Functionalized Atomic Force Microscopy Tips Research Articles

Atomic force spectroscopy with magainin 1 functionalized tips and biomimetic supported lipid membranes.

Antimicrobial peptides are molecules synthesized by living organisms as the first line of defense against bacteria, fungi, parasites, or viruses. Since their biological activity is based on destabilization of the microbial membranes, a study of direct interaction forces between antimicrobial peptides and biomimetic membranes is very important for understanding the molecular mechanisms of their action. Herein, we use atomic force spectroscopy to probe the interaction between atomic force microscopy (AFM) tips functionalized with magainin 1 and supported lipid bilayers (SLBs) mimicking electrically uncharged membranes of normal eukaryotic cells and negatively charged membranes of bacterial cells. The investigations performed on negatively charged SLBs showed that the magainin 1 functionalized AFM tips are quickly adsorbed into the SLBs when they approach, while they adhere strongly to the lipid membrane when retracted. On contrary, same investigations performed on neutral SLBs showed mechanical resistance of the lipid membrane to the tip breakthrough and negligible adhesion force at detachment.

Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems.

During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into a nanoscopic laboratory to directly observe and chemically characterize molecular and cell biological systems under physiological conditions. Here, we review key technological improvements that have established AFM as an analytical tool to observe and quantify native biological systems from the micro- to the nanoscale. Native biological systems include living tissues, cells, and cellular components such as single or complexed proteins, nucleic acids, lipids, or sugars. We showcase the procedures to customize nanoscopic chemical laboratories by functionalizing AFM tips and outline the advantages and limitations in applying different AFM modes to chemically image, sense, and manipulate biosystems at (sub)nanometer spatial and millisecond temporal resolution. We further discuss theoretical approaches to extract the kinetic and thermodynamic parameters of specific biomolecular interactions detected by AFM for single bonds and extend the discussion to multiple bonds. Finally, we highlight the potential of combining AFM with optical microscopy and spectroscopy to address the full complexity of biological systems and to tackle fundamental challenges in life sciences.

Understanding Calcium-Mediated Adhesion of Nanomaterials in Reservoir Fluids by Insights from Molecular Dynamics Simulations

Interest in nanomaterials for subsurface applications has grown markedly due to their successful application in a variety of disciplines, such as biotechnology and medicine. Nevertheless, nanotechnology application in the petroleum industry presents greater challenges to implementation because of the harsh conditions (i.e. high temperature, high pressure, and high salinity) that exist in the subsurface that far exceed those present in biological applications. The most common subsurface nanomaterial failures include colloidal instability (aggregation) and sticking to mineral surfaces (irreversible retention). We previously reported an atomic force microscopy (AFM) study on the calcium-mediated adhesion of nanomaterials in reservoir fluids (S. L. Eichmann and N. A. Burnham, Sci. Rep. 7, 11613, 2017), where we discovered that the functionalized and bare AFM tips showed mitigated adhesion forces in calcium ion rich fluids. Herein, molecular dynamics reveal the molecular-level details in the AFM experiments. Special attention was given to the carboxylate-functionalized AFM tips because of their prominent ion-specific effects. The simulation results unambiguously demonstrated that in calcium ion rich fluids, the strong carboxylate-calcium ion complexes prevented direct carboxylate-calcite interactions, thus lowering the AFM adhesion forces. We performed the force measurement simulations on five representative calcite crystallographic surfaces and observed that the adhesion forces were about two to three fold higher in the calcium ion deficient fluids compared to the calcium ion rich fluids for all calcite surfaces. Moreover, in calcium ion deficient fluids, the adhesion forces were significantly stronger on the calcite surfaces with higher calcium ion exposures. This indicated that the interactions between the functionalized AFM tips and the calcite surfaces were mainly through carboxylate interactions with the calcium ions on calcite surfaces. Finally, when analyzing the order parameters of the tethered functional groups, we observed significantly different behavior of the alkanethiols depending on the absence or presence of calcium ions. These observations agreed well with AFM experiments and provided new insights for the competing carboxylate/calcite/calcium ion interactions.

