Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

Conformational dynamics of individual antibodies using computational docking and AFM

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. In this study, we aimed at analyzing the conformational variability of receptors portrayed by monoclonal antibodies that have been individually imaged using atomic force microscopy (AFM). Individual antibodies were chemically coupled to activated mica surface, and they have been imaged using AFM in ambient conditions. The resulting topographical surface of antibodies was used to assemble the three subunits constituting antibodies: two antigen‐binding fragments and one crystallizable fragment using a surface‐constrained computational docking approach. Reconstructed structures based on 10 individual topographical surfaces of antibodies are presented for which separation and relative orientation of the subunits were measured. When compared with three X‐ray structures of antibodies present in the protein data bank database, results indicate that several arrangements of the reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X‐ray structure consequence of the antibody flexibility. We conclude that high‐resolution AFM imaging with appropriate computational reconstruction tools is adapted to study the conformational dynamics of large individual macromolecules deposited on mica. Copyright © 2013 John Wiley & Sons, Ltd.     

Detection and localization of single molecular recognition events using atomic force microscopy

Because of its piconewton force sensitivity and nanometer positional accuracy, the atomic force microscope (AFM) has emerged as a powerful tool for exploring the forces and the dynamics of the interaction between individual ligands and receptors, either on isolated molecules or on cellular surfaces. These studies require attaching specific biomolecules or cells on AFM tips and on solid supports and measuring the unbinding forces between the modified surfaces using AFM force spectroscopy. In this review, we describe the current methodology for molecular recognition studies using the AFM, with an emphasis on strategies available for preparing AFM tips and samples, and on procedures for detecting and localizing single molecular recognition events.     

Single-molecule recognition force spectroscopy of transmembrane transporters on living cells

Atomic force microscopy (AFM) has proven to be a powerful tool in biological sciences. Its particular advantage over other high-resolution methods commonly used is that biomolecules can be investigated not only under physiological conditions but also while they perform their biological functions. Single-molecule force spectroscopy with AFM tip-modification techniques can provide insight into intermolecular forces between individual ligand-receptor pairs of biological systems. Here we present protocols for force spectroscopy of living cells, including cell sample preparation, tip chemistry, step-by-step AFM imaging, force spectroscopy and data analysis. We also delineate critical steps and describe limitations that we have experienced. The entire protocol can be completed in 12 h. The model studies discussed here demonstrate the power of AFM for studying transmembrane transporters at the single-molecule level.     

Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize

P2X receptors are cation-selective channels activated by extracellular ATP. The architecture of these receptors is still not completely clear. Here we have addressed this issue by both chemical cross-linking and direct imaging of individual receptors by atomic force microscopy (AFM). Cross-linking of the P2X(2) receptor produced higher order adducts, consistent with the presence of trimers. The mean molecular volume of the receptor determined by AFM (409 nm(3)) also points to a trimeric structure. P2X(2) receptors bearing His(6) epitope tags were incubated with anti-His(6) antibodies, and the resultant complexes were imaged by AFM. For receptors with two bound antibodies, the mean angle between the antibodies was 123 degrees , again indicating that the receptor is a trimer. In contrast, cross-linking of the P2X(6) receptor did not produce higher order adducts, and the mean molecular volume of the receptor was 145 nm(3). We conclude that P2X(2) receptors are trimers, whereas the P2X(6) receptor subunits do not form stable oligomers.     

Membrane-based actuation for high-speed single molecule force spectroscopy studies using AFM

Atomic force microscopy (AFM)-based dynamic force spectroscopy of single molecular interactions involves characterizing unbinding/unfolding force distributions over a range of pulling speeds. Owing to their size and stiffness, AFM cantilevers are adversely affected by hydrodynamic forces, especially at pulling speeds >10 μm/s, when the viscous drag becomes comparable to the unbinding/unfolding forces. To circumvent these adverse effects, we have fabricated polymer-based membranes capable of actuating commercial AFM cantilevers at speeds ≥100 μm/s with minimal viscous drag effects. We have used FLUENT^®, a computational fluid dynamics (CFD) software, to simulate high-speed pulling and fast actuation of AFM cantilevers and membranes in different experimental configurations. The simulation results support the experimental findings on a variety of commercial AFM cantilevers and predict significant reduction in drag forces when membrane actuators are used. Unbinding force experiments involving human antibodies using these membranes demonstrate that it is possible to achieve bond loading rates ≥10⁶ pN/s, an order of magnitude greater than that reported with commercial AFM cantilevers and systems.     

