network, molecules were arranged in a manner where each node consisted of a specific kind of feature, that is, either all the bright features (light blue rectangle) or all the dark features (violet rectangle) come closer and form a node. Moreover, these nodes also differ in their packing as the dark-featured node measures 0.9 AE 0.2 nm diagonally, whereas the node comprising bright features measures 1.1 AE 0.2 nm across the diagonal. Hence, the two distinct nodes have been identified, which precisely arose from the specific building blocks like glucose or fructose. However, after analyzing the STM data alone, it is not possible to allocate the glucose or fructose units to the specific nodes. For further confirmation regarding the building blocks and their interactions, the research team modeled the structures and observed that the simulated STM image of this model (Figure 9c) fits well with the experimental data. The simulated data confirmed that the distribution of intensity is the key characteristic of a single molecule, which can clearly identify and distinguish the two-monosaccharide building blocks. The brightest intensity observed in the feature is due to the upright-pointing hydroxymethyl chain present in the fructose. Hence, the bright features have been assigned to the fructose building block, and the dark ones are the glucose units. In this study, the disaccharide sucrose has been imaged for the very first time by STM at this molecular resolution, recognizing the constituent subunits, and presented a basic understanding of polysaccharide structural conformations.
The next abundant form of carbohydrate, cellulose, has also been imaged through STM. Cellulose and chitin are the forms of carbohydrates that occur in plethora on the Earth. Anggara et al. imaged and studied the single-cellulose chains at the nanometer scale. [222] They exhibited that glucose (Glc) linked by β-1,4-linkages [223,224] in cellulose plays an important role in the flexibility of the molecule. In a previous report, cellohexaose, a Glc hexasaccharide, in Figure 10a shows behavioral resemblance with the cellulose polymer. [225] Therefore, it has been used as a model for a single-cellulose chain in this report. An automated glycan assembly (AGA) [226] method was used to make different modifications in analogs to alter intramolecular interactions and subsequently the flexibility of cellulose molecules.
This study reported the effect of sequence modification on cellohexaose analogs' on-chain flexibility ( Figure 10). The pristine Cellohexaose, AAAAAA (Figure 10a), and its different analogs, substituted with the different groups, have been compared. The groups substituted in different analogs are as follows: ABAABA, ACAACA, ADAADA, and AFAAFA (written from the nonreducing end) (Figure 10b-e), where A is Glc, B is Glc methylated at OH(3), C is Glc methylated at OH(3) and OH(6), D is Glc carboxymethylated at OH(3), and F is Glc deoxyfluorinated at C(3). These substitutions manipulate the intramolecular interactions including hydrogen bonding between the first and the second as well as between the fourth and fifth Glc units. The parent cellohexaose chains (Figure 10a) exhibited straight geometry, while the substituted cellohexaoses (Figure 10b-e) acquired both straight-and bent-chain geometries. These  analogs showed the different local steric environment (i.e., the bulky carboxymethyl group) (Figure 10d) and the local electronic properties (i.e., the electronegative fluorine group) as per the substituted functional group's characteristics (Figure 10e). It has been observed that the geometrical freedom of the cellulose chain increases with the change in substituted functional groups. The study emphasizes that the flexibility of the molecule can be engineered at the single-linkage level. Possible modifications in the local flexibility motivate further to develop a different bottom-up design of carbohydrate materials. Subsequently, it helps to design and modify molecular machines. [227] 3.2. AFM Studies of Biomolecules

DNA
The innovation of AFM by Binnig brought a prompt possibility of more clear visualization of biomolecules, proteins, and nucleic acids immobilized in or under physiological conditions. In the very early work on biomolecules, nucleic acids were used to establish the sample preparation protocol and optimized imaging conditions to obtain well-resolved images of DNA. [159,[228][229][230] In those early days, Hansma and coworkers put forward a well-studied example of sequencing of single molecules using the AFM. [231] The early studies of DNA through AFM imaging have faced substantial problems regarding sample deposition, which consequently led to low-resolution images and a lack of reproducibility. [229,[232][233][234] Further, imaging has been carried out in air to achieve successful deposition and increase the chances of reproducibility. [235][236][237] Subsequently, visualization of a single biomolecule through AFM has become routine, for exmaple, imaging of the nucleic acid with high resolution in various states as hydrated, uncoated, and dynamic, has been done. [158] Typically, in the case of DNA, immobilization of the sample can be done on the mica surface in various ways like divalent cations, [141] silanization, [238,239] and the use of cationic surfactants [157] and polymers. [240] A large three-kilobase pair of Figure 10. a) STM images of cellohexaose (AAAAAA) and b-e) its analogs (AXAAXA). Cellohexaose contains six Glcs (labeled as A; colored black). The cellohexaose analogs contain two substituted Glcs, as the second and the fifth residues from the nonreducing end that have a single methoxy (-OCH3) at C(3) (labeled as B; colored red), two methoxy groups at C(3) and C(6) (labeled as C; colored green), a single carboxymethoxy (-OCH2COOH) at C(3) (labeled as D; colored