Functionalization of atomic force microscopy cantilevers and tips by activated vapour silanization

Atomic force microscopy (AFM) is a powerful tool in Biology due to the possibility of identifying and locating single molecules at a subnanometer scale. Performing these experiments requires the usage of functionalized AFM tips to which sensing molecules can be attached. In this work the possibility of functionalizing AFM cantilevers and tips using the activated vapour silanization (AVS) process is explored. Functionalized thin films are deposited on commercial silicon nitride chips and their presence is assessed by measuring the variation in the resonance frequency of the cantilever. Subsequently, the presence and functionality of the films is studied through the covalent immobilization of a fluorescein-derived molecule to the amine reactive groups on the surface. With regard to its sensing ability, the functionalized tips are shown to sustain repetitive interactions with a model graphite substrate (HOPG) under relatively harsh conditions, while no apparent damage is observed in the tip. It is also found that the substrate-tip interaction may be modified by covalently attaching a molecule to the latter. Consequently, AVS functionalized AFM tips are found to be robust and reliable candidates for their use in the affinity (or chemical) atomic force microscopy (AF-AFM) mode.

Adhesion force measured by atomic force microscopy for direct carbon fiber-epoxy interfacial characterization

Adhesion force measurement by Atomic force microscopy (AFM) is used to investigate the interfacial interaction of the carbon fiber (CF)/epoxy composite for the first time. The epoxy functionalized AFM tip and three types of carbon fibers with different surface chemistry and morphologies were used in this study. Results show that the Bis (3-aminophenyl) phenyl phosphine oxide (BAPPO) modified CF possesses a much larger adhesion force (72.7 nN) than the as-received and de-sized CF (22.5 nN and 17.9 nN, respectively) due to formation of the chemical bonding between the BAPPO modified CF and the epoxy functionalized tip. Single fiber microbond test demonstrates that the interfacial shear strength (IFSS) of BAPPO modified CF/EP composite is 15% and 22% larger than that of as-received and de-sized CF/EP, respectively. This nanoscale manipulation by AFM provides a new avenue to measure the interfacial adhesion between the CF and epoxy at the molecular level.

Adsorbed Organic Material and Its Control on Wettability

Laboratory core flood and field scale tests have demonstrated that 5–40% more oil can be released from sandstone reservoirs by injecting low salinity water, rather than high salinity fluids, such as seawater or formation water. The effect has been explained by a change in wettability of the minerals that form the pore surfaces, as a result of the decrease in divalent cation concentration. We have previously demonstrated that, even for solvent cleaned core samples, mineral surfaces retain a significant quantity of carbon containing material and this affects wettability and response to changed salinity. Here we quantified the response of sandstone core plug material in its preserved state (i.e., after storage in kerosene) and after the same core plug material was treated with ethanol and ozone to remove adsorbed organic compounds. We used the chemical force microscopy (CFM) mode of atomic force microscopy (AFM) to directly measure the adhesion force for two types of molecules on pore surfaces of individual sand grains that were plucked from an oil reservoir core plug. We functionalized AFM tips with alkane or carboxylate, so they resembled tiny oil droplets and measured adhesion to the sand grain surfaces in artificial seawater (ASW; 35,600 ppm) and in ASW diluted to ∼1,500 ppm (ASW-low). Both before and after the ethanol/ozone treatment, and for both the alkane and the carboxylate functionalized tips, the adhesion was lower in ASW diluted to ∼1,500 ppm than in ASW. For both alkane and carboxylate, the difference in adhesion between ASW and ASW-low was higher before the ethanol/ozone treatment. We attribute this change in response to the salinity dependent force caused mainly by the electric double layer (EDL) at the sand grain surfaces. We interpret the higher adhesion difference, before a very thorough ethanol/ozone treatment, to be a result of the loss of the organic material that was originally adsorbed on these surfaces, which adds to the charge density and thereby to the salinity dependent EDL force. Investigating the same area on the same pore surface, before and after removal of the organic material, demonstrates without doubt that it is organic material that causes the low salinity response, not the underlying mineral surface.