Bacteriorhodopsin folds into the membrane against an external force

Despite their crucial importance for cellular function, little is known about the folding mechanisms of membrane proteins. Recently details of the folding energy landscape were elucidated by atomic force microscope (AFM)-based single molecule force spectroscopy. Upon unfolding and extraction of individual membrane proteins energy barriers in structural elements such as loops and helices were mapped and quantified with the precision of a few amino acids. Here we report on the next logical step: controlled refolding of single proteins into the membrane. First individual bacteriorhodopsin monomers were partially unfolded and extracted from the purple membrane by pulling at the C-terminal end with an AFM tip. Then by gradually lowering the tip, the protein was allowed to refold into the membrane while the folding force was recorded. We discovered that upon refolding certain helices are pulled into the membrane against a sizable external force of several tens of picoNewton. From the mechanical work, which the helix performs on the AFM cantilever, we derive an upper limit for the Gibbs free folding energy. Subsequent unfolding allowed us to analyze the pattern of unfolding barriers and corroborate that the protein had refolded into the native state.     

Atomic force microscopy detection of serological markers of viral hepatites B and C

The possibility of detection of serological markers, containing the hepatitis B surface antigen (HBsAg) and hepatitis C virus core-antigen (HCVcoreAg) in human serum, by a new atomic force microscopy (AFM)-based nanotechnological approach has been demonstrated. The antibodies against the hepatitis B virus surface antigen (anti-HBsAg) and the antibodies against the hepatitis C virus core antigen (anti-HCVcoreAg) were immobilized on an AFM-chip. It was shown that such approach enables to detect HBsAg, HCVcoreAg and the viral fragments containing these antigens in the serum. The comparative analysis of detection of HBsAg- and HCVcoreAg-containing particles by the AFM method versus traditional methods (ELISA, PCR) has demonstrated the 75% coincidence of results between the AFM and two other methods.     

Biomolecular imaging using atomic force microscopy

Atomic force microscopy (AFM) has become a well-established technique for imaging single biomacromolecules under physiological conditions. The exceptionally high spatial resolution and signal-to-noise ratio of the AFM enables the substructure of individual molecules to be observed. In contrast to other methods, specimens prepared for AFM remain in a plastic state, which enables direct observation of the dynamic molecular response, creating unique opportunities for studying the structure–function relationships of proteins and their functionally relevant assemblies. This review presents recent advances in methods and applications of AFM to imaging biological samples. It is clear that AFM will become an increasingly important tool for probing both the structural and kinetic properties of biological macromolecules.     

Atomic force microscope-based single-molecule force spectroscopy of RNA unfolding

Single-molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) has emerged as an important tool for probing biomolecular interaction and exploring the forces, dynamics, and energy landscapes that underlie function and specificity of molecular interaction. These studies require attaching biomolecules on solid supports and AFM tips to measure unbinding forces between individual binding partners. Herein we describe efficient and robust protocols for probing RNA interaction by AFM and show their value on two well-known RNA regulators, the Rev-responsive element (RRE) from the HIV-1 genome and an adenine-sensing riboswitch. The results show the great potential of AFM–SMFS in the investigation of RNA molecular interactions, which will contribute to the development of bionanodevices sensing single RNA molecules.     

Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Biomolecular force measurements and the atomic force microscope

The atomic force microscope (AFM) is a surface-sensitive instrument capable of imaging biological samples at nanometer resolution in all environments including liquids. The sensitivity of the AFM cantilever, to forces in the pico Newton range, has been exploited to measure breakaway forces between biomolecules and to measure folding-unfolding forces within single proteins. By attaching specific antibodies to cantilevers the simultaneous imaging of target antigens and identification of antigen-antibody interactions have been demonstrated.     

Lateral mechanical coupling of stereocilia in cochlear hair bundles

For understanding the gating process of transduction channels in the inner ear it is essential to characterize and examine the functional properties of the ultrastructure of stereociliary bundles. There is strong evidence that transduction channels in hair cells are gated by directly pulling at the so-called tip links. In addition to these tip links a second class of filamentous structures was identified in the scanning and transmission electron microscope: the side-to-side links. These links laterally connect stereocilia of the same row of a hair bundle. This study concentrates on mechanical coupling of stereocilia of the tallest row connected by side-to-side links. Atomic Force microscopy (AFM) was used to investigate hair bundles of outer hair cells (OHCs) from postnatal rats (day 4). Although hair bundles of postnatal rats are still immature at day 4 and interconnecting cross-links do not show preferential direction yet, hair bundles of investigated OHCs already showed the characteristic V-shape of mature hair cells. In a first experiment, the stiffness of stereocilia was investigated scanning individual stereocilia with an AFM tip. The spring constant for the excitatory direction was 2.5 +/- 0.6 x 10(-3) N/m whereas a higher spring constant (3.1 +/- 1.5 x 10(-3) N/m) was observed in the inhibitory direction. In a second set of experiments, the force transmission between stereocilia of the tallest row was measured using AFM in combination with a thin glass fiber. This fiber locally displaced a stereocilium while the force laterally transmitted to the neighboring untouched taller stereocilia was measured by AFM. The results show a weak force interaction between tallest stereocilia of postnatal rats. The force exerted to an individual stereocilium declines to 36% at the nearest adjacent stereocilium of the same row not touched with the fiber. It is suggested that the amount of force transmitted from a taller stereocilium to an adjacent one of the same row depends on the orientation of links. Maximum force transmission is expected to appear along the axis of interconnecting side links. In our studies it is suggested that transmitted forces are small because connecting side links are oriented very close to an angle of 90 degrees with respect of the scan direction (excitatory-inhibitory direction).     