blue), and single fluorine (-F) at C(3) (labeled as F; colored purple). (a-e) Reproduced with permission. [222] Copyrights 2021, National Academy of Sciences. plasmid DNA was imaged by Bustamante et al. [241] using AFM on the mica surface treated with magnesium ions; nickel and zinc also can be included in divalent cation protocols. [241] In this process, the binding efficiency between DNA and the hydrated atomic radii of the cation can be established. [141] Similarly, 1-(3amino-propyl) silatrane (APS) can also be used to treat mica surfaces. [242] A contact mode can offer high scan speed for biomolecules; however, in this type of scanning, the sample must be tightly bound, which exerts resistance to the lateral forces. [157] In the case of tapping-mode AFM techniques, during scanning, the lateral forces are reduced due to intermittent tip-sample contact, which could be a favorable condition for the biological samples to be imaged. [243] Subsequently, the tapping-mode AFM imaging of DNA has been done successfully. [244] For the very first time, Hansma et al. carried out imaging of plasmid DNA in a fluid using tapping mode and observed high-resolution images of DNA in water. [245] In the subsequent years, differently modified AFM came into the limelight to image DNA, FM-AFM. [246] In another technique, local laser heating has been used to actuate the cantilevers, known as photothermal actuation. [247] This technique has been successfully used to envision different biomolecules, such as DNA, [248] the self-assembly of proteins, [249] and live cells. [250] Protein-DNA complexes were visualized for the first time using AFM in 1992, the E.coli-RNA polymerase interaction with DNA [241] and DNA polymerase on M13 phage DNA. [251] These studies have proven that AFM is a unique tool to study protein-nucleic acid interactions. For instance, the research group studied the interaction between DNA and the minichromosome maintenance (MCM) protein using AFM. The binding ssDNA and ssDNA-binding proteins (SSBs) was also studied. For the dynamics of DNA processes involving DNA-SSB complexes, the sample was immobilized onto the mica surface. [252] In the early '90s, Hanssa and group imaged plasmids in a propanol medium (Figure 11a) and observed a better resolution of strands along with the visible narrower widths as compared to the imaging in the air (Figure 11b). [159] In subsequent years, Mou et al. developed another approach to imaging, where a cationic lipid bilayer was used for the absorption of DNA to image in an aqueous buffer medium. The incubation of absorbed DNA was performed at elevated temperatures (50-55°C) in the EDTA environment, which led to uniform and dense packaging of DNA, as shown in Figure 11c. [157] A well-resolved periodicity has been observed with an approximate width of 2 nm, including periodic lateral intervals of 3.4 AE 0.4 nm, which match the known pitch of the double helix. [242] In later years, researchers observed cruciform structures in circular DNA. AFM imaging of plasmid pUC8F14C DNA was performed, and it showed the presence of long extrusions (hairpin arms), indicated by arrows in Figure 11d. Mainly, two features of cruciform were seen, with an extended confirmation, which is typically recognized by an angle between the hairpins and arms at %180°. While in the second class, the arms formed an acute angle (X-type geometry), molecules 1 and 3 (Figure 11e), with DNA strands as sharp bending was observed. [253] Ido et al. exhibited the direct imaging of plasmid DNA in water using FM-AFM [158] with low noise and high sensitivity. Figure 11f shows a typical FM-AFM topographic image of the plasmid DNA in 50 mM NiCl 2 solution, which matches with the previously reported interwound structures of DNA. [159] Here, two types of grooves have been observed distinctly, with the different apparent widths alternately and highlighted with red and blue arrows, respectively (Figure 11f ). These grooves specifically occurred due to the major and minor grooves of B-DNA (the most common form of DNA). The sum of these alternative groove widths was 3.7 nm with a standard deviation of 0.15 nm, which is a typical characteristic of the B-DNA helix pitch. Hence, this has been confirmed the nature of real plasmid DNA under physiological conditions through AFM imaging. Pyne et al. performed AFM measurements using rapid force-distance and amplitude-modulation imaging methods for accurately reproducing B-DNA images with the typical dimensions. Specifically, intramolecular variations in the groove depth of the helical DNA structure have also been observed (Figure 11g, inset: zoomed image), which exhibited a supercoiled structure of DNA of %1.2 μm. The more resolved and periodic structure of the double band has been observed in Figure 11h-i with a specific color contrast (Figure 11h-ii) typically attributed to the two oligonucleotide strands of the double helix. [254] Further, the same research group revealed supercoiled DNA structures as minicircles or also known as looped DNA at plectoneme tips [255] representative of extrachromosomal circular DNA. [256] Figure 12 shows the high-resolution AFM images of negatively supercoiled DNA minicircles followed by atomistic molecular dynamics. The AFM images were recorded in an aqueous solution and show DNA minicircles captured with different conformation to get the sufficient resolution of specific oligonucleotide strands of the double helix. The twist observed here is corroborated by a helical repeat of 10.5 AE 0.5 bp, which is in agreement with a canonical structure of B-DNA. The observed AFM images of surface-bound minicircles at different conformations are shown in Figure 12a,d. Molecular dynamics (MD)generated conformers showed the close resemblance in global structure (Figure 12e). The thermal fluctuations were produced, owing