Recent progressive use of atomic force microscopy in ... · aUniversity of Maribor, Faculty of Medicine, Institute of Biomedical Sciences, Taborska ulica 8, SI-2000 Maribor, Slovenia

Recent progressive use of atomic force microscopy in biomedicalapplicationsUroš Maver a,*, Tomaž Velnar b, Miran Gaberšcek c, Odon Planinšek d, Matjaž Finšgar e

a University of Maribor, Faculty of Medicine, Institute of Biomedical Sciences, Taborska ulica 8, SI-2000 Maribor, Sloveniab University Medical Centre Maribor, Department of Neurosurgery, Ljubljanska ulica 5, SI-2000 Maribor, Sloveniac National Institute of Chemistry, Laboratory for Materials Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Sloveniad University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, SI-1000 Ljubljana, Sloveniae University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory of Analytical Chemistry and Industrial Analysis, Smetanova ulica 17,SI-2000 Maribor, Slovenia

A R T I C L E I N F O

Keywords:AFMAtomic force microscopyForce spectroscopyDrug-tissue interactionInteraction mappingBiomedical applicationsSingle molecule studies

A B S T R A C T

In recent years, a great deal of interest has been focused on the development of novel atomic force mi-croscopy (AFM)-based methods. From first being an unstable method, AFM has emerged as the perfecttool for the study of phenomena at the nanoscale, which includes quantitative single molecule studies.Numerous novel AFMmethods play a crucial role in the invention of novel drugs, their delivery systems,based on either polymers or inorganic/metallic matrices, and in the examination of disease-related tissuechanges. Such contemporary progressive studies are a perfect example of interdisciplinary research, whichresults in exemplary findings and discoveries. This review focuses especially on the literature publishedin the last decade; however the most important earlier discoveries are also included.

© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

Contents

1. Introduction ........................................................................................................................................................................................................................................................... 971.1. Surface properties of solid pharmaceutical forms ...................................................................................................................................................................... 971.2. About the technique ............................................................................................................................................................................................................................. 97

1.2.1. The force between the sample and the tip .................................................................................................................................................................. 971.2.2. Special AFM modes .............................................................................................................................................................................................................. 97

2. Examples of AFM use in drug discovery and development ............................................................................................................................................................... 1002.1. Drug discovery ...................................................................................................................................................................................................................................... 100

2.1.1. The search for novel drug targets .................................................................................................................................................................................. 1002.1.2. Identification of species ................................................................................................................................................................................................... 100

2.2. Pharmaceutical technology .............................................................................................................................................................................................................. 1012.2.1. Drug delivery ....................................................................................................................................................................................................................... 1012.2.2. Determination of the structural properties of pharmaceutical formulations ............................................................................................... 1012.2.3. Biophysical examination .................................................................................................................................................................................................. 1022.2.4. Transport phenomena ...................................................................................................................................................................................................... 102

2.3. Bionanotechnology and nanobiotechnology .............................................................................................................................................................................. 1032.3.1. Study of disease-related changes .................................................................................................................................................................................. 1042.3.2. Receptor-ligand interaction studies ............................................................................................................................................................................. 1052.3.3. Single molecule measurements .................................................................................................................................................................................... 105

2.4. Interaction mapping ........................................................................................................................................................................................................................... 1052.5. Combinations of AFM with other methods ................................................................................................................................................................................ 107

3. Conclusions ......................................................................................................................................................................................................................................................... 107Acknowledgements .......................................................................................................................................................................................................................................... 108References ............................................................................................................................................................................................................................................................ 108

* Corresponding author. Tel.: +386 2 2345 823; Fax: +386 2 2345 820.E-mail address: [email protected] (U. Maver).

http://dx.doi.org/10.1016/j.trac.2016.03.0140165-9936/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Trends in Analytical Chemistry 80 (2016) 96–111

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1. Introduction

1.1. Surface properties of solid pharmaceutical forms

The surface properties of solid pharmaceutical dosage forms,which have a major influence on the final pharmaceutical productand therefore its use, are determined during preformulation and for-mulation studies. In the course of evaluating pharmaceuticalformulations, the respective components can exhibit significantlydifferent properties. Furthermore, the measured properties can belocally specific and therefore do not truly represent the bulk prop-erties of the whole system. Several characterisation methods alsorely on averaging, making measured properties not appropriate fordescribing the separate components. Such component-specific in-formation is crucial for predicting final product performance,enabling a thorough understanding of the overall bulk properties,and can only be obtained when measurements are performed sep-arately at several representative points on the surface and areanalysed as separate entities. Finally, the statistical validity of suchmeasurements is of utter importance, permitting a thorough un-derstanding of the examined materials [1,2].

While the European Pharmacopeia prescribes several tests thatare important for pharmaceutical formulations safety and quality,several other important physicochemical properties arising due tointeractions between the components comprised in the pharmaceu-tical formulation contribute to the final product properties andtherefore require attention [3,4]. The surface roughness, grain size,polymorphism, and surface energy of standalone components andinteraction among them are just some of the surface characteristicsthatmaysignificantly influence thebioavailabilityof thebuilt-in activeingredient and consequently its efficiency [5]. Although variouswell-known methods are being used for the local characterisation ofpharmaceutical formulations (electronmicroscopy, contact anglemea-surement, thermal analysis, x-raypowderdiffraction, andothers), noneof them is able to evaluate the regionally specific properties in a com-plete and reliableway.At themoment, atomic forcemicroscopy (AFM)is themostwell-knownalternative for thoroughqualitative andquan-titative evaluation of pharmaceutical formulations with regard tosurface properties that can affect the final formulation characteris-tics. In addition,AFMis theonlyavailable techniquewith thecapabilityto measure interactions in the pN range, which makes it one of themost important tools for in biomedicine.

1.2. About the technique

The AFM technique was introduced in 1986 by Binning, Quate,and Gerber with the intention of overcoming the drawbacks of its

predecessor, the scanning tunnelling microscope (STM) [6,7]. Themost prominent upgrades are the capability of AFM to also measurenon-conductive samples and to measure the surface topography ofsamples at sub–nanometer resolution [8].

A schematic representation of the basic AFM set-up is shown inFig. 1a. When using AFM a tip attached to a flexible cantilever movesacross the sample surface, measuring the surface morphology onthe atomic scale. The force between the tip and the sample is mea-sured during scanning, bymonitoring the deflection of the cantilever[11].

1.2.1. The force between the sample and the tipIn order to understand the mechanisms behind the interacting

components in a pharmaceutical formulation all the contributingforces must be taken into account. This is especially important whena quantitative analysis of the interaction is required, as in the caseof interactions between biological macromolecules [12]. The forcesbetween the tip and the substrate have short- and long-range con-tributions. When measurements are performed, it is crucial toseparate the contributions of various forces and to eliminate the un-desired ones. This ensures the measurement of only the desiredsample properties and makes further quantitative analysis possi-ble [13,14]. At room conditions, water moisture can condense onthe tip, which is a source of capillary force. As the capillary forcesare relatively strong and can cover the contributions of other forces,they have to be avoided if possible. This is possible by measuringin special water-free conditions, such as in a N2 or Ar atmosphere.

To represent forces on the atomic level, different potentials cor-responding to changes of potential energy at various particlepositions are used. Known empirical models used to illustrate chem-ical bonds include the Lennard-Jones andMorse potentials [9]. Thesemodels quite satisfactorily fit the force regime curve shown in Fig. 1b,which represents the course of the tip-sample interaction. Never-theless, these models are not always applicable due to experimentallimitations such as sample contamination and to many contribu-tions to the measured interaction [9].

1.2.2. Special AFM modesMany different variations of the basic AFM set-up have been in-

troduced over the years of its use. Although most of them areapplicable to all types of samples, not all yield the same amountand quality of results for all experimental set-ups. Proper use of theseversatile measurement variations enables one to study and under-stand processes even at the molecular level [15,16].

1.2.2.1. Contact mode. Contact modewas the first known AFMmode.Here, the tip moves across the surface and deflects according to its

Fig. 1. Atomic force microscopy. a) Schematic presentation of AFM; b) Force regimes governing AFM measurement (this image was drawn considering the explanation in[9] and was partially reproduced from [10]).

97U. Maver et al. / Trends in Analytical Chemistry 80 (2016) 96–111

profile (Fig. 2a). Two types of contact mode measurements areknown, the constant force mode and the constant height mode. Inthe constant force mode, a feedback loop is used tomove the sampleor the tip up and down and keep its deflection constant. The valueof z-movement (z is height) is equal to the height changes in thesurface of the sample. The result of such measurement is informa-tion about the surface topography. Since the tip is in constant contactwith the surface, significant frictional forces appear, which candestroy or sweep soft samples, such as polymers or biological mac-romolecules on the surface [18]. Such an approach was mostly usedfor examining surfaces of solid pharmaceutical formulations [19,20].When small forces are used, this mode can also be used for surfacetopography imaging of fixed cells [21,22].

The other type of contact mode AFM measurement is based ona constant height, but the forces change. In this case, the cantile-ver deflection is measured directly and the deflection force on thetip is used to calculate the distance from the surface. Since no feed-back loop is required for this type of measurement, it is appropriatefor quick scans of samples with small height differences (if heightdifferences are large, the tip will very likely crash onto the surface,resulting in either it being destroyed or in it damaging the samplesurface). With this type of measurement, atomic resolution isachieved at low temperatures and in a high vacuum. Such mea-surements are often used for quick examination of fast changes inbiological structures [15,23,24].

1.2.2.2. Non-contact mode. In non-contactmode, the sample’s surfaceis investigated using big spring constant cantilevers. The tip hoversvery close to the surface (approximately 5–10 nm distant). It nevercomes into contact with it, hence the name non-contact mode(Fig. 2b). A major advantage of this mode is the negligible fric-tional forces, making it capable of measuring biological sampleswithout altering their surface, as well as its speed. The major draw-backs of this mode are the low lateral- and z-resolution whencompared to contact mode. Recently, it was used for a thorough ex-amination of specific regions of cell surfaces [25–27] and for thecharacterisation of single polymer chains [28,29].

1.2.2.3. Lateral force microscopy. In lateral force microscopy (LFM),an AFM is used tomeasure the frictional forces on the surface. Insteadof the z-deflection, the lateral deflection is measured. This mea-

surement requires more robust tips and cantilevers that are notdestroyed in the event of high frictional forces, which are commonin suchmeasurements. LFM enables the determination of the regionson the substrate surface with lower or greater friction. Data ob-tained in the measurement of lateral forces is composed of actualfrictional forces and the influence of the height change of the sample(Fig. 2c in 2d). In order to acquire appropriate friction informa-tion, the contributions of lateral forces and sample topography aremeasured and the latter is computationally subtracted from the totaldata. The resulting image shows the contribution of the surface fric-tion only. Such an approach is especially useful for samples exhibitingmulticomponent (not layer-by-layer) surface coatings, such as ineroding drug delivery systems, as it can reveal the regions of dif-ferent materials in the coating [30]. Appropriate release studiescombined with LFM information can be a basis for development ofcontrolled release dosage forms [31,32].

1.2.2.4. Force modulation. In force modulation, repulsive forcesbetween the sample and the tip are used to investigate the sampleproperties. In this mode, the AFM tip has to be coated with mate-rials that exhibit repulsive forces with the sample surface (for thispurpose, the sample is also sputtered or coated in certain cases).The tip is then oscillated at a high frequency, and moved to a dis-tance at which the repulsive forces act. Such experimental set-upallows high resolution, non-destructive topography measure-ments. If some additional approximations, e.g. in relation to thehomogeneity of the materials and an inert tip surface are consid-ered, the force versus distance curves can be measured. Sampleelasticity can be calculated by using different theoretical models [9].Such measurements are especially interesting for multicompo-nent coatings, because local variations can be calculated as well[33–35]. A schematic representation of the force modulation modeis shown in Fig. 3a.

1.2.2.5. Phase imaging. In phase mode imaging, the phase shift ofthe cantilever oscillation is compared to the driving signal (Fig. 3b).This difference is shown in a separate image window in most con-ventional AFMs. Different samples exhibit various interactions withthe AFM tip and hence the corresponding phase shift is altered. Thesample properties that are influenced by the interactions with theAFM tip include friction, adhesion and high elasticity. Use of this

Fig. 2. Schematic representation of different AFMmodes. a) the contact mode, b) the non-contact mode, c) and d) lateral force microscopy (LFM), where the frictional forcesappear on the cantilever due to different materials and different surface shapes, respectively. Images a), b), c), and d) were drawn after the explanation in [17] and partiallyreproduced with permission from [10].

98 U. Maver et al. / Trends in Analytical Chemistry 80 (2016) 96–111

mode is sometimes the only way to show differences between localregions on the sample [37,38]. In pharmaceutical sciences, this modeis very useful for the examination of the consistency of the outercoatings of solid pharmaceutical formulations and for exposing cracksand other degradation features [39–41].

1.2.2.6. Amplitude modulation mode or dynamic forcemode. Developed based on the non-contact mode, this mode is oftenalso called the intermittent-contact or tapping mode. It eliminatesthe major weaknesses of the non-contact mode, such as the lowlateral resolution and z-resolution. Instead of hovering above thesample, the cantilever vibrates above the surface andmoves throughthe force gradient above the surface, during which it might mo-mentarily touch the surface [42]. Due to interactions of the AFM tipwith the sample surface, the amplitude of vibrations decreases anda phase shift occurs (Fig. 4a). One of these parameters may be chosen(amplitude or phase shift) and kept constant through the feed-back loop by moving either the sample or the tip in the z-direction.This provides information about the surface topography, similar tothe contact mode. In order to measure in the amplitude modula-tion mode, much stiffer cantilevers are needed, which exhibit thesmallest possible damping factors (this factor is commonly re-ferred to as the Q-factor) [43,44].