Hydrodynamic effects in fast AFM single-molecule force measurements

Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture individual receptor–ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few m/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 m/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 m/s pulling speed.Abbrevations AFM atomic force micrcoscopy - pN piconewton - BR bacteriorhodopsin - DFS dynamic force spectroscopy - Ig27 immunoglobulin 27 - If27-8 immunoglobulin 27 octameric construct - BFP biomembrane force probe     

Atomic force microscopy measurements. Measurements of binding strength between a single pair of molecules in physiological solutions

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteogylycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today’s simplest living Metazoa), are proposed to the primary cell adhesive molecules essential for the evolution of the multicellularity.     

Atomic force microscopy investigations of heterogeneities in the adhesion energies measured between pathogenic and non-pathogenic Listeria species and silicon nitride as they correlate to virulence and adherence

Atomic force microscopy (AFM) was used to probe heterogeneities in adhesion energies measured between pathogenic and non-pathogenic species of Listeria and silicon nitride in water at four levels. Adhesion energies were quantified on individual bacterial cells (cell level), bacterial cells that belonged to an individual Listeria strain but varied in their cultures (strain level), bacterial cells that belonged to an individual Listeria species but varied in their strain type (species level) and on bacterial cells that belonged to the Listeria genus but varied in their species type (genus level). To quantify heterogeneities in the adhesion energies, a heterogeneity index (HI) was defined based on quantified standard errors of mean. At the cell level, spatial variations in the adhesion energies were not observed. For the strain, species, and genus levels, the HI increased with increased adhesion energies. At the species level, the HI increased with strain virulence.     

Direct force measurements of the streptavidin-biotin interaction

The interaction between streptavidin and its ligand, biotin, were studied by direct force measurements. The complimentary approaches of surface force apparatus (SFA) and atomic force microscopy (AFM) were used to elucidate both long-range and short-range adhesive interactions of the streptavidin biotin interaction. The high spatial resolution of the SFA provided a detailed profile of the intersurface forces of apposing surfaces functionalized with streptavidin and biotin. Measurements obtained by the SFA corresponded to long and intermediate-range forces that are important in determining ligand receptor association. AFM was used to measure the unbinding force of individual streptavidin biotin complexes. These measurements revealed the short-range interactions (i.e. hydrophobic and hydrogen bonding forces) that stabilize the intermolecular bond.     

A COOH-terminal peptide confers regiospecific orientation and facilitates atomic force microscopy of an IgG1.

An antibody (IgG1) was designed for oriented adherence to a metal-containing surface. This was achieved by adding a metal-chelating peptide, (CP = His-Trp-His-His-His-Pro), to the COOH-terminus of the heavy chain through genetic engineering. Electroporation of the engineered heavy chain gene into cells expressing the complimentary light chain yielded colonies secreting an intact antibody containing the metal-chelating peptide (IgG1-CP) which had high affinity for a nickel-loaded iminodiacetate column. Purified IgG1-CP was bound to nickel-treated mica and imaged by atomic force microscopy (AFM). Antibody lacking the COOH-terminal metal binding peptide failed to produce discernible AFM images. The AFM images of individual IgG1-CP molecules and their calculated dimensions demonstrated that regiospecific binding and uniform orientation of the antibody was imparted by the peptide. The ability to stably orient macromolecules in their native state to a surface may be used advantageously to visualize them.     

Ligand-receptor interactions and membrane structure investigated by AFM and time-resolved fluorescence microscopy

The atomic force microscope (AFM) and the associated dynamic force spectroscopy technique have been exploited to quantitatively assess the interaction between proteins and their binding to specific ligands and membrane surfaces. In particular, we have studied the specific interaction between lung surfactant protein D and various carbohydrates. In addition, we have used scanning AFM and time-resolved fluorescence microscopy to image the lateral structure of different lipid bilayers and their morphological changes as a function of time. The various systems studied illustrate the potential of modern AFM techniques for application to biomedical research, specifically within immunology and liposome-based drug delivery.