to energy transmission through the tip, while AFM imaging contributes toward the variation in the structures within supercoiled DNA. This process further facilitates the molecule to explore its energy landscape even though they are surface bound. [257] Klejevskaja et al. [258] developed self-assembled DNA minicircles containing a G-quadruplex-forming sequence. These minicircles were further explored by AFM, [254] which showed circular DNA loops with contour lengths of %41 and 46 nm (Figure 12f ). The images were taken in the presence of Ni 2þ (Figure 12f,g) and exhibited different shapes of minicircles; closed rings with a single protrusion were observed as an indicator of G-quadruplex formation. The open ring observed in the horseshoe shape is attributed to unfolded ssDNA. "Open" minicircle structures were primarily observed in the absence of salts (Figure 12h). However, the closed structures were observed predominantly with the single protrusions as signature characteristics of G-quadruplex formation in the presence of salt, K þ (Figure 12i). Kwon's research group [259] developed a designer DNA nanostructure that can act as a template for exceptional sensing and has potent viral inhibitory capabilities. A star-shaped DNA architecture, carrying five molecular motifs, was constructed. The resulting multivalent motifs show a high binding affinity toward the specific domain groups present on the Dengue viral surface. By connecting the clusters of ED3 (envelope protein domain III) sites linearly, they further elaborated a star-shaped DNA comprising an interior pentagon connected to five exterior triangles shown in Figure 13a. This leads to the formation of a complete DNA star (Figure 13b). Two scaffolds were further constructed, which are necessary for sensing (Figure 13c). The first comprised two aptamers (bivalent) and the second consisted of a flexible linear DNA scaffold. Subsequently, a DNA star was created using these strands. Next, two additional 2D DNA nanostructures, (i.e., hexagon and heptagon-centered star complexes) were developed for the perfect matching of the 2D pattern as shown in Figure 13c. The hexagon centered star complexes was formed by adding an equivalent triangle to the original five-point star design. Moreover, after one more triangle addition into the hexagon, the heptagon can be developed. The detailed study showed that a hexagon exhibited better sensing capabilities than that heptagon. The credit for better sensing through a hexagon goes to the actual and perfect pattern matching for the detection. In the case of heptagons, a small mismatch of 2D geometry occurs as Figure 11. AFM images of plasmid in a) propanol of 360 Â 360 nm and b) in the air of 500 Â 500 nm. (a,b) Reproduced with permission. [159] Copyrights 1992, American Association for the Advancement of Science. c) High-resolution image of DNA in solution of 100 Â 100 nm. Reproduced with permission. [157] Copyrights 1998, Elsevier. d) AFM images of circular pUC8F14C plasmid DNA with an extruded cruciform indicated with an arrow. e) Various conformations of cruciforms indicated with arrows and numbers. (d,e) Reproduced with permission. [253] Copyrights 2012, American Chemical Society. f ) Highresolution FM-AFM topographic image of the plasmid DNA in aqueous solution (SO mM NiCl 2 ). The red and blue arrows highlight major and minor grooves of B-DNA, respectively. Gray arrows indicate local melting regions. Inset: Molecular structure of the B-DNA. Reproduced with permission. [158] Copyrights 2013, American Chemical Society. g) A plasmid DNA image. Inset: Highresolution AFM topography of the DNA, showing major and minor grooves. h) AFM topography of (i) plasmid DNA and (ii) digitally straightened trace (top) and retrace (bottom) image of (i) compared with a spacefilling representation of the B·DNA crystal structure. (g,h) Reproduced with permission. [254]

Electrical Conductance Measurement of DNA through AFM
DNA molecules exhibit an ability to form a self-assembled network structure, which can be supremely useful in the development of various nanodevices owing to interesting electronic properties. However, different sequences and structures of the DNA have shown contradictory results. [260][261][262] For instance, Otsuka et al. reported that in DNA thin films with a poly(dG)·poly(dC) electrode, ionic conduction occurred through alternating current (ac) in humid conditions of >80%. [263] Terawak et al. demonstrated the typical use of point-contact current imaging AFM for the electrical conductance measurement of a DNA network structure on a mica substrate in high humidity. Moreover, it has been demonstrated that the DNA network shows better conductivity than that of the mica surface. However, no conductance differences were observed in the DNA network and the mica substrate under dry conditions. [264] The research performed by Heim et al. used c-AFM to observe the electrical conductance behavior of DNA molecules, varying prominently with different compositions of the substrate, types of electrical contact used, and the numbers of DNA molecules. It has been observed that the DNA molecule is resistive in nature and showed conductance of about 10 À6 -10 À5 S cm À1 per DNA molecule. While modifying DNA structure by intercalating an organic semiconductor buffer film between the DNA and the metal electrode is a reliable way to improve the contact. However, longer exposure to vacuum or dry nitrogen decreased the conductance. This highlights that traces of water molecules and ions in the hydration shell of the DNA play a vital role in the conduction mechanism. [265] Cohen et al. utilized a c-AFM and emphasized the presence of thiol groups that affect the Color scales for f, h, and i are 2 nm, 2.5 nm, 2.5 nm, respectively. (f-i) Reproduced with permission. [258] Copyrights 1969, Elsevier.