Enhanced Site-Specific Anchoring of Biomolecules for Single-Molecule Force Spectroscopy

Single-molecule force spectroscopy (SMFS) provides unique insights into the mechanical properties and dynamics of biomolecules. In atomic force microscopy (AFM)-based SMFS, non-specific interactions (i.e., passive adsorption) is often used to couple biomolecules to surfaces and AFM tips. However, non-specific interactions lead to artifacts in SMFS data and decrease throughput. Building on prior efforts by other groups, we developed an efficient, hetero-bifunctional coupling of biomolecules via copper-free click chemistry to a PEG-coated substrate and by a strong, but reversible streptavidin-biotin linkage to AFM tips. PEG coating reduces non-specific interactions between the biomolecule and the surfaces (both sample surfaces and AFM tips). Importantly, our protocol substantially reduces the labor involved in functionalizing AFM tips and sample surfaces. Functionalized coverslips can be coated with protein or DNA even weeks after surface modification. Moreover, protein-coated tips and surfaces were reused over multiple days and DNA-coated surfaces were reused over 2 weeks. The use of a streptavidin-biotin bond to attach the biomolecule to the tip facilitates rapid tip-molecule coupling and provides a strong (but reversible) connection between the biotinylated biomolecule and the AFM tip. The reversible bond leaves the biomolecule on the surface and the tip can re-engage the same molecule for repeated unfolding. The overall combination of PEG-coated surfaces, site-specific coupling, and streptavidin-coated tips yielded a dramatic improvement the acquisition rate of high-quality AFM-based SMFS data. In particular, we measured the full dynamic force spectrum of a single, individual polyprotein (NuG2) in just 2 hours and the resulting biophysical parameters agreed with prior literature. We demonstrated the site-specific coupling for stretching individual DNA molecules with both optical traps and AFM. Hence, while this site-specific coupling protocol was developed primarily for AFM-based SMFS, this technique is also applicable to a wide range of surface-coupled, single-molecule assays.

Desorption Mechanism of Asphaltenes in the Presence of Electrolyte and the Extended Derjaguin–Landau–Verwey–Overbeek Theory

Desorption of asphaltenes from silica-coated quartz crystals upon exposure to a series of saline solutions was studied through the measurements of quartz crystal microbalance with dissipation (QCM-D), atomic force microscopy (AFM), and contact angle. Interestingly, it was found that the mass loading and thickness of asphaltene film decreased during the injection of sodium chloride solution at the concentrations ranging from 1 to 10 mM, with the surface tending to be hydrophilic, whereas the mass loading and film thickness increased gradually when the concentration increased from 10 to 1000 mM, with the surface inclined to be hydrophobic. It was also found that the electrostatic force had a great effect on this process due to the interactions between the charged interfaces of oil/water and water/solid. Besides, some additional interactions may arise under small distance at the presence of the electrolyte solution, and therefore, a direct force-measuring technique was introduced, in which the functionalized AFM tips felt a solid surface to model the interactions among three phases of oil, water, and solid. Based on the computed results of disjoining pressure isotherms, the theory of Derjaguin-Landau-Verwey-Overbeek (DLVO) was extended, taking into account of the participation of hydration forces which played an important role at short range. These structural forces mostly originated from the overlap of the hydrated layers under a variety of salinity concentrations, resulting in the balance of resultant interactions.

A Flexible Nanoarray Approach for the Assembly and Probing of Molecular Complexes

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers’ rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.