Amplitude modulation mode is the most often used AFMmodein biomedical applications due to its high resolution, the nearly non-destructive nature of the imaging, and its applicability in air andalso in liquid conditions [45,46]. Many researchers have used thistechnique to clarify or confirm their results and to show high-resolution images of their samples, which in bio-related sciencesis often the only way to prove their claims [47,48].

For the last couple of years, many researchers have been tryingto explore the material characteristics even further by using the am-plitude modulation mode while introducing multi-frequencymeasurements [49]. Since improving the spatial resolution, data ac-quisition times, and imaging of material properties are perennialgoals in amplitude modulation AFM, multi-frequency measure-ments seem to be very promising for future research in biomedicine[50,51]. At present, the best results were gathered by exciting anddetecting several frequencies of the tip oscillation. These frequen-cies are usually associated with either the higher harmonics of theoscillation or the Eigen modes of the cantilever [52,53]. The mostrecent studies using the mentioned approach include, e.g., themapping of the nanomechanical properties of living cells [54–56].

1.2.2.7. Force spectroscopy. Upon contact with the sample surface,the tip experiences a force, which is monitored as a change in thedeflection of the cantilever [6]. This force is a function of the tip-sample separation and the material properties of the tip and thesample and can be used to investigate the characteristics of thesample, the tip, or the medium in between [57]. The AFM force mea-surement procedure for gathering force curves is schematicallydepicted in Fig. 4b.

In addition to determining interaction forces between the tip anddifferent (mostly model) surfaces, AFM can also produce two-dimensional chemical affinity maps by modifying the cantilever tipwith specific molecules [58]. Such tip modifications enable the char-acterisation of differently responding regions on thematerial surface,resulting in a better understanding and, consequently, applicationof the examined materials [59]. In this way even quantitative data

Fig. 3. Force modulation mode. a) the working principle of the force modulation mode (parts of this image were reproduced with the permission of C. Roduit [36]) and b)a schematic representation of the phase imaging mode (this image was prepared in light of the explanation in [17]).

Fig. 4. Amplitude modulation mode. a) a schematic representation of the amplitude modulation mode and b) a typical force curve. When approaching the surface, the can-tilever is in an equilibrium position (1) and the curve is flat. As the tip approaches the surface (2), the cantilever is pushed up to the surface – being deflected upwards,which is seen as a sharp increase in the measured force (3). Once the tip starts retracting, the deflection starts to decrease and passes its equilibrium position at (4). Movingaway from the surface, the tip snaps in due to interaction with the surface and the cantilever is deflected downwards (5). Once the tip-sample interactions are terminateddue to increased distance, the tip snaps out and returns to its equilibrium position (6). Parts of the images were reproduced from [10].


can be gathered, which can be used to identify the forces involvedin the specific biological systems [60–62].

Mapping the functional groups and examining their interac-tions with different materials is of significant importance forproblems ranging from lubrication and adhesion to the recogni-tion of biological systems and other biomedical applications[15,63–65]. Changing the environmental conditions during themea-surement has also been used extensively to monitor changes in theinteractions among different functional groups and surfaces to sim-ulate the behaviour of the materials upon exposure to a realenvironment [4,59,66].

Nowadays, the ultimate use of AFM is for single-molecule rec-ognition, which can be achieved by applying force spectroscopy.Although the study of single molecules by AFM started several yearsago [67], breakthrough discoveries have only been made recently[68–72].

The recent advancement of AFM force spectroscopy has allowedthe introduction of single molecule force measurements, e.g. dif-ferent biological macromolecules like chitosan, RNA, andmonoclonalantibodies [60,73–76].

2. Examples of AFM use in drug discovery and development

Several reports point out the applicability of AFM and its dif-ferent modes in different areas of pharmaceutical researchbiomedicine and biophysics [12,39,77]. To demonstrate actual proofof the unprecedented features of AFM, which are right at the limitof experimental capabilities, we have structured the novel re-search reports into several sub-sections with regard to the emphasisof the research possibilities.

2.1. Drug discovery

In recent years, numerous research articles regarding drug dis-covery through use of AFM have appeared [12,78,79]. The uniqueability of AFM to provide structural information about moleculesat the single molecule level enables thorough study of theirbehaviour at simulated physiological conditions [79].

2.1.1. The search for novel drug targetsEdwardson and Henderson reviewed the possibilities to use AFM

for the study of novel drug targets, such as proteins (Fig. 5 showsthe possible effect of novel drug candidates on a chosen mem-brane protein) and DNA and the folding of modular proteins usingsingle molecule force spectroscopy [79,80].

AFM-based drug discovery is possible either by testing the in-teractions of drugs with model receptors, or by testing the contactof the novel drug candidate with target cell membranes, which oftenpose an impenetrable barrier for them [81,82]. Such drugs oftenrequire the use of costly delivery systems. Alternatively, novel can-didates can be studied on carefully designed model systems, whichcan indicate the actual potential of the respective drugs prior to costlypre-formulation [83]. Peetla et al. discussed the importance of theuse of differentmodel membranes and the biophysical study of drug-membrane interactions in drug discovery. They mentioned AFM asone of the possible benchmark methods for successful “fishing” forappropriate drug candidates, which need to pass such lipid mem-branes. Additionally, they pointed out the significance of theseinteractions and the interactions of a model membrane withnanocarriers upon the incorporation of potent drugs [84]. In Fig. 6,clear morphological changes on model membranes can be ob-served, following interaction with nanoparticles.

There is no evolution and progress without proper develop-ment and monitoring of the latest technological advancements inany scientific field. The latter is especially true in drug discovery,where several new nano- and micro- technological advancements

are pushing the boundaries of the applicability of new drugs[12,78,85]. The progress in AFM development and the addition ofseveral new modes have already proven the usability of AFM forthe identification and examination of drug candidates and the de-velopment of new delivery technologies [12]. Both are often requiredto transform biological potential into medical reality, thus contrib-uting to the acceleration of the drug discovery and formulationdevelopment process [12].

The classic therapeutic design involves combinatorial chemis-try and system biology-based molecular synthesis, aided by bulkpharmacological assays. To increase the efficiency of therapeuticdiscovery and delivery, one should understand the therapeutic–effector interactions and their cell and tissue responses at themolecular level [12]. Lal et al. reviewed recent advances in the useof multidimensional scanning probe techniques, especially AFMfor drug discovery [86]. Multidimensional AFM introduces simul-taneous measurement at different tip oscillations (often referredto as higher harmonics). Each of these higher harmonics can yielddifferent information with regard to the sample characteristics bywhich the final measurement output becomes even more thor-ough and conclusive [86–88]. This was evident in the research paperof La et al., where multidimensional AFM measurement for defin-ing targets was used for designing therapeutics and monitoringtheir efficacy [12].

2.1.2. Identification of speciesMany diseases evolve as a consequence of unwanted morpho-

logical changes in healthy tissue. The identification of species in thebody causing disease-related symptoms and the response of thehuman body to these changes are crucial parameters for a success-ful struggle against disease or for the development of effective cures.AFM has proven to be one of our best shots in the identification ofdisease-related structural changes [78,89].

Fig. 5. AFM in search for novel drug targets. AFM imaging of human aquaporin-1(AQP1). (a) deglycosylated hAQP1 2D crystals before carboxypeptidase Y treat-ment. (b) after carboxypeptidase Y treatment. (c) The symmetrised correlation averageshows a windmill-like structure of 1.5 nm height on one side and a pronounced de-pression with a windmill-shaped peripheral (1) and central protrusions (2) on theother side. (d) In the symmetrised correlation average of the deglycosylated and de-carboxylated hAQP1, the high windmill-like structure was unchanged. On the lowerside, the central protrusion (2) had disappeared, while the peripheral one (1) ap-peared to be unaffected by the digestion. (e) Differences are evident in this differencemap between the undigested and digested hAQP1 topographs [see the contours in(d)]. (Figure reproduced from [79]).


Legleiter et al. used ex situ AFM to gain insight into the physi-cochemical processes involving antibodymorphology, with importantimplications for the key factors involved in neurodegenerativechanges, as the underlying cause of Alzheimer’s disease [89]. In theirstudy, AFMwas used to study antibody aggregation, considering therole of the antibody epitope specificity of antibodies as potentialinhibitors of fibril formation. Ono et al. reviewed recent develop-ments in the synthesis of molecules that are used to inhibit theformation of α–Synuclein (αS) fibrils as well as the oligomerisationof αS [90]. These molecules were confirmed as small units of proteinaggregates in the brain that constitute histopathological featuresof Parkinson’s disease [91]. Although themechanisms bywhich thesenew compounds inhibit the formation of αS fibrils and destabilisealready formed fibrils are still unclear, they could represent the futureof novel therapeutics for Parkinson’s disease, as well as for otherneurodegenerative diseases exhibiting similar pathophysiologicalchanges in the brain [89,92,93].

2.2. Pharmaceutical technology

The possibility of following different physiological and pharma-cological responses in a simulated environment in real time, whilechanging either the parameters or composition of the liquidmediumused, is one of the most important features AFM can deliver [94,95].Combining the above-mentioned with quantitative measurementof forces between interacting species, resulting in knowledge aboutsample hardness, elasticity, miscibility between components, andthe degree of aggregation, makes AFM a very valuable addition tothe usual set of techniques used in preformulation studies [4,96].

2.2.1. Drug deliveryDue to its non-destructive nature, AFM can be used for re-

search of soft systems under various controlled environmentalconditions. Such studies can serve as a unique foundation for thein vitro development of novel drug delivery systems [39,77,97,98].Tumer et al. report on the main use of AFM and its success in pro-gressive drug delivery development. They focus especially on thepossible aid of AFM in preformulation and formulation studies [97].

One of the still unanswered questions regarding the delivery ofdrugs to and through the skin concerns the particle size limit atwhich the ability to penetrate the stratum cornea ceases [99]. Prowet al. reviewed recent advances in the use of different nano- andmicro- particles for skin drug delivery and also presented some oftheir unpublished data from clinical studies. AFMwas put made thefocus of this review because of its ability to evaluate molecular in-teractions between the particles used and the biological systemsthat are the basis of understanding the efficiency and possible sideeffects of skin delivery formulation [99]. The understanding of in-teractions between nanoparticles and common structures of skin,e.g. furrows, hair follicles, eccrine ducts, etc., is critical to the im-provement of percutaneous drug delivery and in the design of skindrug delivery systems, like lipid nanoparticles, as well as dendrimersthat exhibit the capacity for customised pharmacokinetics [99,100].

2.2.2. Determination of the structural properties of pharmaceuticalformulations

Excipient behaviour after exposure to certain environmental con-ditions during drug production or use is one of the most importantcharacteristics for the pharmaceutical technology and industry[101–103]. The production of novel formulations depends on ob-taining laboratory-scale analyses, which can have a costly outcome,if not conducted properly. Wu et al. used AFM to help identify char-acteristics of two transition points of trechalose, a commonly usedcryo-protectant in lyophilised dosage forms for parenteral appli-cation [104]. Their measurements were able to clearly differentiatebetween the samples’ glass transition and the loss of crystallinity.When combined with thermal analysis, such studies could serve asa perfect platform for testing excipient suitability in the mixturefor the final formulation [104]. Another related research area whereAFM already proved able to explain several final formulation in-compatibilities is the study of material miscibility [103]. Lauer et al.studied the possibility of developing a method for predicting thedrug/polymer miscibility and stability of solid dispersions using amelt-based mixing method. Their method was developed using acombination of Raman spectroscopy and AFM to differentiatebetween homogenously and heterogeneously mixed drug/polymercombinations [103]. Knowledge of miscibility is the basis for thepreparation of stable formulations and the avoidance of subse-quent possible incompatibilities of the final products. Theirexperimental set-up serves as a great starting point for further AFM–based miscibility evaluation.

Metastable pharmaceutical formulations are often used in phar-maceutical technology due to their very useful characteristics. Soliddispersions are one of the more commonly used formulations to

Fig. 6. AFM in search for novel drug targets – different modes. Surface morpholo-gy of the endothelial cell model membrane (EMM) following interaction with NPs.Langmuir–Schaeffer films were transferred onto a silicon substrate following inter-action with NPs for 20 min and the imaging was carried out using AFM in tappingmode in air. (a) EMM alone, (b) EMM following interaction with RNPs, and (c) EMMfollowing interaction with TAT200-RNPs. The EMMwas transferred at the SP 29mN/mfor (a), whereas (b) and (c) were transferred at the SP 31 and 36 mN/m, respective-ly. The corresponding zoom images for (a), (b), (c) are (d), (e), (f). The height scalesfor the images were: a = 3 nm; b, c = 150 nm. (Figure reprinted with permission from[84]. Copyright (2015) American Chemical Society.).


improve the solubility of drugs [105]. The crucial characteristics con-nected with the final solid dispersion behaviour aremostly the resultof the drug crystallite size and the homogeneity of its dispersionthrough the material. AFM proved very successful in both theimaging of different regions of the solid dispersion and in the de-termination of crystallite sizes with a high degree of efficiency. Thecorrelation of the latter with dissolution testing was helpful in pre-dicting the dissolution behaviour of solid dispersions made of drugsand different polyethylene glycols (PEGs) [105]. Pegylated sur-faces have a long history of use in biomedicine. Although they arepart of many drug delivery systems and their general characteris-tics are already known, additional knowledge of the correlationbetween their structure and their behaviour in different circum-stances is still needed to extend their potential even further[106,107]. One of the efforts to expand such knowledge was per-formed by Sant et al. [108]. They confirmed the importance ofpolymer architecture in determining the surface properties of thefinal material or product (Fig. 7) and hence the protein binding andcellular interactions of nanoparticles. AFM was also used to showthe reducedmacrophage uptake of grafted copolymer nanoparticles(NPs) as compared to multiblock copolymer grafted NPs. Al-though the uptakemechanisms involved could not be fully explainedby AFM only, this is a nice indication of how AFM-based studies canbe used to gain further insight into material properties [108].