www.advancedsciencenews.com www.small-structures.com conductance of DNA molecules. Here, ds DNA was embedded in between the ss DNA and then connected with the metal substrate and Au NPs. Approximately, 220 nA current flow has been observed at 2 V through dsDNA. The present thiol groups contributed significantly in an ion conductance mechanism. Therefore, without thiols, ds-DNA monolayer can transport low current and with thiols can transport significantly high current. This confirms that ds-DNA shows current conduction in appropriate environments. [266] The presence of π-electron cores makes DNA a good candidate for long-distance charge transport. The study has reported that a design of a specific base sequence poly(dG)·poly(dC) can be a promising semiconducting nanowire as specific base-stacking characteristics may alter the charge transport capabilities. Guanine (G) shows an easy oxidation process and can become a hole carrier. Once charges are generated on the DNA chain, electron hopping occurs among the discrete G sites and hence makes poly(dG)·poly(dC) a good hole conductor. [262] The study of ds-DNA through controlled AFM measurements by Cohen et al. [267] demonstrated thorough charge transportation, as the characterized S-shaped I-V curves depict currents >220 nA at 2 V. Figure 14a,b,e,f shows complementary and noncomplementary measurement (c and d) strands connected to the gold nanoparticles (Au NPs). The topographic images shown in a, c, and e have been taken at zero voltage applied. While, b, d, and f were measured at 4 V bias, ds-DNA exhibited current at the positions where it was connected to GNPs; however, no current has been observed for ss-DNA monolayer. Figure 14g-l shows an I-V curve measured by establishing different configurations between the metalized tip and the substrate. In Figure 14g, the metal tip and one of the GNPs were brought in contact with each other without pressing it (see the inset), where the raised current was observed as the voltage exceeded %1 V. The maximum current has been recorded, 220 nA at 2 V; however, beyond this value, current cannot be measured due to preamplifiers' range limitation. The asymmetry occurred between contacts and molecules; hence, it generates asymmetric curves. The observed S-shaped curve shows the typical resistance of %60 MΩ at the bias range of À1-1 V. The apparent The native structure of the star consists of five "scaffold" strands (S-1 to S-5) to form the internal edges, ten "edge" strands to connect internal and external edges, five "fix" strands to connect the external edges, and one "close" strand to cap all the external edges of each triangle. AFM confirmed the formation of a star-shape DNA comprising an interior pentagon connected to five exterior triangles. c) Schematic showing the design of bivalent, flexible, linear, hexagon-centered, and heptagon-centered control sensors. AFM imaging verified the formation of the 2D scaffolds. (a-c) Reproduced with permission. [259] Copyrights 2019, Springer Nature. stability at %2 V results in the consecutive order of resistance. The appeared flat curve (Figure 14h) was attributed to the conductance measured on the ss-DNA monolayer without pressing it (inset: a similar force is applied, as shown in Figure 14a,c). The contact between the tip and the surface generates hysteresis due to the retraction force-distance curve. The flat curve confirmed the insulating behavior through multiple measurements at the monolayers' different positions. Figure 14i shows an I-V curve measured on a bare Au surface for reference. The contact between a metal tip and a metal substrate reflects an ohmic nature from in which the resistance was determined (%500 ). Similarly, Figure 14j shows the current in the order of 1 μA as, during measurement, one of the GNPs pressed ss-DNA strongly (see Inset). That has established contact between the substrate and the pressed ssDNA monolayer. However, Figure 14k,l is attributed to the collection of different measurement curves performed at various GNPs in combination with different samples and tips. Simultaneously acquired topography and current maps. a,b,e,f ) A sample with GNPs connected to a dsDNA c,d) on a sample with GNPs connected to noncomplementary strands. g) This measurement was performed on a metal particle without pressing on it, as shown in the inset. h) I-V curve was measured on the ssDNA monolayer without pressing it. i) A measurement that was taken on a bare gold surface showing j) I-V curve measured on a metal particle while pressing it strongly to the metal substrate. k and l) Two sets of I-V curves that were measured on different GNPs on different samples and tips. (a-l) Reproduced with permission. [267] Copyrights 2005, National Academy of Sciences.
www.advancedsciencenews.com www.small-structures.com GNPs play a vital role in the conductance measurement studies of DNA. Hence, to define the actual effect of different sizes of GNPs on conductance measurements, this research group experimented with varying GNPs' sizes. [268] Figure 15a-c shows the topographical and conductance measurement studies of ds-DNA molecules, where topographic images showed the sample having GNPs of different sizes (insets) on the ss-DNA monolayer background. The current behaviors of "standing" ds-DNA connected to different sizes, 5, 10, and 20 nm of GNPs, have been compared using I-V measurements. Figure 15d-f shows more than 220 nA current in all cases, which revealed the ability of ds-DNA to transport such current as supported by many other researchers. [266,269,270] In the typical S-shaped I-V curves having a bandgap of %2 V, the max current observed was 220 nA with a few MΩ resistance at high currents. This study observed similar general behavior for all GNP sizes in I-V measurements. The study concluded that current flows through the same number of molecules no matter what the diameter of GNPs is.
Moving further, the effect of defects on the conductivity of DNAs has been studied by Stern et al., who used a c-AFM system to measure the conductivity of the DNA-based nanowires. These wires are Au coated and the narrowest, which exhibited a longrange conductivity that revealed the effect of defects on the conductivity. The thicker the coating of Au, the less the number of defects, which in turn increases the conductive length. In the conductivity measurement by AFM, the contact with wires protruding from under the Au electrode has been established with a c-AFM tip, which plays a vital role and acts as a second mobile electrode at different distances for the current measurement ( Figure 16a). The I-V measurements were performed during contacting different points on each wire and its surroundings as shown in Figure 1b,d. Subsequently, the conductive length of wires, the actual length of the wire between the Au electrode border and the most distant point that showed conductivity has been determined (Figure 16c). Examples of individual I-V curves measured at three different points have been shown in the insets. Due to AFM tip convolution, %50 nm is the minimum distance from the Au border for the conductivity measurement. Hence, 50 nm is the least measured conductive length. [271] Recently, Wang et al. demonstrated surface morphological information, and current images of λ DNA molecules were obtained through c-AFM. The current flowing through the different forms of DNA such as stretched DNA, the random distribution of DNA, and the DNA networks have been compared and shown in Figure 16e-j. The current flowing through the stretched DNA, random distribution of DNA, and DNA networks have been observed as À1.92, À8.26, and À30.97 pA, respectively (Figure 16k). DNA molecule itself is highly resistive, and hence, the current measured for a single DNA molecule was relatively small, À1.92 pA. However, in the random orientation, DNAs were scattered and crossed together, which ultimately increased the electrical conductivity of DNA. While forming a DNA network, more DNA molecules were added together; subsequently, higher conductivity has been observed in this case than that in the two cases mentioned earlier. [272] Such measurements of the electrical characteristics of DNA create appreciable anticipation in the fields of nanoelectronics and biosensors.