Using Atomic Force Microscopy to Evaluate the Nanostructure and Nanomechanics of Warm Mix Asphalt

AbstractIn this paper, various Atomic Force Microscopy (AFM) techniques are utilized to study the effects of different warm mix asphalt (WMA) additives on the nanostructure and microstructure as well as the adhesive and cohesive properties of an asphalt binder. To achieve this objective, a control asphalt binder was modified with three different types of WMA additives, which included: Advera (a synthetic zeolite), Evotherm, and Sasobit (a commercial wax). The AFM tapping-mode imaging technique was used to examine the nanostructure and microstructure of the different asphalt material considered. In addition, AFM force spectroscopy experiments were conducted using chemically functionalized AFM tips to measure the nanoscale level adhesive and cohesive forces for the control and WMA asphalt binders before and after moisture conditioning. The results of the AFM imaging showed that while the Sasobit additive has reduced the width of the so-called “bee-like” structures within the asphalt binder, the other WMA ad...

In situ probing the interior of single bacterial cells at nanometer scale

We report a novel approach to probe the interior of single bacterial cells at nanometre resolution by combining focused ion beam (FIB) and atomic force microscopy (AFM). After removing layers of pre-defined thickness in the order of 100 nm on the target bacterial cells with FIB milling, AFM of different modes can be employed to probe the cellular interior under both ambient and aqueous environments. Our initial investigations focused on the surface topology induced by FIB milling and the hydration effects on AFM measurements, followed by assessment of the sample protocols. With fine-tuning of the process parameters, in situ AFM probing beneath the bacterial cell wall was achieved for the first time. We further demonstrate the proposed method by performing a spatial mapping of intracellular elasticity and chemistry of the multi-drug resistant strain Klebsiella pneumoniae cells prior to and after it was exposed to the ‘last-line’ antibiotic polymyxin B. Our results revealed increased stiffness occurring in both surface and interior regions of the treated cells, suggesting loss of integrity of the outer membrane from polymyxin treatments. In addition, the hydrophobicity measurement using a functionalized AFM tip was able to highlight the evident hydrophobic portion of the cell such as the regions containing cell membrane. We expect that the proposed FIB–AFM platform will help in gaining deeper insights of bacteria–drug interactions to develop potential strategies for combating multi-drug resistance.

How Naturally Adsorbed Material on Minerals Affects Low Salinity Enhanced Oil Recovery

Laboratory core flood and field scale tests have demonstrated that about 5 to 40% more oil can be released from sandstone reservoirs by injecting low salinity water, rather than high salinity fluids such as seawater or formation water. The effect has been explained by a change in wettability of the minerals that form the pore wall, as a result of the decrease in divalent cation concentration. Using X-ray photoelectron spectroscopy, we have demonstrated that even for solvent cleaned core samples, mineral surfaces retain a significant quantity of carbon containing material. Thus, pore wall wettability is more likely dominated by tightly adsorbed organic material than by the character of the underlying minerals. To test this hypothesis, we used the chemical force mapping (CFM) mode of atomic force microscopy (AFM) to directly measure adhesion forces on individual quartz grains that were plucked from core plugs. We functionalized AFM tips with model oil compounds so they would represent tiny oil droplets, and we measured their ability to adhere to surfaces as salinity changed. We examined grains from a sandstone core plug that had been cut into segments, which had been stored in kerosene or solvent cleaned. On all samples, surfaces were more oil wet (higher adhesion) in artificial seawater (ASW; 35,600 ppm) than in ASW diluted with ultrapure deionized water to ∼1,500 ppm. XPS demonstrated that solvent cleaned surfaces had less adsorbed organic material than the kerosene stored sample. AFM measurements showed that the low salinity effect, namely the change in adhesion caused by decreasing salinity, was twice as high on kerosene stored samples as on solvent cleaned surfaces. The organic material that is adsorbed on the pore surfaces in the preserved sandstone offer very sticky anchor points for adhering oil molecules. This suggests that in reservoirs, even hydrophilic minerals located at the pore-fluid interface have tightly adhering hydrocarbons and the low salinity response depends on the behavior of this adsorbed material.