Amorphous materials, either drugs or excipients, are an inter-esting form of metastable formulation in different biomedicalapplications. Mao et al. developed a general method to induce ametastable or amorphous form of an active pharmaceutical ingre-dient (API) by chemical confinement under high undercoolingconditions [109]. In their research, AFM was used to study the pro-duced film structure and morphology related to the changed formand improved characteristics. The results can be used as the basisfor explaining the aggregation and deposition of Aspirin® on amodelbilayer surface, serving for further development of carrier systemsinvolving the drug and phospholipid bilayers [109]. Using AFM, theauthors proved that the advantage of such structures lies in the in-creased solubility and bioavailability achieved by preparation of ametastable form of API.

Currently, a great deal of interest has been focused on the ap-plicability of several AFM modes in studying specially designedpharmaceutical formulations to address and explore its suitabilityfor improving formulation design [110–112]. Veerapandian and Yunreviewed AFM applications in pharmaceutical and biopharmaceuticalareas with a special emphasis on the investigation of the crystalgrowth, polymorphism, particles, granules, and coating morphol-ogy of the solid dosage form for the optimisation of solid dosageform behaviours [4]. They additionally highlighted in situ

biopharmaceutical investigation of cell-macromolecular surface,drug–drug, drug-cell, drug-DNA, DNA-protein, drug-enzyme, andantigen-antibody interactions, as well as the disease mechanismsfor a better understanding of the physiological mechanisms asso-ciated with disease in order to rationalise drug design anddevelopment [4].

2.2.3. Biophysical examinationThis special area of biomedicine addresses the physical aspects

of the underlying mechanisms among interacting species occur-ring between biological species and medicines.

Liao et al. have used AFM nanoindentation to determine hard-ness, stiffness, and creep in a single measurement for four materialscommonly found in medicine in tablet form [20]. The proposedmethod worked equally well with both soft and hard materials. Asimilar approach was used by Masterson and Cao, who imple-mented AFM nanoindentation to evaluate the hardness of theindividual particles of various pharmaceutical solids includingsucrose, lactose, and ascorbic acid, and the influence of ibuprofenon the measured parameters [96]. Their research was focused es-pecially on the effects of data variation regarding indentation sizeand peak load on hardness. The results showed acceptable repro-ducibility and indicated that data variation may appear primarilyfrom the inhomogeneous nature of the samples, pointing out theimportance of sample preparation [96,113,114].

Zhang et al. determined the contact adhesion forces between anAFM tip and lactose in an environment with low humidity. This tech-nique can identify the submicron heterogeneity of organic solidsin terms of their molecular energy states (such as ordered and dis-ordered lactose) [115]. Another interesting study regarding theexcipient lactose was performed by Bunker et al., who electricallycharged single particles of lactose by contact mode AFM scanningand verified the increased electrical charge by measuring the dif-ference in the long-range electrostatic forces with force spectroscopymeasurements [116]. Such an approach is especially interesting inthe case of the preparation of pharmaceutical formulations requir-ing a mixture of different components to interact with each other,such as in the case of tablets whose final hardness can be signifi-cantly affected by shattered interaction forces between differentconstituents. With regard to formulation testing, AFM can addi-tionally be used as a measure for the determination of segregationprocesses and their extent in different mixtures and conditions[117,118]. Duong et al. performed AFM force vs. distance measure-ments for samples with various amounts of water in the mixture.They tried to determine the correlation between the segregationprocess and moisture-induced cohesion, which often causes inho-mogeneities in pharmaceutical formulations [117].

The adhesive and cohesive forces and their intertwining in amixture of a drug and excipients often result in industrial-scale prob-lems in themanufacturing process. The AFM colloid probe techniquehas emerged as a tool for obtaining a quantitative assessment ofthe cohesive and adhesive forces and was tested to evaluate the co-hesive and adhesive balance within dry powder inhaler formulationscontaining an active ingredient (budesonide, salbutamol sul-phate) and alpha-lactose monohydrate [119]. Begat et al. developedspecial cohesive-adhesive balance (CAB) graphs to allow a directcomparison of the interaction forces occurring in model carrier-based formulations. Their novel approach could be very interestingfor the evaluation of cohesive-adhesive balances in dry powder for-mulations and for a further understanding of powder behaviour forvariousmixtures, which is crucial for structure activity relation (SAR)evaluation on a laboratory or industrial scale [119].

2.2.4. Transport phenomenaThe transport phenomena play an important role in the devel-

opment of drug delivery systems, where the behaviour of the

Fig. 7. Amplitude modulation “tapping” mode AFM images of NPs. The upper panelsshow topography and the lower panels show the corresponding phase images; allimages were acquired in air. Scan size: 250 × 250 (nm × nm); PLA (a), PEG1%-g-PLA(b), PEG5%-g-PLA (c), PLA-PEG-PLA)n (d). (Figure reproduced from [108] with per-mission.).


developedmaterials upon contact with the body fluids plays a majorrole in their efficiency determination. AFM provides the tools forstudying such phenomena with a molecular resolution, while ad-ditionally allowing measurements in specially designed liquidenvironments, simulating physiological ones [77,120]. Burgos et al.studied how adhesion forces were affecting the surface energy gra-dients, resulting in directed single molecule diffusion [121]. Suchinformation and its validity could be a novel basis for the devel-opment of controlled delivery systems, where the complex anddifficult to explain dissolution mechanisms are necessary for suc-cessful therapy [122]. An attempt to use the study of transportphenomena to improve drug delivery systems was made by Peetlaet al., who studied the role of lipids in drug transport. Lipid-related drug transport is especially critical in cancer chemotherapyto overcome drug resistance [123]. Biophysical interaction studieson cell membrane lipids might therefore be helpful in improvingdrug transport and efficacy through drug discovery and/or drug de-livery approaches by overcoming the lipid barrier in resistant cells.AFM contributed greatly to this study as it was the main methodof surface characterisation before and after the interactions oc-curred [123].

One of the most promising uses of force spectroscopy in phar-maceutical physics is its use for interaction mapping amongbiological species (membranes, proteins, DNA, etc.) with novel orexisting pharmaceutical formulations. Jadhav et al. pointed out therole of cell adhesion in diverse biological processes that occur inthe dynamic setting of the vasculature, including inflammation andcancer metastasis [124]. Consequently, elucidating the molecularand biophysical nature of cell adhesion requires a multidisci-plinary approach combining the synthesis and preparation of in vitromodels, with fundamental knowledge of the hydrodynamic flow,molecular kinetics, cell mechanics, and biochemistry [124,125]. Ex-perimental work at the nanoscale to determine the lifetime,interaction distance, and the strain responses of adhesion receptor-ligand bonds has been accelerated by the introduction of AFM,combined with the use of biomolecular force probes [126]. Al-though such research efforts started several years ago, our currentknowledge in this area is still far from complete [124,127]. The pro-gress of such studies is key to the development of mathematicalmodels of cell adhesion that incorporate the appropriate biologi-cal, kinetic, andmechanical parameters leading to reliable qualitativeor even quantitative predictions, which could increase the successrate of diagnostics and fighting diseases [128–130]. Jadhav et al.explain multiscale mathematical models which can be employedto predict optimal drug carrier-cell binding through isolated pa-rameter studies and engineering optimisation schemes, which willbe essential for developing effective drug carriers for the deliveryof therapeutic agents to the afflicted sites of the host [124,131,132].

2.3. Bionanotechnology and nanobiotechnology

One of the main reasons for the staggering progress inbionanotechnology and nanobiotechnology in recent years is the in-troduction of several experimental methods, corroborated bytheoretical considerations, into the study of biological phenome-na [133–136].

Jain explains how several technologies, including the prepara-tion of nanoparticles and nanodevices in the form of nanobiosensorsand nanobiochips, may be used to improve drug discovery and de-velopment [135]. Novel and upgraded nanoscale assays cancontribute not only to more efficient drug discovery, but can alsocontribute significantly to cost-saving in screening campaigns.Looking at the number of research reports in the field of health-related nanotechnology, it is clear that the future prospects for theapplication of nanotechnology in healthcare and for the develop-ment of personalised medicine appear to be promising [135].

With a special interest on new bioassays, based on technolog-ical advancements in recent years, Hong and Root reviewed severalnew research reports regarding the use of single–molecule assaysand evaluated their suitability for drug development. One of the keyadvantages they pointed out was the small amount of sample neededto perform such studies [137]. Biological samples are often expen-sive due to their costly production and cleaning procedures. Indeed,only small amounts of the examined material are needed for thepreparation of AFM samples. In order to avoid unwanted contami-nations of any kind and resulting unusable measurements, strictroutines need to be followed [113].

Kada et al. prepared an exemplary review of recent AFM devel-opment in various biological sciences, where knowledge of thestructure and characteristics at the molecular level can be of utterimportance [136]. Some examples of recent advances regarding thevisualisation of biological macromolecules and the measurementsof molecular recognition forces are shown in Fig. 8. Probably oneof the most impressive additions to the study of biological samplesis the combination of AFMwith fluorescence microscopy [138,139].This might be the future of biological studies at the molecular level.

Bionanotechnology combines nanotechnological advance-ments with the knowledge of the biological world, where biologicalmacromolecules serve different natural and artificial purposes [140].Regarding this, Campolongo et al. reviewed the potential of differ-ent DNA–based structures and templates that can be used in

Fig. 8. High-resolution topographical imaging of biomolecular assemblies. (a) 3 kbp(base pairs) pDNA on a mica surface imaged in Ni2+-containing buffer solution. Scalebar 150 nm. (b) Left: crystalline arrangement of human rhinovirus on a lipid bilayercontaining receptors. Inset: Fourier spectrum and average lattice. Right: dense packingof virus particles with regular patterns of small protrusions ≈0.5 nm high and ≈3 nmin diameter. (c) Topographical image of a purple membrane to which a single an-tibody is bound (left) and a three-dimensional representation (right). (Figurereproduced from [136] with permission.).


nanomedicine [141]. The second rise to fame for DNA (the first onewas its discovery, isolation, and cracking) was certainly the realisationthat its structural characteristics made it a perfect template or evena backbone for the design of several novel and extraordinarynanostructures [142,143]. These new DNA-based structures haveopened a whole new avenue for research towards different poly-meric topologies. Such topologies, in turn, possess uniquecharacteristics that translate into specific therapeutic and diagnos-tic strategies. Study of such phenomena at the nanoscale is onlypossible by introducing AFM–based techniques into research[141,144,145].

2.3.1. Study of disease-related changesDiseases lead to various pathophysiological changes in the body,

whether these are structural or compositional. Either way, they canultimately be used as a marker for the early detection of the un-derlying disease. Therefore researchers often focus their studies onthe identification of such markers. Stolz et al. used the indenta-tion type of force microscopy to monitor age-related morphologicaland biomechanical changes in the hips of normal and osteoar-thritic mice [146]. Changes in the micro- and nano-stiffness wereobserved well before the morphological changes could be de-tected with the currently used diagnostic methods. Such researchis very encouraging for further studies regarding the early detec-tion or even prevention of osteoarthritis [146]. Additionally, it wasproven that AFM could be a valuable addition to the early detec-tion of pathological changes that can be observed in tissue, as inthe case of physical trauma, burns, etc., or expressed in the struc-tural deformations of certain biomolecules in the human body[147,148]. The study and understanding of these changes is of utterimportance for our knowledge of the physiological mechanismsleading to disease-related symptoms. This would not only allowbetter symptom treatment, but would also form the basis for suc-

cessful prevention of the disease overall. Oberhauser et al. used AFMto study the interaction strengths between the different domainsof fibronectin, which is a protein comprising up to 15 type IIIdomains [15,149]. Their research was focused on the assessmentof the possibility of detecting conformational changes with forcespectroscopy. Force pulling experiments showed that the range offorces required to unfold the weakest and strongest domains rangesbetween 80 and 200 pN. Moreover, they studied the interconnec-tion between the domains from which the protein is built. It wasfound that several regions affect the behaviour of the others, indi-cating a far more complex nature of such proteins, comprising a largenumber of domains [149]. AFM proved to be suitable for the studyof these finemechanical changes appearing in the quaternary proteinstructure. Such single molecule studies could hold the key to earlydetection of conformational changes leading to neurodegenerativediseases, such as Parkinson’s or Alzheimer’s disease [150–152].

Due to its versatile functions in the body and the relative com-plexity of its structure, fibronectin is an interesting protein forresearch [153–155]. Although its main functions are connectedmostly with cell adhesion, growth, migration, and differentiation,errors in its expression can lead to severe complications and evencancer [156,157]. Hill et al. reviewed the use of AFM for imagingof the cell surface and the simultaneous measurement of ligand-receptor interactions on the cell surface, in order to assess fibronectinbehaviour [158].