Protein
A single protein molecule, the smallest biological sample, can be visualized and studied at the subatomic scale through the AFM technique with more reliability. [273,274] This technique can appropriately appraise the morphology and properties of a protein molecule and its complexes at a lower nanometer scale. [275,276] Mostly, the globular shapes of proteins restrict the study of specific morphologies. In the early days, the topography of proteins with special resolution has been achieved by experimenting with various sample preparation methods, [277,278] capturing the image. [279] Using c-AFM is a critical task since it continuously varies different parameters of the instrument, which harms the substrate/sample. Therefore, contact mode imaging has been replaced by tapping mode. [245,280] Later, the morphological studies progressed up to the resolution of 0.5 nm laterally and 0.1 nm vertically. [281,282] Studying the conformational behavior of proteins is quite important for the basic and advanced studies of proteins and their interactions with others. [283,284] With the employment of AFM, one can study their characteristics. [285,286] In the early studies, immunoglobulin molecule was scanned in the air/fluid and under physiological conditions. It has been observed that there is no significant difference in the volume of IgG molecule, no matter whether it is scanned in the air or fluid, meaning IgG proteins have the same water content in the air or fluid. [287] Tapping-mode AFM also has been used in producing high-resolution images of native protein surfaces. The research group selected a hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans. [288] For imaging, freshly cleaved HOPG was used as a substrate. It has been noted that the topography obtained through the scanning was stable during the multiple scans in the different solvent environments (Figure 17aÀc). The images clearly exhibited the structural features of a sample as the hexagonal units, which is in agreement with the previously observed images, which were obtained by 3D electron microscopy and also by c-AFM. [289] Oesterhelt's group [290] studied the unfolding path for a typical protein. For this, purple membrane patches from Halobacterium salinarum were examined using combined AFM and single-molecule force spectroscopy. While imaging, initially, the bacteriorhodopsin (BR) molecules were localized and afterward were extracted from the membrane; vacancies created after this extraction were imaged again. Hence, it has been concluded that, upon extraction, the unfolding of the helices occurred due to the force exerted. In the typical experiment, a native purple membrane was adsorbed onto a freshly cleaved mica substrate. The clear AFM image of the cytoplasmic purple membrane surface was observed at the submolecular level (Figure 17d). Topographically, hexagons of the trimeric BR molecules were observed. The structural information of a single protein along with its subunits has been remarkably seen in the image (Figure 17d). Subsequently, the AFM stylus was pointed over a protein and pushed with a contact force of 1 nN for about 1 s. This led to the adsorption of protein onto the tip. While retracting the tip back, the force extension spectra (Figure 17e [291] The structurally almost similar moieties, aquaporins (AQPs) from E. coli, the orthodox water channel AqpZ, and the glycerol uptake facilitator GlpF, were chosen for the experiment. [292] The typical structure of both proteins is composed of four water/glycerol conducting channels, which contain six transmembrane helices and two half-spanning helices. High-resolution AFM imaging of individual GlpF tetramers, firmly fixed in the lipid bilayer (Figure 18a), exhibited the periplasmic surface prompting out four prominent protrusions, specifically of height 1.58 AE 0.28 nm, which is in agreement with protruding loops present on its crystal structure. Figure 18d shows a surface morphological image of AqpZ molecule implanted in a lipid bilayer as the former molecule; here also a periplasmic surface with the protrusions having height of 0.93 AE 0.17 nm were observed which is comparatively lower than that of former. The collective motion of the whole tetramer has been observed in the HS-AFM movie, which revealed that the coordinate system was transferred into the center of mass while the remaining loop motions were credited to the random thermal movement. The probability of finding the loop positions has been observed through the image sequences (Figure 18b,e), leading to extracting the free energy surface underlying the loop motion easily. The free energy landscapes underlying the motions of loop C (the loop connecting transmembrane helices 3 and 4) of GlpFs and AqpZs were mapped (Figure 18c,f ) respectively. Specifically, the loop C recognized from these energy landscapes defines further the lateral stiffness of the respective molecules. By studying these results, the flexibility of protein molecules present on the surface of individual membrane proteins can be easily deduced by topography obtained by HS-AFM and evaluating their thermal motion.
Moving further, HS-AFM has also been used for the visualization of a single protein molecule like ferritin, fibrinogen, human serum albumin (HSA), and IgG. Ferritin molecule shows a spherical globular shape (Figure 18g). The surface morphology studies of fibrinogen observed an extended trinodular structure with outer nodes related to D domains and the central (a-c) Reproduced with permission. [288] Copyrights 1999, Elsevier. d) Topographic image of the cytoplasmic surface of a wild-type purple membrane. BR assembles in hexagonal trimers (white circle). e) Force spectrum was recorded. The interaction between tip and surface, marked with discontinuous changes in the force, indicates a formation of a molecular bridge between tip and sample. f ) After the adhesive force peaks were recorded, structural changes were recorded, exhibiting a single monomer missing (white circle). (d-f ) Reproduced with permission. [290] Copyrights 2000, American Association for the Advancement of Science.
www.advancedsciencenews.com www.small-structures.com node-correlated E domain of the protein structure (Figure 18h). Similarly, the image of HSA has shown globules with a triangular arrangement of domains (Figure 18i). AFM images of IgG have confirmed the globular structure of the molecule. However, in some images, the classical IgG tri-nodular structure also has been observed (see white arrows in Figure 18j). [293] The study has addressed the problem of protein molecule adsorption, especially on the graphite surface. Since this is the issue being faced while using proteins in the field of biomaterials or substrates for sensors, high-resolution force-volume AFM was used to reveal the topography of a purple membrane (PM) from Halobacterium salinarum. The membrane typically consists of bacteriorhodopsin (BR) and lipids. The topographical image recorded at lateral resolution of 1-1.5 nm exhibited BR trimers, specifically, three major and three minor protrusions have been observed clearly. [112] Other types of protein such as metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP), and lactoferrin (LF) also play a crucial role in the regulation of inflammation and oxidative stress in vertebrates. The morphological visualization can be beneficial to understanding the inflammatory and oxidative stress mechanism. Initially, the morphological studies of the (a-f ) Reproduced with permission. [291] Copyrights 2015, American Chemical Society. g-j) Montage of AFM images of ferritin, fibrinogen, HSA, and IgG molecules adsorbed on GM-HOPG. (g-j) Reproduced with permission. [293] Copyrights 2016, Elsevier.