The use of functionalized AFM tips as molecular sensors in the detection of pesticides

Atomic force spectroscopy, a technique derived from Atomic Force Microscopy (AFM), allowed us to distinguish nonspecific and specific interactions between the acetolactate synthase enzyme (ALS) and anti-atrazine antibody biomolecules and the herbicides imazaquin, metsulfuron-methyl and atrazine. The presence of specific interactions increased the adhesion force (Fadh) between the AFM tip and the herbicides, which made the modified tip a powerful biosensor. Increases of approximately 132% and 145% in the Fadh values were observed when a tip functionalized with ALS was used to detect imazaquin and metsulfuron-methyl, respectively. The presence of specific interactions between the atrazine and the anti-atrazine antibody also caused an increase in the Fadh values (approximately 175%) compared to those observed when using an unfunctionalized tip. The molecular modeling results obtained with the ALS enzyme suggest that the orientation of the biomolecule on the tip surface could be suitable for allowing interaction with the herbicides imazaquin and metsulfuron-methyl.

Measurements of single molecular affinity interactions between carbohydrate-binding modules and crystalline cellulose fibrils

Combining atomic force microscopy (AFM) recognition imaging and single molecule dynamic force spectroscopy (SMDFS), we studied the single molecule affinity interactions between the carbohydrate-binding module (CBM) and plant cell wall cellulose using the CBM3a (from Clostridium thermocellum) and CBM2a (from Cellvibrio japonicus) functionalized AFM tips. The binding efficiencies of the CBMs to the cellulose were determined by the binding areas on the crystalline cellulose fibrils surface using the recognition imaging. Several dynamic and kinetic parameters, such as the reconstructed free energy change, energy barrier and bond lifetime constant, were also obtained based on the measured single molecule unbinding forces, which are used to illuminate the affinity of the CBMs binding to the natural and single cellulose surface from a totally different aspect. It was found that CBM3a has a little higher binding efficiency and affinity than CBM2a to both natural and extracted cellulose surfaces and both the CBMs have higher affinities to the natural cell wall cellulose compared to the extracted single cellulose. The in-depth understanding of the binding mechanisms of the CBM-cellulose interactions of this study may pave the way for more efficient plant cell wall degradation and eventually facilitate biofuel production.

Single molecular dissection of the ligand binding property of epidermal growth factor receptor

The understanding of ligand binding interactions is an important component of understanding the fundamental mechanism of receptor function. In this study, the binding abilities of EGF and TGF-α to EGFR on human bladder cancer (T24) cells were investigated by single molecular force spectroscopy (SMFS) based on atomic force microscopy (AFM). By approaching the specifically functionalized AFM tips to the T24 cell surface and subsequent retraction, specific unbinding events of the EGF/EGFR complexes and TGF-α/EGFR complexes were investigated. Further, the unbinding forces and the kinetic off rate constants that govern the bond stabilities were calculated through varying the external mechanic forces applied. Meanwhile, the distances from the energy minimum to the transition states along the separation paths of the EGF/EGFR complexes and TGF-α/EGFR complexes were deduced. This study at single-molecule level may enrich our understanding of the ligand binding properties of EGFR and provide some new information to the development of improved EGFR inhibitors. In addition, the results present new insight into the study of the energy landscape of the dissociation of ligand-EGFR system.

Nanoscale analysis of the effects of antibiotics and CX1 on a Pseudomonas aeruginosa multidrug-resistant strain

Drug resistance is a challenge that can be addressed using nanotechnology. We focused on the resistance of the bacteria Pseudomonas aeruginosa and investigated, using Atomic Force Microscopy (AFM), the behavior of a reference strain and of a multidrug resistant clinical strain, submitted to two antibiotics and to an innovative antibacterial drug (CX1). We measured the morphology, surface roughness and elasticity of the bacteria under physiological conditions and exposed to the antibacterial molecules. To go further in the molecules action mechanism, we explored the bacterial cell wall nanoscale organization using functionalized AFM tips. We have demonstrated that affected cells have a molecularly disorganized cell wall; surprisingly long molecules being pulled off from the cell wall by a lectin probe. Finally, we have elucidated the mechanism of action of CX1: it destroys the outer membrane of the bacteria as demonstrated by the results on artificial phospholipidic membranes and on the resistant strain.