It was found that AFM not only enables the detection of cell mor-phology and stiffness, but additionally allows the application ofdiscrete forces to single smooth muscle cells and the observationof subsequent responses, which can serve as a basis for study ofcomplex mechanisms involved in the arteriolar myogenic re-sponse (Fig. 9) [158]. The introduction of controlled and localisedforces with AFM seems to be a valuable tool for detecting disease-related changes in the response of tissue. These changes, either in

Fig. 9. Example modes of AFM operation. (a) (i) A native, or non-functionalised, AFM probe enables the collection of detailed topographic information from viable cells. (ii)A detail of the underlying cytoskeleton. (iii) A calculated topography map showing the relative height of the cell. Such images are usually achieved in the contact or tappingmode operation of AFM, where the probe is controlled to scan across the cell surface with a preset contact force (ii). (b) (i) AFM probes functionalised were with ligandmolecules to enable the detection of individual ligand–receptor interactions on the cell surface. (ii) The probe is then lifted from the cell surface to break the ligand–receptor bonds physically. (iii) The data obtained can be used to resolve the characteristic binding force of a specific ligand–receptor interaction. (c) (i) The tips of the AFMprobes can be replaced by microspheres. (ii) Bringing the fibronectin–coated microspheres into contact with the vascular smooth muscle cell (VSMC) surface induces theformation of focal-adhesion-like structures. The AFM probe can then be controlled to apply either pulling or pushing forces. (iii) As the contact time continues, increasingforce is required to detach FN-coated microspheres from the VSMC surface. (Figure reproduced from [158] with permission.).


the protein structure or in the environmentally induced behaviouralchanges of other species, can be used as an indication of an occur-ring disease [159,160]. Various techniques have been used in recentyears to characterise the protein misfolding that facilitates the ag-gregation process, leading to several diseases [161–163]. Additionally,no real emphasis has been placed on the relation between the con-formational transition and the increase in the intermolecularinteractions between the constituents of the misfolded proteins.McAllister et al. applied AFM to follow the interaction betweenprotein molecules as a function of pH [164]. They performed in-teraction mappings for three unrelated and structurally distinctproteins, a-synuclein, amyloid b-peptide (Ab), and lysozyme. It wasshown that the attractive force between homologous protein mol-ecules is minimal at physiological pH and increases dramatically atacidic pH. Moreover, they found that the dependence of the pullingforces is sharp, suggesting a pH-dependent conformational transi-tion within the protein. In addition, protein self-assembly intofilamentous aggregates studied by AFM imaging was shown to befacilitated at pH values corresponding to the maximum of pullingforces [164]. This work is fundamental for studying the relationbetween conformational changes in single protein structures in re-lation to the disease that they cause. There are also other studiesrelating pH to changes in the behaviour of macromolecules that in-dicate the potential AFM can have in similar research [165–170].

Several methods for the detection of microbial pathogens arecommonly known and have been used for decades. Although dif-ferent improvements have been introduced to these methods, noveltechnological advancements led to another upsurge, some wouldeven say revolution, of methods for the early detection of patho-gens [171,172]. Kaittanis et al. reviewed some of the possiblenanoarrays that use AFM-based methods as the sole method of de-tecting microbial pathogens [173]. Another potential use of AFM inthe detection of pathogens is the use of specifically functionalisedAFM tips to detect possible infection even on samples of living tissue[174].

2.3.2. Receptor-ligand interaction studiesAlthough the receptor-ligand binding kinetics have been thor-

oughly studiedwith differentmathematical models, the experimentalwork is increasingly often performed using different variations offorce spectroscopy measurements and combinations of the men-tioned approaches [175–177]. Such efforts are a basis forunderstanding the underlying phenomena of histopathological tissuechanges in disease, either for their early detection or for the de-velopment of novel therapies. Selectins have often been studied byAFM-based measurements in recent years [178–180]. They have at-tracted much interest in the biomedical community due to theirinvolvement in inflammation and their role in the “rolling” actionattributed to leukocytes during the leukocyte adhesion cascade. Thelatter is the subject of thorough research on nanodevices, increas-ing the “rolling” of cancer cells away from the tumour [181–184].Several research efforts elucidating the mechanisms of this rollingtype of interaction have been performed over the last decade[185–187]. Although different strategies have been used to studythese interactions (single molecule characterisation [131], the ki-netics of their interactions with leukocytes [188], and cell adhesionforces [128]), all of them have in common the search for interaction-based disease-related changes by AFM force measurements.

2.3.3. Single molecule measurementsProbably the most advanced study with AFM is the examina-

tion of single molecule behaviour in its natural environment[189–192]. Huber and Sakmar reviewed recent advances in molec-ular and structural studies of G protein-coupled receptors, whichare some of the most studied receptors in recent years due to theirinvolvement in many biological functions [176,193]. The mecha-

nisms of their activity with regard to different circumstances on themolecular scale is still somewhat blurry, which is why AFM has oftenbeen employed to study the conformational, structural, and posi-tional changes of these seven-trans-membrane domain receptors.There has been increased interest in the use of novel molecular tech-niques to study the behaviour of these proteins in their biologicalenvironments while exposed to different species from their signal-ling machineries [193,194]. The latter shows how biochemicalresearch is becoming increasingly exact and quantitative [193]. Bydecreasing the gap between molecular and systems biology, suchefforts suggest a way forward from the flatlands tomulti-dimensionalquantitative data collection and an understanding of biologicalsystems [193]. Single molecule AFM measurements can mainly bedivided into studies of different interacting species (in either realor model systems), and the identification of novel molecules in theirbiological environment without damaging their native structure [66].Dupres et al. addressed crucial challenges in modern cell biologythat are mostly based on understanding how cell-surface mol-ecules are organised and how these characteristics change due tothe alternating conditions in the environment [195–197].

Information regarding these two issues is central to our under-standing of cell adhesion and pathogen interactions. The past yearshave witnessed rapid progress in the use of AFM to map the dis-tribution of single polysaccharides and proteins on live cells and tomeasure their molecular interactions (Fig. 10) [194,195]. Both typesof measurement are attracting many researchers due to their im-portance in gaining further insight into the behaviour and responseof biological molecules to environmental changes [196,197].

The astonishing power of AFM with regard to single moleculemeasurements was recently shown by Frei et al. [198,199]. They usedAFM to measure Au-N rupture forces using AFM in force spectros-copy mode together with conductance measurements in variousamines. It was found that bond rupture force depends on the mo-lecular backbone, which is in accordance with density functionaltheory-based adiabatic molecular junction elongation and rupturecalculations [198,199] (Fig. 11). Such an approach not only showsthat several additional measurements can be implemented in AFMby using force spectroscopy mode measurement to simultane-ously gain additional insight into material/molecule characteristics,but it also indicates that novel research should search for answersby combining ideas from different fields of research.

2.4. Interaction mapping

One of the well-appreciated contributions of AFM to science inthe last decade is its ability to probe the interaction forces betweendifferent molecules, particles, or surfaces [200–203]. The limita-tion of this approach is the possibility to simulate or replicate theactual environment in which themeasured interactions play a crucialrole and therefore have a huge impact on the final product or onthe desired information. Recently, high yield intracellular deliveryhas been one of the main targets of novel research in relation totargeted drug delivery. Therefore, many research groups have soughtnovel and more sophisticated ways to deliver drugs intracellularly[204]. Because this is a complex task, a great deal of interest hasbeen focused on the study of interactions of the cell wall with dif-ferent existing and newly developed particles or other deliverysystems [204]. AFM is able to evaluate cell morphology and stiff-ness [205–207]. Additionally, it enables discrete forces to be appliedto single cells and their constituents, while the responses are ob-served [158,208]. It can be concluded that AFM has become aubiquitous tool to image nanoscale structures and to estimate certainmechanical characteristics of biological entities, ranging from DNAto tissues [126,209–211]. Van Vliet and Hinterdorfer reviewed recentadvances in in situ investigations of drug-induced changes in cellstructure, membrane stability, and receptor interaction forces [212].


They reviewed the first attempts at AFM use in the progressiveoptimisation of delivery systems by testing them together with theirtargeted physiological structures (Fig. 12). They suggest that al-though further efforts are needed to grasp all of the possibilities AFMintroduces to research, a large portion of the initial problems have

already been eliminated, thus allowing for world-changing re-search [212].

Quantitative interaction mappings between species interactingin real systems form the basis for an understanding of their ap-pearance [200,213,214]. Allison et al. have demonstrated that AFM

Fig. 10. AFM in single molecule measurements. Mapping fibronectin attachment proteins (FAPs) on Mycobacterium bovis bacillus Calmette-Guérin (BCG). (a) A schematicrepresentation of the experimental set-up, showing a fibronectin-modified AFM tip and the mycobacterial cell wall. (b) Low- (inset; 2 μm × 2 μm) and high-resolution de-flection images of a living M. bovis BCG cell. (c) Adhesion force map (gray scale: 200 pN), and (d) adhesion force histogram with representative force curves, recorded onthe cell surface using a fibronectin-tip. (Figure reproduced from [195] with permission.).

Fig. 11. AFM in measuring rupture forces of the Au-N bond. (A) Calculated total energy curves from adiabatic trajectories for 1,4-benzenediamine (red), 1,4-butanediamine(blue), and 4,4′-bipyridine (green) shown as a function of displacement. The bars shown at right indicate the asymptotic values. (B) Calculated applied force curves for thesame molecules shown against displacement display the same trend as the experimental results. (C) Junction structure showing a 1,4–benzenediamine junction at 0.1 nmelongation relative to the local energy minimum. (D) Zoom in of the Au—N bond indicating bond angle with respect to pulling direction. (E) Sample structure used to in-vestigate a single Au—N bond force profile for 1,4-benzenediamine bound to a gold electrode with the bond aligned to the pulling direction. (Figure reprinted with permissionfrom [199]. Copyright (2015) American Chemical Society.).


can be used to measure forces between adenine-coated tips andthymine-coated surfaces in order to develop the methodology tostudy the required forces for unfolding immunoglobulin [215]. Inaddition, they scanned albumin surfaces using an AFM tip with at-tached anti-human serum albumin via a polyethylene glycol linker.With the mentioned experimental set-up, they proved the exis-tence of recognition between the antihuman serum albumin andserum albumin due to their interactions (Fig. 13). When lysosomewas bound on the antibody, the recognition disappeared again, whichwas clearly shown by means of AFM [215]. Several other studiesbase their interaction mappings upon using AFM tips with at-tached species of interest on one side and the other counterpart ofthe interacting pair on the other [216–218].

2.5. Combinations of AFM with other methods

In recent years, there has been a tendency to combine the in-formation that can be gathered with an AFM set-up with otherknown techniques, such as infrared spectroscopy (IR), nuclear mag-netic resonance (NMR), thermal analysis (TGA), mass spectrometry(MS) and others [219–223]. Such combinations allow the assess-ment of additional information regarding locally confined propertiesof nanomaterials. Several research groups are making additions tothe basic commercially available AFMs due to research specific mea-surements, requiring nonstandard components.

An interesting example is the use of AFM in determination ofsurface free energy, an important parameter in the calculation ofinterstitial phenomena. A great deal of effort has been put into theexperimental assessment of this parameter, which could be com-bined with theoretical considerations [115,224]. AFM wassuccessfully used in combination with a contact angle–based tech-nique for the determination of surface free energy by Traini et al.[225–227].

Probably the most interesting recent evolution of AFM is the in-troduction of very fast scanning techniques, which allowed a realtime visualisation of a walking myosin molecule and of mem-brane proteinmotion [192,228,229], as well as visualising living cellsin nanoresolution [230–232].

3. Conclusions

The present review addresses the basic concepts of AFM mea-surements and the recent progress of AFM use in relation tobiomedical applications. The technical aspects are summarised ina compact form, while theoretical considerations, which are not nec-essary for an understanding thereof, are omitted. The increasingnumber of research papers using AFM as a crucial part in the studyof different phenomena indicates intensified scientific awarenessof its capabilities.

As shown, AFM enables a vast range of research options. Its ex-emplary resolution and ability to measure forces in the pN rangemake it a valuable tool for analysis in relation to biomedicalapplications.

Fig. 12. Characterisation of drug and gene delivery vehicles and mechanisms en-hances vehicle development. (a) contact-mode error signal image of lipoplex, aliposome containing DNA (DNA image, inset). Confocal optical microscopy of cellstransfected with these lipoplexes for 1 h shows that the lipoplexes are constrainednear the cell membrane in the presence of endocytosis inhibitors (b), whereas cellstransfected in the presence of membrane fusion inhibitors do not show suchlocalisation (c). Scale bars in (b) and (c) =10 μm. Inset scale bar = 200 nm. (Figurereproduced from [212] with permission.).

Fig. 13. A time course of events to illustrate force-recognition imaging. (a) An antibody to lysozyme tethered to the AFM tip is scanned in a liquid environment over a surfaceof immobilised lysozyme molecules. In this experiment, a low concentration of lysozyme is added to the liquid system. In I, the antibody on the tip has not bound freelysozyme and is reacting with surface-bound lysozyme molecules. This is clearly seen in (b) and part of (c), where recognition imaging is evident by the distortion of theimages. In II, at the black arrow, the antibody on the tip has bound solution lysozyme and the image in part of (c) and all of (d) switches from recognition to topographicimaging. (Figure reproduced from [215] with permission.).


The amount of newly published work with AFM regarding noveldrug discoveries, their delivery, or their relation to the tissue theytry to mend is astonishing and heralds a bright future for AFM-based research in this area.

Acknowledgements

The authors would like to acknowledge the financial support forthis project received from the Slovenian Research Agency (grantnumbers: Z1-6737 and J2-6760).

References

[1] P. Armitage, G. Berry, J.N.S. Matthews, Statistical Methods in Medical Research,fourth ed., Blackwell Science, Malden, MA, 2001.