www.advancedsciencenews.com www.small-structures.com mentioned proteins have been done independently; subsequently, the specific interactions between proteins have been studied through visualizations. [294] AFM images of MPO, CP, and LF molecules adsorbed on graphite modifier (GM)-HOPG (Figure 19a-c) have confirmed globular structures of molecules, the enlarged molecules have been observed in insets of the respective images, and the height observed in the images is in exact corroboration with the results reported in other studies. [295,296] While studying interactions between MPO-CP proteins, (Figure 19d), it has been observed that topographically, both proteins show globular shapes with different heights (examples are enlarged in the insets I-III). Further, the chains observed (inset IV) in images have revealed that the complex formation has taken place through the specific binding sites of MPO and CP, in agreement with the previous studies. [297] Moreover, the interaction studies between LF-MPO proteins have been shown in Figure 19e. Morphologically, globular shapes have been observed with the different heights corresponding to the respective protein molecule (encircled with a solid (LF) and dashed (MPO) line). Here, there has not been single evidence of complex formation occurring in AFM images, which goes hand in hand with the early observations. [298] In addition, an interaction between LF and CP also showed globules present with varying heights. An enlarged image at the bottom of Figure 19f concluded the complex formation as the molecule seen is quite bigger than that of the individual protein molecules.
Hence, it has been demonstrated that there is no any direct communication between MPO and LF molecules. This subsequently, led to understanding the mechanism for the regulation of inflammation and oxidation stress and can be typically driven by interactions between metalloproteins such as MPO, CP, and LF. Jiao et al. demonstrated structural morphology and supramolecular assembly of the septin molecule, cytoskeletal GTP-binding proteins. [299] Experimentally, septins were deposited onto hydrophobic epoxy resolution, where clear individual rods and filaments were observed. When mica was used as a substrate, the assembly of septin rods into several micrometer long-paired filaments was observed. This precisely indicates that mica can be a good substrate for assembling septin rods into higher-order structures. Further, to study assembly formation with respect to environmental factors, such as monovalent-ion (KCI) concentration, bulk septin concentration and pH images were recorded. These studies concluded that the filament formation depends on salt concentrations; as it increases, an electrostatic shielding of repulsive charges (a-f ) Reproduced with permission. [294] Copyrights 2018, Elsevier.
www.advancedsciencenews.com www.small-structures.com is favored on the surface and/or between adjacent septin filaments and pairing. In this condition, ultimately, ionic strengths increase more than that in physiological bulk conditions. Meaning, high salt concentration on the mica substrate supports the assembly formation of septin molecules. This study has demonstrated that the environmental factors affect the assembly formation of septin molecules, which is useful information while performing optical approaches typically in in vivo investigations. Pfreundschuh et al. discussed multiparametric AFM imaging in combination with the probe functionalized with specific chemical groups, ligands, or even live cells, which simultaneously can image the sample and quantify receptor interaction. [300] In this study, AFM tips functionalized with Ni 2þ -nitrilotriacetate (NTA) groups have been used to detect the specific interaction sites on the self-assembling soluble spindle assembly abnormal protein 6 homologue (SAS-6), a protein implicated in centriole duplication ( Figure 20). Each SAS-6 molecule consists of two independent dimerization interfaces. Typically, N-terminal domains show head-to-head dimerization and the C-terminal domains are the two-stranded coiled rods (Figure 20a, inset). The topographical AFM image showed a dense network of SAS-6 cartwheels (Figure 20b,c). FD-based AFM was used to detect the His6-tags; it has been observed that cartwheels show a spike corresponding to the C-terminal of the SAS-6 end and were engineered by His6tag at the end (Figure 20d). Adhesive forces were studied by adhesion maps obtained from the corresponding AFM topographs (Figure 20e,f ). The shape observed for the individual FD-curves revealed adhesive forces of %120 pN and rupture distances from 4 to 22 nm. The observations showed that the tip detected only a small proportion of the tagged C-terminal ends. This is probably due to 1) very less accessibility of the His-tag to the tip; 2) very short contact time, or 3) not achieving the typical transient conformation of the NTA on the tip. In conclusion, this study has revealed that multiparametric imaging of biological materials can be done along with studying their typical biological interactions.

Carbohydrates
Carbohydrate is an important molecule on the cell surface, which communicates with both proteins and lipids and acts as a key constituent in recognizing cell-to-cell signaling. [301] Imaging of carbohydrates by AFM was a quite difficult task in the contact mode, since the capillary forces generate large surface adhesion forces of up to 100 nN, leading to the displacement of the sample. [302] In the early research, polysaccharides were imaged under nonaqueous solutions, such as butanol or other alcohols. This could subsequently reduce the tip-sample adhesive interactions, and hence imaging of molecules, that is, xanthan, acetan, hylan, hyaluronan, and the network structures of gellan and κ-carrageenan became possible. [303,304] Mica was used as a substrate to obtain a high-resolution image of scleroglucan and linear-to-circular triple-helix transition. [305] Later, Gunning et al. Figure 20. High-resolution FD-based AFM of SAS-6 cartwheel structures. a) Structural model of the SAS-6 cartwheel, b) FD-based AFM topograph illustrating the typical structure of SAS-6, c) high-resolution topography of SAS-6 oligomers typically revealed C-terminal His6-tag at the distal ends of the coiled-coil rods, d) high-resolution AFM topography of SAS-6 specimens, e) mapping of specific (red circles) and unspecific (gray circles) adhesion forces ranging from 100 to 200 pN, and f ) FD detecting an adhesion event in the adhesion map. Red-colored FD curves (S1-S4) detect specific adhesion separated from the tip-support contact region (<4 nm). The black-colored FD curve detects an unspecific adhesion event within the tip-support contact region (U1). (a-f ) Reproduced with permission. [300] Copyrights 2014, American Chemical Society.