Structural-Mechanical Characterization of Nanoparticle Exosomes in Human Saliva, Using Correlative AFM, FESEM, and Force Spectroscopy

All living systems contain naturally occurring nanoparticles with unique structural, biochemical, and mechanical characteristics. Specifically, human saliva exosomes secreted by normal cells into saliva via exocytosis are novel biomarkers showing tumor-antigen enrichment during oral cancer. Here we show the substructure of single human saliva exosomes, using a new ultrasensitive low force atomic force microscopy (AFM) exhibiting substructural organization unresolvable in electron microscopy. We correlate the data with field emission scanning electron microscopy (FESEM) and AFM images to interpret the nanoscale structures of exosomes under varying forces. Single exosomes reveal reversible mechanical deformation displaying distinct elastic, 70-100 nm trilobed membrane with substructures carrying specific transmembrane receptors. Further, we imaged and investigated, using force spectroscopy with antiCD63 IgG functionalized AFM tips, highly specific and sensitive detection of antigenCD63, potentially useful cancer markers on individual exosomes. The quantitative nanoscale morphological, biomechanical, and surface biomolecular properties of single saliva exosomes are critical for the applications of exosomes for cancer diagnosis and as a model for developing new cell delivery systems.

Nanoscale Evaluation of Moisture Damage in Polymer Modified Asphalts

The moisture damage in polymer modified asphalts has been studied for decades, yet the effects of chemical functional groups on moisture sensitivity are not known. In this study, a nanoscale experiment is conducted to measure these effects in terms of adhesive/cohesive forces using an atomic force microscopy (AFM). A base asphalt binder and two polymers such as styrene-butadiene (SB) and styrene-butadiene-styrene (SBS) modified asphalts are used to prepare an AFM sample on glass substrates. The AFM samples are conditioned under dry and wet conditions. In the AFM, these samples are probed by silicon nitrite ( Si3 N4 ) , carboxyl ( —COOH ) , methyl ( — CH3 ) , and hydroxyl ( —OH ) functionalized AFM tips and nanoscale pull-off or adhesion/cohesion forces between asphalt and tip molecules are measured. Based on the ratio of wet to dry adhesion/cohesion forces, it is shown that the polymer modification makes binders less susceptible to moisture damage. Among the four tips, the —COOH tip shows almost no differ...

Analysis of Force Interactions between AFM Tips and Hydrophobic Bacteria Using DLVO Theory

Microbial adhesion to surfaces and interfaces is strongly influenced by their structure and physicochemical properties. We used atomic force microscopy (AFM) to measure the forces between chemically functionalized AFM tips and two bacterial species exhibiting different cell surface hydrophobicities, measured as the oil/water contact angle (theta): Acinetobacter venetianus RAG-1 (theta = 56.4 degrees ) and Rhodococcus erythropolis 20S-E1-c (theta = 152.9 degrees ). The forces were measured as the AFM tips, coated with either hydrophobic (octadecane) or hydrophilic (undecanol) groups, approached the bacterial cells in aqueous buffer. The experimental force curves between the two microbial cells and functionalized AFM probes were not successfully described by the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid stability. To reconcile the discrepancy between theory and experiments, two types of extended DLVO models were proposed. The first modification considers an additional acid-base component that accounts for attractive hydrophobic interactions and repulsive hydration effects. The second model considers an additional exponentially decaying steric interaction between polymeric brushes in addition to the acid-base interactions. These extended DLVO predictions agreed well with AFM experimental data for both A. venetianus RAG-1, whose surface consists of an exopolymeric capsule and pili, and R. erythropolis 20S-E1-c, whose surface is covered by an exopolymeric capsule. The extended models for the bacteria-AFM tip force-distance curves were consistent with the effects of steric interactions.

Probe Tips Functionalized with Colloidal Nanocrystal Tetrapods for High‐Resolution Atomic Force Microscopy Imaging

Probe Tips Functionalized with Colloidal Nanocrystal Tetrapods for High‐Resolution Atomic Force Microscopy Imaging