[2] X. Zhou, D.K. McClish, N.A. Obuchowski, Statistical Methods in DiagnosticMedicine, second ed., Wiley, Hoboken, N.J., 2011.

[3] C. European Pharmacopoeia, E. Council of European pharmacopoeia, Councilof Europe, Strasbourg, 2014.

[4] M. Veerapandian, K. Yun, Study of atomic force microscopy in pharmaceuticaland biopharmaceutical interactions – a mini review, Curr. Pharmaceut. Anal.5 (2009) 256–268.

[5] H. van deWaterbeemd, D.A. Smith, K. Beaumont, D.K. Walker, Property-baseddesign: optimization of drug absorption and pharmacokinetics, J. Med. Chem.44 (2001) 1313–1333.

[6] G. Binnig, C.F. Quate, C. Gerber, Atomic force microscope, Phys. Rev. Lett. 56(1986) 930.

[7] G. Binnig, H. Rohrer, C. Gerber, E. Weibel, Surface studies by scanning tunnelingmicroscopy, Phys. Rev. Lett. 49 (1982) 57.

[8] S.S. Wong, A.T. Woolley, T.W. Odom, J.L. Huang, P. Kim, D.V. Vezenov, et al.,Single-walled carbon nanotube probes for high-resolution nanostructureimaging, Appl. Phys. Lett. 73 (1998) 3465–3467.

[9] J.N. Israelachvili, Intermolecular and Surface Forces, Academic Press, 2010.[10] U. Maver, A. Vesel, K. Stana-Kleinschek, M. Gaberscek, M. Mozetic, T. Maver,

et al., Polymer Characterization with the Atomic Force Microscope, INTECHOpen Access Publisher, 2013.

[11] S.H. Cohen, M.T. Bray, M.L. Lightbody, Atomic Force Microscopy/ScanningTunneling Microscopy, Plenum Press, New York, 1994.

[12] R. Lal, M.F. Arnsdorf, Multidimensional atomic force microscopy for drugdiscovery: a versatile tool for defining targets, designing therapeutics andmonitoring their efficacy, Life Sci. 86 (2010) 545–562.

[13] B. Shah, V.K. Kakumanu, A.K. Bansal, Analytical techniques for quantificationof amorphous/crystalline phases in pharmaceutical solids, J. Pharm. Sci. 95(2006) 1641–1665.

[14] T. Uchihashi, M.J. Higgins, S. Yasuda, S.P. Jarvis, S. Akita, Y. Nakayama, et al.,Quantitative force measurements in liquid using frequency modulation atomicforce microscopy, Appl. Phys. Lett. 85 (2004) 3575–3577.

[15] A.F. Oberhauser, Single-Molecule Studies of Proteins, Springer, New York, 2013.[16] E.J.G. Peterman, G.J.L. Wuite, Single Molecule Analysis: Methods and Protocols,

Humana Press, New York, 2011.[17] S.H. Cohen, M.L. Lightbody, Foundation for Advances in Medicine and Science.,

Atomic ForceMicroscopy/Scanning TunnelingMicroscopy 3, Kluwer Academic/Plenum Publishers, New York, 1999.

[18] B. Bhushan, D. Luo, S.R. Schricker, W. Sigmund, S. Zauscher, Handbook ofNanomaterials Properties, Springer Verlag, Heidelberg, 2014.

[19] H. Fuchs, B. Bhushan, Biosystems Investigated by Scanning Probe Microscopy,Springer, Heidelberg; New York, 2010.

[20] X.M. Liao, T.S. Wiedmann, Characterization of pharmaceutical solids byscanning probe microscopy, J. Pharm. Sci. 93 (2004) 2250–2258.

[21] T. De, A.M. Chettoor, P. Agarwal, M.V. Salapaka, S. Nettikadan, Immobilizationmethod of yeast cells for intermittent contact mode imaging using the atomicforce microscope, Ultramicroscopy 110 (2010) 254–258.

[22] C. Le Grimellec, E. Lesniewska, M.-C. Giocondi, E. Finot, V. Vié, J.-P. Goudonnet,Imaging of the surface of living cells by low-force contact-mode atomic forcemicroscopy, Biophys. J. 75 (1998) 695–703.

[23] H. Yamashita, K. Inoue, M. Shibata, T. Uchihashi, J. Sasaki, H. Kandori, et al.,Role of trimer-trimer interaction of bacteriorhodopsin studied by opticalspectroscopy and high-speed atomic force microscopy, J. Struct. Biol. 184(2013) 2–11.

[24] T. Ando, T. Uchihashi, T. Fukuma, High-speed atomic force microscopy fornano-visualization of dynamic biomolecular processes, Prog. Surf. Sci. 83(2008) 337–437.

[25] S. Morita, R. Wiesendanger, E. Meyer, F.J. Giessibl, Noncontact Atomic ForceMicroscopy, Springer, Berlin; New York, 2002.

[26] J. Seifert, J. Rheinlaender, P. Novak, Y.E. Korchev, T.E. Schaffer, Comparison ofatomic force microscopy and scanning ion conductance microscopy for livecell imaging, Langmuir 31 (2015) 6807–6813.

[27] F. Braet, E. Wisse, AFM imaging of fenestrated liver sinusoidal endothelial cells,Micron 43 (2012) 1252–1258.

[28] K. Sugihara, J. Kumaki, Visualization of two-dimensional single chainconformations solubilized in a miscible polymer blend monolayer by atomicforce microscopy, J. Phys. Chem. B 116 (2012) 6561–6568.

[29] A.E. Cherian, F.C. Sun, S.S. Sheiko, G.W. Coates, Formation of nanoparticles byintramolecular cross-linking: following the reaction progress of single polymerchains by atomic forcemicroscopy, J. Am. Chem. Soc. 129 (2007) 11350–11351.

[30] C.-S. Ha, J.A. Gardella, Surface chemistry of biodegradable polymers for drugdelivery systems, Chem. Rev. 105 (2005) 4205–4232.

[31] A.J. Kulik, M. Lekka, K. Lee, G. Pyka-Fosciak, W. Nowak, Probing fibronectin-antibody interactions using AFM force spectroscopy and lateral forcemicroscopy, Beilstein J. Nanotechnol. 6 (2015) 1164–1175.

[32] A.J. Weymouth, T. Hofmann, F.J. Giessibl, Quantifying molecular stiffness andinteraction with lateral force microscopy, Science 343 (2014) 1120–1122.

[33] B. Codan, G. Del Favero, V. Martinelli, C.S. Long, L. Mestroni, O. Sbaizero,Exploring the elasticity and adhesion behavior of cardiac fibroblasts by atomicforce microscopy indentation, Mater. Sci. Eng. C Mater. Biol. Appl. 40 (2014)427–434.

[34] Y. Luo, M. Buchsenschutz-Gobeler, W. Arnold, K. Samwer, Local elasticity andmobility of twin boundaries in martensitic films studied by atomic forceacoustic microscopy, New J. Phys. 16 (2014).

[35] A.M. Jakob, J. Buchwald, B. Rauschenbach, S.G. Mayr, Nanoscale-resolvedelasticity: contact mechanics for quantitative contact resonance atomic forcemicroscopy, Nanoscale 6 (2014) 6898–6910.

[36] C. Roduit, AFM figures. <http://www.freesbi.ch/fr/illustration/figures>, CreativeCommons Attribution, 2010.

[37] D.V. Vezenov, A.V. Zhuk, G.M. Whitesides, C.M. Lieber, Chemical forcespectroscopy in heterogeneous systems: intermolecular interactions involvingepoxy polymer, mixed monolayers, and polar solvents, J. Am. Chem. Soc. 124(2002) 10578–10588.

[38] A. Noy, C.H. Sanders, D.V. Vezenov, S.S. Wong, C.M. Lieber, Chemically-sensitiveimaging in tapping mode by chemical force microscopy: relationship betweenphase lag and adhesion, Langmuir 14 (1998) 1508–1511.

[39] J. Sitterberg, A. Ozcetin, C. Ehrhardt, U. Bakowsky, Utilising atomic forcemicroscopy for the characterisation of nanoscale drug delivery systems, Eur.J. Pharm. Biopharm. 74 (2010) 2–13.

[40] Z. Ye, X. Zhao, Phase imaging atomic force microscopy in the characterizationof biomaterials, J. Microsc. 238 (2010) 27–35.

[41] D. Platz, E.A. Tholen, C. Hutter, A.C. von Bieren, D.B. Haviland, Phase imagingwith intermodulation atomic force microscopy, Ultramicroscopy 110 (2010)573–577.

[42] D. Parrat, F. Sommer, J.M. Solleti, T.M. Due, Imaging modes in atomic-forcemicroscopy, J. Trace Microprobe Techn. 13 (1995) 343–352.

[43] G. Haugstad, Atomic Force Microscopy: Understanding Basic Modes andAdvanced Applications, John Wiley & Sons, Hoboken, N.J., 2012.

[44] C. Basdogan, A. Varol, I. Gunev, B. Orun, Numerical simulation of nano scanningin intermittent-contact mode AFM under Q control, Nanotechnology 19 (2008).

[45] F. Liu, C. Zhao, F. Mugele, D. Ende, Amplitude modulation atomic forcemicroscopy, is acoustic driving in liquid quantitatively reliable?,Nanotechnology 26 (2015) 385703.

[46] X. Hu, P. Egberts, Y. Dong, A. Martini, Molecular dynamics simulation of am-plitudemodulationatomic forcemicroscopy,Nanotechnology26 (2015)235705.

[47] D. Ebeling, S.D. Solares, Amplitude modulation dynamic force microscopyimaging in liquids with atomic resolution: comparison of phase contrasts insingle and dual mode operation, Nanotechnology 24 (2013) 135702.

[48] S. Santos, N.H. Thomson, High resolution imaging of immunoglobulin Gantibodies and other biomolecules using amplitude modulation atomic forcemicroscopy in air, Methods Mol. Biol. 736 (2011) 61–79.

[49] S.D. Solares, S. An, C.J. Long, Multi-frequency tapping-mode atomic forcemicroscopy beyond three eigenmodes in ambient air, Beilstein J. Nanotechnol.5 (2014) 1637–1648.

[50] S. Santos, V. Barcons, J. Font, A. Verdaguer, Unlocking higher harmonics inatomic force microscopy with gentle interactions, Beilstein J. Nanotechnol. 5(2014) 268–277.

[51] J.Q. Qian, Y. Li, Y.Z. Li, Theory of higher harmonics imaging in tapping-modeatomic force microscopy, Chin. Phys. B 19 (2010).

[52] O. Sahin, C.F. Quate, O. Solgaard, A. Atalar, Resonant harmonic response intapping-mode atomic force microscopy, Phys. Rev. B 69 (2004).

[53] O. Sahin, A. Atalar, Simulation of higher harmonics generation in tapping-modeatomic force microscopy, Appl. Phys. Lett. 79 (2001) 4455–4457.

[54] R. Garcia, E.T. Herruzo, The emergence of multifrequency force microscopy,Nat. Nanotechnol. 7 (2012) 217–226.

[55] A. Raman, S. Trigueros, A. Cartagena, A.P.Z. Stevenson, M. Susilo, E. Nauman,et al., Mapping nanomechanical properties of live cells using multi-harmonicatomic force microscopy, Nat. Nanotechnol. 6 (2011) 809–814.

[56] F. Kienberger, A. Dulebo, J. Preiner, G. Kada, C. Rankl, L. Chtcheglova, et al.,Second harmonic atomic force microscopy imaging of live and fixedmammalian cells, Ultramicroscopy 109 (2009) 1056–1060.

[57] H.-J. Butt, B. Cappella, M. Kappl, Force measurements with the atomic forcemicroscope: technique, interpretation and applications, Surf. Sci. Rep. 59 (2005)1–152.

[58] A. Ebner, L. Wildling, R. Zhu, C. Rankl, T. Haselgrubler, P. Hinterdorfer, et al.,Functionalization of probe tips and supports for single-molecule recognitionforce microscopy, Top. Curr. Chem. 285 (2008) 29–76.

[59] F. Kienberger, A. Ebner, H.J. Gruber, P. Hinterdorfer, Molecular recognitionimaging and force spectroscopy of single biomolecules, Acc. Chem. Res. 39(2006) 29–36.

[60] M. Kocun, M. Grandbois, L.A. Cuccia, Single molecule atomic force microscopyand force spectroscopy of chitosan, Colloids Surf. B. Biointerfaces 82 (2011)470–476.


http://refhub.elsevier.com/S0165-9936(15)30206-5/sr0010

























































































http://www.freesbi.ch/fr/illustration/figures

































































[61] P.R. Selvin, T. Ha, Single-Molecule Techniques: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2008.

[62] M.I. Giannotti, M. Rinaudo, G.J. Vancso, Force spectroscopy of hyaluronan byatomic forcemicroscopy: from hydrogen-bonded networks toward single-chainbehavior, Biomacromolecules 8 (2007) 2648–2652.

[63] D.J. Taatjes, J. Roth, Cell imaging Techniques: Methods and Protocols, 2nd ed.,Humana Press, New York, 2013.

[64] A. Noy, C.D. Frisbie, L.F. Rozsnyai, M.S. Wrighton, C.M. Lieber, Chemical forcemicroscopy – exploiting chemically-modified tips to quantify adhesion, friction,and functional-group distributions in molecular assemblies, J. Am. Chem. Soc.117 (1995) 7943–7951.