www.advancedsciencenews.com www.small-structures.com imaged a single-xanthan molecule in the tapping-mode AFM on a mica substrate. [306] Structural changes in the molecule, polysaccharide succinoglycan, have been studied, where the rigid, flexible chains and aggregates of the molecule were observed. The rigid chains were the dimers of the flexible single helices. However, only flexible single helices appeared when imaging was carried out under a salt solution (0.01 m KCl). [307] Grandbois et al. studied the surface chemistry of a single polysaccharide. The molecules were covalently embedded between a surface and the tip, followed by applying a force to stretch the molecule up to its detachment. It has been observed that at 2 nN, the silicon-carbon bond was broken, while the sulfur-gold bond lysed at 1.4 nN. [308] To comprehend the basic interaction between lectin and carbohydrates, and know the vital role in the process of cellular communications, Touhami et al. studied lectin, concanavalin A (Con-A), and carbohydrate, oligoglucose saccharide interactions through the AFM. Experimentally, the AFM probes were modified with Con-A and thiol-terminated hexasaccharide molecules. [309] Figure 21a shows the topography of the hexasaccharide-terminated substrates as a uniform and continuous monolayer free from any aggregates. For the further appraisal of the interaction between Con A and the hexasaccharide-terminated molecule, the substrates were incubated first with lectin; subsequently, the images were recorded. Figure 21b shows that the surface morphology of the substrate has been completely changed as a credit of Con-A adsorption. The surface roughness in the presence of small protrusions has been observed, which arose due to the appearance of Con-A tetramers. To further crosscheck the role of specific receptor-ligand interactions in the observed morphological changes, the experiment was carried out in a slightly tricky way, where the binding sites of Con-A were blocked with mannose; consequently, the probe became hydroxyl-terminated. The related Figure 21c,d has not exhibited any protruding structures on the surface in the presence of hydroxyl-terminated probes. Hence, it has been assumed that these features sprang up only after the specific lectin-carbohydrate interactions. AFM mapping confirmed that the hexasaccharide-terminated substrates interact specifically with the lectin. This kind of carbohydrate probe can be further useful in mapping the cell surface and its interactions.
Later, to study the effect of annealing on polysaccharides, the Iijima group performed annealing of xanthan gum Figure 21. AFM topographic images (3 Â 3 μm; z-range, 10 nm) recorded in aqueous solution for a) hexasaccharide-terminated substrate, b) a hexasaccharide-terminated substrate after adsorption of Con-A, c) a hexasaccharide-terminated substrate after adsorption of Con-A in the presence of 100 mM of D-mannose, and d) a hydroxyl-terminated substrate after adsorption of Con-A. (a-d) Reproduced with permission. [309] Copyrights 2003, American Chemical Society.
www.advancedsciencenews.com www.small-structures.com molecules. [310] The morphological changes were observed with respect to varying annealing times through AFM. For imaging, xanthan gum molecules were extended on the mica substrate in the mono-or double layers. Figure 22a shows topographs of nonannealed and annealed xanthan molecules, and Figure 22b-d shows the images of annealed xanthan molecules at 40°C for 1, 6, and 24 h respectively. In the nonannealed image, the network structure did not appear, whereas the same has been clearly seen in the images of the annealed molecule. Precisely, molecules formed a heterogeneous assembly after 1 h. However, the morphology recorded after 6 h of annealing showed heterogeneous distribution. After 24 h annealing, the xanthan molecule reflected homogeneous assembly. Hence, it can be stated that the network structure has not been observed in nonannealed molecules; however, the same has been seen in the annealed ones.
Here, the AFM topographic study has dealt with the oscillational change of the network structure. Moreover, one can conclude that the aggregates present in the nonannealed molecules get dissociated upon annealing and a homogeneous structure has been formed after 24 h of annealing at 40°C. The study has clarified the gelation process of xanthan gum hydrogels.