[65] C.D. Frisbie, L.F. Rozsnyai, A. Noy, M.S. Wrighton, C.M. Lieber, Functional-groupimaging by chemical force microscopy, Science 265 (1994) 2071–2074.

[66] A. Mendez-Vilas, L. Labajos-Broncano, J. Perera-Nunez, M.L. Gonzalez-Martin,Are the soft, liquid-like structures detected around bacteria by ambientdynamic atomic force microscopy capsules?, Appl. Environ. Microbiol. 77(2011) 3102–3114.

[67] H. Clausen-Schaumann, M. Seitz, R. Krautbauer, H.E. Gaub, Force spectroscopywith single bio-molecules, Curr. Opin. Chem. Biol. 4 (2000) 524–530.

[68] D. Dulin, B.A. Berghuis, M. Depken, N.H. Dekker, Untangling reactionpathways through modern approaches to high-throughput single-moleculeforce-spectroscopy experiments, Curr. Opin. Struct. Biol. 34 (2015) 116–122.

[69] N.Wang, H. Liu, J. Hao, X. Bai, H. Li, Z. Zhang, et al., Single molecular recognitionforce spectroscopy study of a DNA aptamer with the target epithelial celladhesion molecule, Analyst 140 (2015) 6226–6229.

[70] S. Maity, M. Mazzolini, M. Arcangeletti, A. Valbuena, P. Fabris, M. Lazzarino,et al., Conformational rearrangements in the transmembrane domain of CNGA1channels revealed by single-molecule force spectroscopy, Nat. Commun. 6(2015) 7093.

[71] S. Casalini, A.C. Dumitru, F. Leonardi, C.A. Bortolotti, E.T. Herruzo, A. Campana,et al., Multiscale sensing of antibody-antigen interactions by organic transistorsand single-molecule force spectroscopy, ACS Nano 9 (2015) 5051–5062.

[72] O.E. Farrance, E. Paci, S.E. Radford, D.J. Brockwell, Extraction of accuratebiomolecular parameters from single-molecule force spectroscopyexperiments, ACS Nano 9 (2015) 1315–1324.

[73] S.J. Attwood, A.M. Simpson, R. Stone, S.W. Hamaia, D. Roy, R.W. Farndale, et al.,A simple bioconjugate attachment protocol for use in single molecule forcespectroscopy experiments based on mixed self-assembled monolayers, Int.J. Mol. Sci. 13 (2012) 13521–13541.

[74] M.E. Drew, A. Chworos, E. Oroudjev, H. Hansma, Y. Yamakoshi, A tripodmolecular tip for single molecule ligand-receptor force spectroscopy by AFM,Langmuir 26 (2010) 7117–7125.

[75] H.A. Heus, E.M. Puchner, A.J. van Vugt-Jonker, J.L. Zimmermann, H.E. Gau,Atomic force microscope-based single-molecule force spectroscopy of RNAunfolding, Anal. Biochem. 414 (2011) 1–6.

[76] M.P. Nicholas, L. Rao, A. Gennerich, Covalent immobilization of microtubuleson glass surfaces for molecular motor force measurements and other single-molecule assays, Methods Mol. Biol. 1136 (2014) 137–169.

[77] Y.T.A. Tumer, C.J. Roberts, M.C. Davies, Scanning probe microscopy in the fieldof drug delivery, Adv. Drug Deliv. Rev. 59 (2007) 1453–1473.

[78] J. Legleiter, T. Kowalewski, Tapping, pulling, probing: atomic force microscopyin drug discovery, Drug Discov. Today Technol. 1 (2004) 163–169.

[79] J.M. Edwardson, R.M. Henderson, Atomic force microscopy and drug discovery,Drug Discov. Today 9 (2004) 64–71.

[80] C. Albrecht, K. Blank, M. Lalic-Multhaler, S. Hirler, T. Mai, I. Gilbert, et al., DNA:a programmable force sensor, Science 301 (2003) 367–370.

[81] L. Li, R. Liu, F. Xu, Y. Zu, Z. Liu, Study on the interactions between anti-cancerdrug oxaliplatin and DNA by atomic force microscopy, Micron 76 (2015) 46–51.

[82] M. Iijima, M. Yoshimura, T. Tsuchiya, M. Tsukada, H. Ichikawa, Y. Fukumori,et al., Direct measurement of interactions between stimulation-responsive drugdelivery vehicles and artificial mucin layers by colloid probe atomic forcemicroscopy, Langmuir 24 (2008) 3987–3992.

[83] C. Peetla, A. Stine, V. Labhasetwar, Biophysical interactions with model lipidmembranes: applications in drug discovery and drug delivery, Mol. Pharm.6 (2009) 1264–1276.

[84] C. Peetla, K.S. Rao, V. Labhasetwar, Relevance of biophysical interactions ofnanoparticles with a model membrane in predicting cellular uptake: studywith TAT peptide-conjugated nanoparticles, Mol. Pharm. 6 (2009) 1311–1320.

[85] D.A. LaVan, D.M. Lynn, R. Langer, Moving smaller in drug discovery anddelivery, Nat. Rev. Drug Discov. 1 (2002) 77–84.

[86] A.M.Whited, P.S.H. Park, Atomic force microscopy: a multifaceted tool to studymembrane proteins and their interactions with ligands, Biochim. Biophys. Acta2014 (1838) 56–68.

[87] M. Li, L. Liu, N. Xi, Y. Wang, Nanoscale monitoring of drug actions on cellmembrane using atomic force microscopy, Acta Pharmacol. Sin. 36 (2015)769–782.

[88] F.J. Giessibil, Higher-harmonic atomic force microscopy, Surf. Interface Anal.38 (2006) 1696–1701.

[89] J. Legleiter, D.L. Czilli, B. Gitter, R.B. DeMattos, D.M. Holtzman, T. Kowalewski,Effect of different anti-A beta antibodies on A beta fibrillogenesis as assessedby atomic force microscopy, J. Mol. Biol. 335 (2004) 997–1006.

[90] K. Ono, M. Hirohata, M. Yamada, α-synuclein assembly as a therapeutic targetof Parkinson’s disease and related disorders, Curr. Pharm. Des. 14 (2008)3247–3266.

[91] K. Ono, M. Hirohata, M. Yamada, alpha-synuclein assembly as a therapeutictarget of parkinson’s disease and related disorders, Curr. Pharm. Des. 14 (2008)3247–3266.

[92] A. S Mostaert, R. Crockett, G. Kearn, I. Cherny, E. Gazit, L. C Serpell, et al.,Mechanically functional amyloid fibrils in the adhesive of amarine invertebrateas revealed by Raman spectroscopy and atomic force microscopy, Arch. Histol.Cytol. 72 (2009) 199–207.

[93] S.P. Jarvis, T. Fukuma, A.S. Mostaert, L.C. Serpell, Revealing molecular-levelsurface structure of amyloid fibrils in liquid bymeans of frequencymodulationatomic force microscopy, Nanotechnology 19 (2008).

[94] P.J. Thurner, Atomic force microscopy and indentation force measurement ofbone, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1 (2009) 624–649.

[95] S.M. Akrami, H. Nakayachi, T. Watanabe-Nakayama, H. Asakawa, T. Fukuma,Significant improvements in stability and reproducibility of atomic-scaleatomic force microscopy in liquid, Nanotechnology 25 (2014) 455701.

[96] V.M. Masterson, X.P. Cao, Evaluating particle hardness of pharmaceutical solidsusing AFM nanoindentation, Int. J. Pharmaceut. 362 (2008) 163–171.

[97] C. Lamprecht, P. Hinterdorfer, A. Ebner, Applications of biosensing atomic forcemicroscopy in monitoring drug and nanoparticle delivery, Expert Opin. DrugDeliv. 11 (2014) 1237–1253.

[98] E. Spyratou, E.A. Mourelatou, M. Makropoulou, C. Demetzos, Atomic forcemicroscopy: a tool to study the structure, dynamics and stability of liposomaldrug delivery systems, Expert Opin. Drug Deliv. 6 (2009) 305–317.

[99] T.W. Prow, J.E. Grice, L.L. Lin, R. Faye, M. Butler, W. Becker, et al., Nanoparticlesand microparticles for skin drug delivery, Adv. Drug Deliv. Rev. 63 (2011)470–491.

[100] J. Madan, N. Dhiman, S. Sardana, R. Aneja, R. Chandra, A. Katyal, Long-circulating poly(ethylene glycol)-grafted gelatin nanoparticles customized forintracellular delivery of noscapine: preparation, in-vitro characterization,structure elucidation, pharmacokinetics, and cytotoxicity analyses, AnticancerDrugs 22 (2011) 543–555.

[101] S. Bozdağ, K. Gümüş, Ö. Gümüş, N. Ünlü, Formulation and in vitro evaluationof cysteamine hydrochloride viscous solutions for the treatment of cornealcystinosis, Eur. J. Pharm. Biopharm. 70 (2008) 260–269.

[102] G. Oliveira, M.A. Wahl, J.F. Pinto, Characterization of laminar extrudatesmanufactured at room temperature in the absence of solvents for the deliveryof drugs, Int. J. Pharmaceut. 454 (2013) 90–98.

[103] M.E. Lauer, O. Grassmann, M. Siam, J. Tardio, L. Jacob, S. Page, et al., Atomicforce microscopy-based screening of drug-excipient miscibility and stabilityof solid dispersions, Pharm. Res. 28 (2011) 572–584.

[104] J. Wu, M. Reading, D. Craig, Application of Calorimetry, sub-ambient atomicforce microscopy and dynamic mechanical analysis to the study of frozenaqueous trehalose solutions, Pharm. Res. 25 (2008) 1396–1404.

[105] Q. Zhu, L.S. Taylor, M.T. Harris, Evaluation of the microstructure ofsemicrystalline solid dispersions, Mol. Pharm. 7 (2010) 1291–1300.

[106] H. Lee, D. Sung, M. Veerapandian, K. Yun, S.W. Seo, PEGylatedpolyethyleneimine grafted silica nanoparticles: enhanced cellular uptake andefficient siRNA delivery, Anal. Bioanal. Chem. 400 (2011) 535–545.

[107] S. Essa, J.M. Rabanel, P. Hildgen, Effect of polyethylene glycol (PEG) chainorganization on the physicochemical properties of poly(D, L-lactide) (PLA)based nanoparticles, Eur. J. Pharm. Biopharm. 75 (2010) 96–106.

[108] S. Sant, S. Poulin, P. Hildgen, Effect of polymer architecture on surfaceproperties, plasma protein adsorption, and cellular interactions of pegylatednanoparticles, J. Biomed. Mater. Res. A 87A (2008) 885–895.

[109] G.Z. Mao, D.Z. Chen, H. Handa, W.F. Dong, D.G. Kurth, H. Mohwald, Depositionand aggregation of aspirin molecules on a phospholipid bilayer pattern,Langmuir 21 (2005) 578–585.

[110] R. Ashayer, P.F. Luckham, S. Manimaaran, P. Rogueda, Investigation of themolecular interactions in a pMDI formulation by atomic force microscopy, Eur.J. Pharm. Sci. 21 (2004) 533–543.

[111] N.K. Mishra, K.B. Joshi, S. Verma, Inhibition of human and bovine insulin fibrilformation by designed peptide conjugates, Mol. Pharm. 10 (2013) 3903–3912.

[112] D. Weber, Y. Pu, C.L. Cooney, Quantification of lubricant activity of magnesiumstearate by atomic force microscopy, Drug Dev. Ind. Pharm. 34 (2008) 1097–1099.

[113] K. El Kirat, I. Burton, V. Dupres, Y.F. Dufrene, Sample preparation proceduresfor biological atomic force microscopy, J. Microsc. 218 (2005) 199–207.

[114] J.A. Hodges, S. Saffarian, Sample preparation for single virion atomic forcemicroscopy and super-resolution fluorescence imaging, J. Vis. Exp. (2014)e51366, doi:10.3791/51366.

[115] J. Zhang, S. Ebbens, X. Chen, Z. Jin, S. Luk, C. Madden, et al., Determination ofthe surface free energy of crystalline and amorphous lactose by atomic forcemicroscopy adhesion measurement, Pharm. Res. 23 (2006) 401–407.

[116] M. Bunker, M. Davies, M. James, C. Roberts, Direct observation of single particleelectrostatic charging by atomic force microscopy, Pharm. Res. 24 (2007)1165–1169.

[117] N.H. Duong, E. Shen, T. Shinbrot, F. Muzzio, Segregation in granular materialsand the direct measurement of surface forces using atomic force microscopy,Powder Technol. 145 (2004) 69–72.

[118] B.S. Pizzorno, E.R. Dourado, M.B. deMoraes, R.A. Simão, L.F.M. Leite, Segregationand crystallization of waxes on the surface of asphalt binders as observed byatomic force microscopy, Pet. Sci. Technol. 32 (2014) 2738–2745.

[119] P. Begat, D.A.V. Morton, J.N. Staniforth, R. Price, The cohesive-adhesive balancesin dry powder inhaler formulations I: direct quantification by atomic forcemicroscopy, Pharm. Res. 21 (2004) 1591–1597.














































































































































































[120] L.A. Lyon, A.B. South, Direct observation of microgel erosion via in-liquid atomicforce microscopy, Chem. Mater. 22 (2010) 3300–3306.

[121] P. Burgos, Z.Y. Zhang, R. Golestanian, G.J. Leggett, M. Geoghegan, Directed singlemolecule diffusion triggered by surface energy gradients, ACS Nano 3 (2009)3235–3243.

[122] R. Chen, L. Li, J. Zhao, Single chain diffusion of poly(ethylene oxide) in itsmonolayers before and after crystallization, Langmuir 26 (2010) 5951–5956.

[123] C. Peetla, R. Bhave, S. Vijayaraghavalu, A. Stine, E. Kooijman, V. Labhasetwar,Drug resistance in breast cancer cells: biophysical characterization of anddoxorubicin interactions with membrane lipids, Mol. Pharm. 7 (2010) 2334–2348.

[124] S. Jadhav, C.D. Eggleton, K. Konstantopoulos, Mathematical modeling of celladhesion in shear flow: application to targeted drug delivery in inflammationand cancer metastasis, Curr. Pharm. Des. 13 (2007) 1511–1526.

[125] B. Larijani, C.A. Rosser, R. Woscholski, Chemical Biology: Applications andTechniques, John Wiley & Sons, Chichester, England; Hoboken, NJ, 2006.

[126] T.D. Silverstein, B. Gibb, E.C. Greene, Visualizing protein movement on DNAat the single-molecule level using DNA curtains, DNA Repair (Amst) 20 (2014)94–109.

[127] S.M. Lindsay, Y.L. Lyubchenko, N.J. Tao, Y.Q. Li, P.I. Oden, J.A. Derose, et al.,Scanning-tunneling-microscopy and atomic-force microscopy studies ofbiomaterials at a liquid-solid interface, J. Vac. Sci. Technol. A 11 (1993)808–815.

[128] B.T. Marshall, M. Long, J.W. Piper, T. Yago, R.P. McEver, C. Zhu, Direct observationof catch bonds involving cell-adhesionmolecules, Nature 423 (2003) 190–193.

[129] G.A. Meininger, The central importance of the cytoskeleton for increased cellstiffness in cardiovascular disease. Focus on “Diabetes increases stiffness oflive cardiomyocytes measured by atomic force microscopy nanoindentation,Am. J. Physiol. Cell Physiol. 307 (2014) C908–C909.

[130] D.J. Taatjes, A.S. Quinn, J.H. Rand, B.P. Jena, Atomic force microscopy: highresolution dynamic imaging of cellular and molecular structure in health anddisease, J. Cell. Physiol. 228 (2013) 1949–1955.

[131] W. Hanley, O. McCarty, S. Jadhav, Y. Tseng, D. Wirtz, K. Konstantopoulos, Singlemolecule characterization of P-selectin/ligand binding, J. Biol. Chem. 278 (2003)10556–10561.

[132] A. Trache, J.P. Trzeciakowski, L. Gardiner, Z. Sun, M. Muthuchamy, M. Guo, et al.,Histamine effects on endothelial cell fibronectin interaction studied by atomicforce microscopy, Biophys. J. 89 (2005) 2888–2898.

[133] J. James, B. Crean, M. Davies, R. Toon, P. Jinks, C.J. Roberts, The surfacecharacterisation and comparison of two potential sub-micron, sugar bulkingexcipients for use in low-dose, suspension formulations in metered doseinhalers, Int. J. Pharmaceut. 361 (2008) 209–221.

[134] P. Tamamis, E. Kasotakis, G. Archontis, A. Mitraki, Combination of theoreticaland experimental approaches for the design and study of fibril-formingpeptides, Methods Mol. Biol. 1216 (2014) 53–70.

[135] K.K. Jain, The role of nanobiotechnology in drug discovery, Pharm. Biotechnol.(2009) 37–43.

[136] G. Kada, F. Kienberger, P. Hinterdorfer, Atomic force microscopy inbionanotechnology, Nano Today 3 (2008) 12–19.

[137] F. Hong, D.D. Root, Downscaling functional bioassays by single-moleculetechniques, Drug Discov. Today 11 (2006) 640–645.

[138] J. Oreopoulos, R.F. Epand, R.M. Epand, C.M. Yip, Peptide-induced domainformation in supported lipid bilayers: direct evidence by combined atomicforce and polarized total internal reflection fluorescence microscopy, Biophys.J. 98 (2010) 815–823.

[139] M. Duman, M. Pfleger, R. Zhu, C. Rankl, L.A. Chtcheglova, I. Neundlinger, et al.,Improved localization of cellular membrane receptors using combinedfluorescence microscopy and simultaneous topography and recognitionimaging, Nanotechnology 21 (2010) 115504.

[140] A. Demming, Bionanotechnology: arrays in the future prospects of the field,Nanotechnology 25 (2014) 360201.

[141] M.J. Campolongo, S.J. Tan, J.F. Xu, D. Luo, DNA nanomedicine: engineering DNAas a polymer for therapeutic and diagnostic applications, Adv. Drug Deliv. Rev.62 (2010) 606–616.

[142] R. Dahm, Discovering DNA: friedrich Miescher and the early years of nucleicacid research, Hum. Genet. 122 (2008) 565–581.

[143] E. Paredes, X. Zhang, H. Ghodke, V.K. Yadavalli, S.R. Das, Backbone-branchedDNA building blocks for facile angular control in nanostructures, ACS Nano7 (2013) 3953–3961.

[144] A.K. Mazur, M. Maaloum, Atomic force microscopy study of DNA flexibilityon short length scales: smooth bending versus kinking, Nucleic Acids Res. 42(2014) 14006–14012.

[145] K. Li, S. Du, S. Van Ginkel, Y. Chen, Atomic force microscopy study of theinteraction of DNA and nanoparticles, Adv. Exp. Med. Biol. 811 (2014) 93–109.

[146] M. Stolz, R. Gottardi, R. Raiteri, S. Miot, I. Martin, R. Imer, et al., Early detectionof aging cartilage and osteoarthritis in mice and patient samples using atomicforce microscopy, Nat. Nanotechnol. 4 (2009) 186–192.

[147] A. Beljebbar, S. Dukic, N. Amharref, M. Manfait, Screening of biochemical/histological changes associated to C6 glioma tumor development by FTIR/PCAimaging, Analyst 135 (2010) 1090–1097.

[148] X. Liu, Y.-P. Zhao, Donut-shaped fingerprint in homologous polypeptiderelationships–A topological feature related to pathogenic structural changesin conformational disease, J. Theor. Biol. 258 (2009) 294–301.

[149] A.F. Oberhauser, C. Badilla-Fernandez, M. Carrion-Vazquez, J.M. Fernandez, Themechanical hierarchies of fibronectin observed with single-molecule AFM, J.Mol. Biol. 319 (2002) 433–447.

[150] J.S. Jiménez, Protein-DNA interaction at the origin of neurological diseases:a hypothesis, J. Alzheimers Dis. 22 (2010) 375–391.

[151] M. Innocenti, E. Salvietti, M. Guidotti, A. Casini, S. Bellandi, M.L. Foresti, et al.,Trace Copper(II) or Zinc(II) ions drastically modify the aggregation behaviorof amyloid-β1–42: an AFM study, J. Alzheimers Dis. 19 (2010) 1323–1329.

[152] A. Nazem, G.A. Mansoori, Nanotechnology solutions for Alzheimer’s disease:advances in research tools, diagnostic methods and therapeutic agents, J.Alzheimers Dis. 13 (2008) 199–223.

[153] T. Gudzenko, C.M. Franz, Studying early stages of fibronectin fibrillogenesisin living cells by atomic force microscopy, Mol. Biol. Cell 26 (2015) 3190–3204.

[154] S.T. Souza, L.C. Agra, C.E. Santos, E. Barreto, J.M. Hickmann, E.J. Fonseca,Macrophage adhesion on fibronectin evokes an increase in the elastic propertyof the cell membrane and cytoskeleton: an atomic force microscopy study,Eur. Biophys. J. 43 (2014) 573–579.

[155] A. Gelmi, M.J. Higgins, G.G. Wallace, Quantifying fibronectin adhesion withnanoscale spatial resolution on glycosaminoglycan doped polypyrrole usingAtomic Force Microscopy, Biochim. Biophys. Acta 2013 (1830) 4305–4313.

[156] F. Andre, E. Ruiz-Garcia, V. Scott, C. Machavoine, J.M. Bidart, L. Lacroix, et al.,Gene expression profiling identifies Fibronectin 1 and CXCL9 as candidatebiomarkers for breast cancer screening, Br. J. Cancer 102 (2010) 462–468.

[157] S. Waalkes, H. Eggers, A. Grosshennig, J. Hennenlotter, F. Atschekzei, W.Trankenschuh, et al., Fibronectin 1 mrna expression correlates with risk ofrecurrence in renal cell cancer, Eur. Urol. Suppl. 10 (2011) 235.

[158] M.A. Hill, Z. Sun, L. Martinez-Lemus, G.A. Meininger, New technologies fordissecting the arteriolar myogenic response, Trends Pharmacol. Sci. 28 (2007)308–315.

[159] K.H. Jeong, T.W. Lee, C.G. Ihm, J.Y. Moon, G.J. Lee, H.K. Park, et al., Real-timemonitoring of the effects of telmisartan on angiotensin II-induced mechanicalchanges in live mesangial cells using atomic force microscopy, Kidney BloodPress. Res. 35 (2012) 573–582.

[160] M. Li, L. Liu, N. Xi, Y. Wang, Z. Dong, O. Tabata, et al., Imaging and measuringthe rituximab-induced changes of mechanical properties in B-lymphoma cellsusing atomic force microscopy, Biochem. Biophys. Res. Commun. 404 (2011)689–694.

[161] T. Flatmark, J. Leandro, N. Simonsen, J. Saraste, P. Leandro, Phenylketonuriaas a protein misfolding disease: the mutation pG46S in phenylalaninehydroxylase promotes self-association and fibril formation, Biochim. Biophys.Acta 1812 (2011) 106–120.

[162] P. Hammarstrom, Protein folding, misfolding and disease Preface, FEBS Lett.583 (2009) 2579–2580.

[163] K.L. Lim, J.M.M. Tan, E.S.P. Wong, Protein misfolding and aggregation inParkinson’s disease, Antioxid. Redox Signal. 11 (2009) 2119–2134.

[164] C. McAllister, M.A. Karymov, Y. Kawano, A.Y. Lushnikov, A. Mikheikin, V.N.Uversky, et al., Protein interactions and misfolding analyzed by AFM forcespectroscopy, J. Mol. Biol. 354 (2005) 1028–1042.

[165] T. Maver, K. Stana-Kleinschek, Z. Peršin, U. Maver, Functionalization of AFMtips for use in force spectroscopy between polymers andmodel surfaces, Mater.Tehnol. 45 (2011) 205–211.

[166] U. Maver, T. Maver, A. Žnidaršic, Z. Peršin, M. Gaberšcek, K. Stana-Kleinschek,Use of AFM force spectroscopy for assessment of polymer response toconditions similar to the wound, during healing, Mater. Tehnol. 45 (2011)259–263.

[167] U. Maver, A. Znidarsic, M. Gaberscek, An attempt to use atomic forcemicroscopy for determination of bond type in lithium battery electrodes, J.Mater. Chem. 21 (2011) 4071–4075.

[168] A. Jacquot, C. Sakamoto, A. Razafitianamaharavo, C. Caillet, J. Merlin, A. Fahs,et al., Dynamic modulation of fimbrial extension and FimH-mannose bindingforce on live bacteria under pH changes: a molecular atomic force microscopyanalysis, J. Biomed. Nanotechnol. 10 (2014) 3361–3372.

[169] M. Richter, D. Steinhilber, R. Haag, R. von Klitzing, Visualization of real-timedegradation of pH-responsive polyglycerol nanogels via atomic forcemicroscopy, Macromol. Rapid Commun. 35 (2014) 2018–2022.

[170] Y. Yokoyama, K. Yamada, Y. Higashi, S. Ozaki, H. Wang, N. Koito, et al.,Dependence of purple membrane bump curvature on pH and ionic strengthanalyzed using atomic force microscopy combined with solvent exchange, J.Phys. Chem. B 118 (2014) 9322–9328.

[171] N. Sergeev, M. Brevnov, M. Furtado, TaqMelt – a novel real time PCR methodfor highly multiplexed pathogen detection assays, J. Mol. Diagn. 12 (2010) 882.

[172] H. Artelsmair, F. Kienberger, A. Tinazli, R. Schlapak, R. Zhu, J. Preiner, et al.,Atomic force microscopy-derived nanoscale chip for the detection of humanpathogenic viruses, Small 4 (2008) 847–854.

[173] C. Kaittanis, S. Santra, J.M. Perez, Emerging nanotechnology-based strategiesfor the identification of microbial pathogenesis, Adv. Drug Deliv. Rev. 62 (2010)408–423.

[174] T.-R.J. Tzeng, Y.R. Cheng, R. Saeidpourazar, S.S. Aphale, N. Jalili, Adhesin-specificnanomechanical cantilever biosensors for detection of microorganisms, J. HeatTransfer 133 (2011) 11012–11015.

[175] M.A. Jobst, C. Schoeler, K. Malinowska, M.A. Nash, Investigating receptor-ligandsystems of the cellulosome with AFM-based single-molecule forcespectroscopy, J. Vis. Exp. (2013) e50950, doi:10.3791/50950.

[176] J. Lin, H. Xu, Y. Wu, M. Tang, G.D. McEwen, P. Liu, et al., Investigation of freefatty acid associated recombinant membrane receptor protein expression inHEK293 cells using Raman spectroscopy, calcium imaging, and atomic forcemicroscopy, Anal. Chem. 85 (2013) 1374–1381.














































































































































































[177] S. Sasuga, R. Abe, O. Nikaido, S. Kiyosaki, H. Sekiguchi, A. Ikai, et al., Interactionbetween pheromone and its receptor of the fission yeast Schizosaccharomycespombe examined by a force spectroscopy study, J. Biomed. Biotechnol. 2012(2012) 804793.