In the bioapplications study, Fructo-oligosaccharide was used to treat lung injuries, which has been isolated from Polygonatum Cyrtonema Hua (PFOS) [311] and analyzed using AFM. In the typical experiment, PFOS was purified and extracted in two fractions PFOS-1and PFOS-2 with different molecular weights. The AFM image of PFOS-1 at a lower concentration (5 μg mL À1 ) revealed a short-chain structure with a branch (Figure 23a). Simultaneously, the same molecular image at a relatively high concentration (50 μg mL À1 ) was reflected as a snowflake aggregate structure (Figure 23b). However, the topography of PFOS-2 exhibited a structure with longer chains due to precise branching at lower concentration (5 μg mL À1 ) ( Figure 23c). As the concentration of PFOS-2 increased, an entangled macromolecular assembly with more branches was noticed (Figure 23d), which may result from the presence of the high number of hydroxyl groups in the oligosaccharide molecules. This porous and highly branched structure may possess good water solubility. The presence of hydrogen bonds plays a crucial role in aggregation, which subsequently decides the surface properties and biological activities of PFOS-1 and PFOS-2. Accordingly, further biological studies were carried out and concluded, wherein the fraction of PFOS with a short-branch chain shows prompt healing of lung injury and respiratory issues. The fibrous structure of the pectin molecules and plant cell wall polysaccharides has been noticed by Morris et al. [312] Chitosan is a natural, nontoxic, biodegradable polysaccharide, and chitosan-folate conjugates were developed to treat lung cancer. Different formulations of chitosan were developed and their morphologies have been studied using AFM. The topographical studies of the different formulations showed a spherical shape, uniform, and smooth surfaces free from any kind of pinholes or cracks demonstrating quite favorable morphology for drug delivery application. [313] A recent study verified that changes in the surface morphology of chitosan  led to altering basic properties of the thin film upon intercalation of chitin nanofibrils in it. [314] The modified structure exhibited that the properties like tensile strength, elongation at break, hydrophobicity, specific electrical conductivity, hydrophobicity, and biocompatibility have been redefined to a better extent. The 3D AFM image of the chitosan thin film shows a uniform and homogeneous film (Figure 23e). The change in the morphology was seen by adding even lower concentration (0.5 wt%) of chitin nanofibrils into chitosan solution, and anisometric shapes appeared (Figure 23f ). The subsequent addition of chitin led to it being more effective in changing the morphology (Figure 23g). The observed anisometric structures have been spread throughout the film probably due to the shear stress developed during the formation of composites. [315] Chitin nanofibrils possess a relatively high specific surface area and efficiently adsorb chitosan from an aqueous solution. Chitin and chitosan possess high thermodynamic affinity and lead to better absorption of chitosan on chitin with rearrangement of the molecular chains. [316] Moving ahead, the addition of more chitin nanofibrils in the composite film reflects increased roughness of the film with heterogeneity and the presence of large structural elements in Figure 23h is observed. It has been concluded that as the filler concentration increases, the disturbance in the oriented lamellar structure increases, forming a rigid structural network of chitin nanofibrils. Further, biological assays have affirmed that the composite film with 5 wt% of chitin nanofibrils shows better antiproliferative activity against fibroblasts.

Summary and Perspective
In the interdisciplinary field of biophysics, tremendous advances in the application of tools are being explored. Various techniques such as X-ray powder diffraction (XRD), NMR, and fluorescent spectroscopy are being employed to analyze biomolecules, right from their fundamental properties including chemical and physical sciences to intricate biomolecule structures and their interactions. In the curious pathway toward molecular high-speed imaging with the simultaneous assessment of electrical properties and molecular specifications, SPM has emerged as one of the essential techniques for biomolecules' appraisal. However, due to some experimental limitations, such as sample preparation, or sometimes, instrumental limitations, SPM has been rarely used for the successful analysis of biological materials. Here, in this review, in the beginning, we have precisely mentioned the literature, which has addressed experimental challenges, mainly sample preparation. This is the prominent cause of restricting SPM for widespread applications, especially in biophysics. As the biomolecules are soft, mobile, and the intricate structures exhibit timely conformational changes, the implementation of SPM for characterizing biomaterials is a quite challenging task. Research groups worldwide are sincerely working on this challenge, using specific sample preparation methods to immobilize the sample under physiological conditions. The methods may include the use of covalent linkers for typical binding between sample molecules and the substrate, which creates electrostatic interactions. These schemes further facilitate the simultaneous evaluation of other properties of biomolecules along with imaging such as dynamic conformational interactions and electrical characteristics. On the other hand; the instrumental limitations are also being addressed keenly concerning the issues related to scanning mode, experimental settings/ environment, and probe material choice, which can limit the structural deformation of the sample. This review is focused on the biophysical appraisal of DNA, proteins, and carbohydrates, addressing researchers' innovative approach to conquering the hurdles in studying biomolecular  [311] Copyrights 2021, Elsevier. 3D images of surfaces of e) chitosan film and f-h) composite films containing 0.5, 5.0, and 30.0 wt% of chitin nanofibrils respectively. The areas of the studied surfaces were 1 Â 1 μm. (e-h) Reproduced with permission. [314] Copyrights 2021, Elsevier. surface chemistry and its interactions. During the direct visualization and typical characteristics measurements, the fundamental properties, including structural conformations of biomolecules, get modified with an experimental environment, and instrumental settings as biological entities respond quickly to external stimuli. Besides that, instrumental sensitivity, tunneling distances, thermal drift, humidity, probe dampening by solvent, and changes in pH also play a crucial role in the typical characteristic's appraisal. This review has specifically identified these issues and has mentioned inherent sensitivity of the biomolecules toward these factors, which, subsequently, affect the molecules' characterization studies. A few examples of electrical conductance measurement studies have been mentioned here and have represented exceptional behavior of biomolecules exhibiting specific electrical characteristics. These are accountable for various modern applications like drug delivery, biomolecular devices, artificial intelligence, and the spintronics field. Present literature studies make researchers aware of the different characteristics of biomolecules, which can be used in various modern techniques. The persistent need for understanding the complex structures of biomolecules and the thirst for getting a clear idea regarding the dynamic biological processes will always demand a new tool for analyzing biomolecules at the molecular level. SPM can be one of the methodological techniques, which can firmly assist researchers to analyze the biomolecules in different aspects; additionally, the instrument can be modified in different experimental settings. The combination of the SPM technique with other analytical instruments led to broadening the horizon of SPM practices. For instance, the integration of SPM techniques has been done with microfluidics, [317,318] a mechanical sensor too, that contributed to studying the mechanical properties of living cells. [319,320] To execute this successfully, researchers should have a better understanding of SPM fundamentals and experimental details. This review will make an important contribution to the understanding and implementation of SPM in different ways to attain meticulous experimentation and analysis. Subsequently, researchers will get an idea regarding bundles of possibilities influencing the measurement systems of interest. In this way, this review will pave the way for researchers to spring up with various potential ideas to broaden the use of the SPM technique in the biomedical research arena.