[178] R.H. Eibl, V.T. Moy, Atomic force microscopy measurements of protein-ligandinteractions on living cells, in: G.U. Nienhaus (Editor), Protein-LigandInteractions, Humana Press, 2005, pp. 439–449.

[179] T. Tsapikouni, Y.F. Missirlis, P-selectin/ligand unbinding force measured withatomic force microscopy: comparison of two chemical protocols for thetethering of single molecules, J. Mol. Recognit. 24 (2011) 847–853.

[180] J. Fritz, A.G. Katopodis, F. Kolbinger, D. Anselmetti, Force-mediated kineticsof single P-selectin/ligand complexes observed by atomic force microscopy,Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 12283–12288.

[181] R. Karnik, S. Bose, S.K. Das, J.M. Karp, A semianalytical model to study the effectof cortical tension on cell rolling, Biophys. J. 99 (2010) 3870–3879.

[182] R. Karnik, S. Hong, H. Zhang, Y. Mei, D.G. Anderson, J.M. Karp, et al.,Nanomechanical control of cell rolling in two dimensions through surfacepatterning of receptors, Nano Lett. 8 (2008) 1153–1158.

[183] R. Karnik, C.H. Lee, S. Bose, K.J. Van Vliet, J.M. Karp, Examining the lateraldisplacement of HL60 cells rolling on asymmetric p-selectin patterns, Langmuir27 (2011) 240–249.

[184] J.M. Karp, D. Sarkar, P.K. Vemula, G.S.L. Teo, D. Spelke, R. Karnik, et al., Chemicalengineering of mesenchymal stem cells to induce a cell rolling response,Bioconjug. Chem. 19 (2008) 2105–2109.

[185] R. Langer, R. Karnik, S. Hong, H. Zhang, Y. Mei, D.G. Anderson, et al.,Nanomechanical control of cell rolling in two dimensions through surfacePatterning of receptors, Nano Lett. 8 (2008) 1153–1158.

[186] E. Evans, A. Leung, V. Heinrich, C. Zhu, Mechanical switching and couplingbetween two dissociation pathways in a P-selectin adhesion bond, Proc. Natl.Acad. Sci. U.S.A. 101 (2004) 11281–11286.

[187] K.K. Sarangapani, T. Yago, A.G. Klopocki, M.B. Lawrence, C.B. Fieger, S.D. Rosen,et al., Low force decelerates L-selectin dissociation from P-selectin glycoproteinligand-1 and endoglycan, J. Biol. Chem. 279 (2004) 2291–2298.

[188] W.D. Hanley, D. Wirtz, K. Konstantopoulos, Distinct kinetic and mechanicalproperties govern selectin-leukocyte interactions, J. Cell Sci. 117 (2004)2503–2511.

[189] X. Shi, X. Zhang, T. Xia, X. Fang, Living cell study at the single-molecule andsingle-cell levels by atomic force microscopy, Nanomedicine 7 (2012) 1625–1637.

[190] M. Moretti, C. Canale, C. Canale, M. Francardi, S. Dante, F. De Angelis, et al.,AFM characterization of biomolecules in physiological environment by anadvanced nanofabricated probe, Microsc. Res. Tech. 75 (2012) 1723–1731.

[191] A. Eleta-Lopez, J. Etxebarria, N.-C. Reichardt, R. Georgieva, H. Bäumler, J.L.Toca-Herrera, On the molecular interaction between albumin and ibuprofen:an AFM and QCM-D study, Colloids Surf. B. Biointerfaces 134 (2015) 355–362.

[192] N. Kodera, T. Ando, The path to visualization of walking myosin V by high-speed atomic force microscopy, Biophys. Rev. 6 (2014) 237–260.

[193] T. Huber, T.P. Sakmar, Escaping the flatlands: new approaches for studyingthe dynamic assembly and activation of GPCR signaling complexes, TrendsPharmacol. Sci. 32 (2011) 410–419.

[194] H. Nia, C. Ortiz, A. Grodzinsky, Aggrecan: approaches to study biophysical andbiomechanical properties, in: K. Balagurunathan, H. Nakato, U.R. Desai (Editors),Glycosaminoglycans, Springer New York, 2015, pp. 221–237.

[195] V. Dupres, D. Alsteens, G. Andre, C. Verbelen, Y.F. Dufrene, Fishing singlemolecules on live cells, Nano Today 4 (2009) 262–268.

[196] D. Alsteens, A. Beaussart, S. Derclaye, S. El-Kirat-Chatel, H.R. Park, P.N. Lipke,et al., Single-cell force spectroscopy of als-mediated fungal adhesion, Anal.Methods 5 (2013) 3657–3662.

[197] D. Alsteens, V. Dupres, E. Dague, C. Verbelen, G. André, G. Francius, et al.,Imaging chemical groups and molecular recognition sites on live cells usingAFM, in: B. Bhushan, H. Fuchs (Editors), Applied Scanning Probe Methods XIII,Springer Berlin Heidelberg, 2009, pp. 33–48.

[198] M. Frei, S.V. Aradhya, M.S. Hybertsen, L. Venkataraman, Linker dependent bondrupture force measurements in single-molecule junctions, J. Am. Chem. Soc.134 (2012) 4003–4006.

[199] M. Frei, S.V. Aradhya, M. Koentopp, M.S. Hybertsen, L. Venkataraman,Mechanics and chemistry: single molecule bond rupture forces correlate withmolecular backbone structure, Nano Lett. 11 (2011) 1518–1523.

[200] E. Balnois, G. Raj, C. Baley, Y. Grohens, Probing cellulose/polylactic acidinteractions in model biocomposite by colloidal force microscopy, ColloidsSurf. A Physicochem. Eng. Asp. 352 (2009) 47–55.

[201] C. Formosa, V. Lachaize, C. Gales, M.P. Rols, H. Martin-Yken, J.M. Francois, et al.,Mapping HA-tagged protein at the surface of living cells by atomic forcemicroscopy, J. Mol. Recognit. 28 (2015) 1–9.

[202] A. Rigato, F. Rico, F. Eghiaian, M. Piel, S. Scheuring, Atomic force microscopymechanical mapping of micropatterned cells shows adhesion geometry-dependent mechanical response on local and global scales, ACS Nano 9 (2015)5846–5856.

[203] D. Ebeling, B. Eslami, J. Solares Sde, Visualizing the subsurface of soft matter:simultaneous topographical imaging, depth modulation, and compositionalmapping with triple frequency atomic force microscopy, ACS Nano 7 (2013)10387–10396.

[204] H. Hillaireau, P. Couvreur, Nanocarriers’ entry into the cell: relevance to drugdelivery, Cell. Mol. Life Sci. 66 (2009) 2873–2896.

[205] L.R. Nyland, D.W. Maughan, Morphology and transverse stiffness of Drosophilamyofibrils measured by atomic force microscopy, Biophys. J. 78 (2000)1490–1497.

[206] H. Zoellner, N. Paknejad, K. Manova, M.A. Moore, A novel cell-stiffness-fingerprinting analysis by scanning atomic force microscopy: comparison offibroblasts and diverse cancer cell lines, Histochem. Cell Biol. (2015)doi:10.1007/s00418-015-1363-x.

[207] N. Gavara, R.S. Chadwick, Relationship between cell stiffness and stress fiberamount, assessed by simultaneous atomic force microscopy and live-cellfluorescence imaging, Biomech. Model. Mechanobiol. (2015) doi:10.1007/s10237-015-0706-9.

[208] T. Watanabe-Nakayama, S. Machida, I. Harada, H. Sekiguchi, R. Afrin, A. Ikai,Direct detection of cellular adaptation to local cyclic stretching at the singlecell level by atomic force microscopy, Biophys. J. 100 (2011) 564–572.

[209] R. Roy, J.P. Desai, Determination of mechanical properties of spatiallyheterogeneous breast tissue specimens using contact mode atomic forcemicroscopy (AFM), Ann. Biomed. Eng. 42 (2014) 1806–1822.

[210] J.T. Morgan, V.K. Raghunathan, S.M. Thomasy, C.J. Murphy, P. Russell, Robustand artifact-free mounting of tissue samples for atomic force microscopy,Biotechniques 56 (2014) 40–42.

[211] L. Penuela, F. Wolf, R. Raiteri, D. Wendt, I. Martin, A. Barbero, Atomic forcemicroscopy to investigate spatial patterns of response to interleukin-1betain engineered cartilage tissue elasticity, J. Biomech. 47 (2014) 2157–2164.

[212] K.J. Van Vliet, P. Hinterdorfer, Probing drug-cell interactions, Nano Today 1(2006) 18–25.

[213] J.R. Couchman, M.R. Austria, A. Woods, Fibronectin-cell interactions, J. Invest.Dermatol. 94 (1990) 7S–14S.

[214] P.J. McKeown-Longo, Fibronectin-cell surface interactions, Rev. Infect. Dis. 9(Suppl. 4) (1987) S322–S334.

[215] D.P. Allison, P. Hinterdorfer, W.H. Han, Biomolecular force measurements andthe atomic force microscope, Curr. Opin. Biotechnol. 13 (2002) 47–51.

[216] A. Ebner, L. Wildling, R. Zhu, C. Rankl, T. Haselgrübler, P. Hinterdorfer, et al.,Functionalization of probe tips and supports for single-molecule recognitionforce microscopy, in: P. Samorì (Editor), STM and AFM Studies on(Bio)molecular Systems: Unravelling the Nanoworld, Springer Berlin/Heidelberg, 2008, pp. 29–76.

[217] A. Ebner, P. Hinterdorfer, H.J. Gruber, Comparison of differentaminofunctionalization strategies for attachment of single antibodies to AFMcantilevers, Ultramicroscopy 107 (2007) 922–927.

[218] A. Ebner, L. Wildling, A.S.M. Kamruzzahan, C. Rankl, J. Wruss, C.D. Hahn, et al.,A new, simple method for linking of antibodies to atomic force microscopytips, Bioconjug. Chem. 18 (2007) 1176–1184.

[219] C. Mayet, A. Dazzi, R. Prazeres, J.-M. Ortega, D. Jaillard, In situ identificationand imaging of bacterial polymer nanogranules by infrared nanospectroscopy,Analyst 135 (2010) 2540–2545.

[220] J.A. Sidles, J.L. Garbini, K.J. Bruland, D. Rugar, O. Züger, S. Hoen, et al., Magneticresonance force microscopy, Rev. Mod. Phys. 67 (1995) 249.

[221] W.P. King, S. Saxena, B.A. Nelson, B.L. Weeks, R. Pitchimani, Nanoscale thermalanalysis of an energetic material, Nano Lett. 6 (2006) 2145–2149.

[222] D.M. Price, M. Reading, A. Hammiche, H.M. Pollock, Micro-thermal analysis:scanning thermal microscopy and localised thermal analysis, Int. J. Pharmaceut.192 (1999) 85–96.

[223] O.S. Ovchinnikova, M.P. Nikiforov, J.A. Bradshaw, S. Jesse, G.J. Van Berkel,Combined atomic force microscope-based topographical imaging andnanometer-scale resolved proximal probe thermal desorption/electrosprayionization–mass spectrometry, ACS Nano 5 (2011) 5526–5531, doi:10.1021/nn200939e.

[224] Y. Wei, R.A. Latour, Correlation between desorption force measured by atomicforce microscopy and adsorption free energy measured by surface plasmonresonance spectroscopy for peptide-surface interactions, Langmuir 26 (2010)18852–18861.

[225] P.G. Rogueda, R. Price, T. Smith, P.M. Young, D. Traini, Particle synergy andaerosol performance in non-aqueous liquid of two combinations metered doseinhalation formulations: an AFM and Raman investigation, J. Colloid InterfaceSci. 361 (2011) 649–655.

[226] D. Traini, P.M. Young, P. Rogueda, R. Price, In vitro investigation of drugparticulates interactions and aerosol performance of pressurisedmetered doseinhalers, Pharm. Res. 24 (2007) 125–135.

[227] D. Traini, P.M. Young, P. Rogueda, R. Price, The use of AFM and surface energymeasurements to investigate drug-canister material interactions in a modelpressurized metered dose inhaler formulation, Aerosol Sci. Technol. 40 (2006)227–236.

[228] T. Ando, T. Uchihashi, N. Kodera, D. Yamamoto, A. Miyagi, M. Taniguchi, et al.,High-speed AFM and nano-visualization of biomolecular processes, PflugersArch. 456 (2008) 211–225.

[229] N. Kodera, D. Yamamoto, R. Ishikawa, T. Ando, Video imaging of walkingmyosinV by high-speed atomic force microscopy, Nature 468 (2010) 72–76.

[230] M. Shibata, T. Uchihashi, T. Ando, R. Yasuda, Long-tip high-speed atomic forcemicroscopy for nanometer-scale imaging in live cells, Sci. Rep. 5 (2015) 8724.

[231] A.P. Nievergelt, B.W. Erickson, N. Hosseini, J.D. Adams, G.E. Fantner, Studyingbiological membranes with extended range high-speed atomic forcemicroscopy, Sci. Rep. 5 (2015) 11987.

[232] A. Rajendran, M. Endo, H. Sugiyama, State-of-the-art high-speed atomic forcemicroscopy for investigation of single-molecular dynamics of proteins, Chem.Rev. 114 (2014) 1493–1520.












































































































































































Recent progressive use of atomic force microscopy in ... · aUniversity of Maribor, Faculty of Medicine, Institute of Biomedical Sciences, Taborska ulica 8, SI-2000 Maribor, Slovenia

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