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During the processes involved in pharmaceutical manufacturing, particulate matter may be introduced into a product from a variety of sources and at different points in the manufacturing process. Companies design quality at the beginning of the process to ensure against defects and strive to manufacture products that meet the pharmacopeial standard of being “practically/essentially free” of particles, which can be challenging, though necessary. As particulate matter recalls are predominantly associated with parenteral products, most companies employ a quality risk management program to identify critical parameters or conditions that could affect product quality or patient safety and incorporate systemic and procedural controls to mitigate or reduce the probability of their occurrence. Yet, determining where particulates are most likely to enter the process, what types of materials are most vulnerable, and how the size and number of particles might affect product quality can be very complex. Visual inspection and sampling of the manufactured drug product are designed to control the risk of particulate contamination; building prevention controls will ensure sustainability. This concept paper highlights the necessity of a more thorough understanding of the failure mechanisms that result in particle contamination across a range of products, such as elastomeric components and glass, and processes, such as the formulation and filling of injectables. The goal is to identify process steps within the end-to-end manufacturing process that are most critical to particle generation and entering of visible particles into the final drug product. LAY ABSTRACT: This concept paper highlights the necessity of a more thorough understanding of the failure mechanisms that result in particle contamination across a range of products, such as elastomeric components and glass, and processes, such as the formulation and filling of injectables. The goal is to identify process steps within the end-to-end manufacturing process that are most critical to particle generation and entering of visible particles into the final drug product.

Background: The serotonin transporter is the site of action of antidepressants and amphetamines. Results: Single molecular force spectroscopy allowed for mapping the energy landscape involved in MFZ2-12/SERT binding. Conclusion: Our data indicate that the outer vestibule imposes a barrier on the entry of MFZ2-12 into the SERT substrate binding site. Significance: Our results provide a useful framework for a further exploration of antidepressant binding., The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: koff values were in good agreement with those derived from uptake experiments; in contrast, the kon values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction.

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe 2 O 3 ), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.

Minor group human rhinoviruses (HRVs) attach to members of the low-density lipoprotein receptor family and are internalized via receptor-mediated endocytosis. The attachment of HRV2 to the cell surface, the first step in infection, was characterized at the single-molecule level by atomic force spectroscopy. Sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra, which suggests that multiple receptors bound to the virus in a timely manner. Unbinding forces required to detach the virus from the cell membrane increased within a time frame of several hundred milliseconds. The number of receptors involved in virus binding was determined, and estimates for on-rate, off-rate, and equilibrium binding constant of the interaction between HRV2 and plasma membrane-anchored receptors were obtained.

Atomic force microscopy is a technique capable to study biological recognition processes at the single-molecule level. In this work we operate the AFM in a force-scan based mode, the jumping mode, where simultaneous topographic and tip-sample adhesion maps are acquired. This approach obtains the unbinding force between a well-defined receptor molecule and a ligand attached to the AFM tip. The method is applied to the avidin-biotin system. In contrast with previous data, we obtain laterally resolved adhesion maps of avidin-biotin unbinding forces highly correlated with single avidin molecules in the corresponding topographic map. The scanning rate 250 pixel s(-1) (2 min for a 128 x 128 image) is limited by the hydrodynamic drag force. We are able to build a rupture-force distribution histogram that corresponds to a single defined molecule. Furthermore, we find that due to the motility of the polymer used as spacer to anchor the ligand to the tip, its direction at rupture does not generally coincide with the normal to the tip-sample, this introduces an appreciable error in the measured force.

Covalent binding of bioligands to atomic force microscope (AFM) tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a approximately 6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly "scan" a large surface area while the tip is at or near the sample surface. In the standard coupling scheme, amino groups are first generated on the AFM tip. In the second step, these amino groups react with the amino-reactive ends of heterobifunctional PEG linkers. In the third step, the 2-pyridyl-S-S groups on the free ends of the PEG chains react with protein thiol groups to give stable disulfide bonds. In the present study, this standard coupling scheme has been critically examined, using biotinylated IgG with free thiols as the bioligand. AFM tips with PEG-tethered biotin-IgG were specifically recognized by avidin molecules that had been adsorbed to mica surfaces. The unbinding force distribution showed three maxima that reflected simultaneous unbinding of 1, 2, or 3 IgG-linked biotin residues from the avidin monolayer. The coupling scheme was well-reproduced on amino-functionalized silicon nitride chips, and the number of covalently bound biotin-IgG per microm2 was estimated by the amount of specifically bound ExtrAvidin-peroxidase conjugate. Coupling was evidently via disulfide bonds, since only biotin-IgG with free thiol groups was bound to the chips. The mechanism of protein thiol coupling to 2-pyridyl-S-S-PEG linkers on AFM tips was further examined by staging the coupling step in bulk solution and monitoring turnover by release of 2-pyridyl-SH which tautomerizes to 2-thiopyridone and absorbs light at 343 nm. These experiments predicted 10(3)-fold slower rates for the disulfide coupling step than actually observed on AFM tips and silicon nitride chips. The discrepancy was reconciled by assuming 10(3)-fold enrichment of protein on AFM tips via preadsorption, as is known to occur on comparable inorganic surfaces.

Atomic force microscopy (AFM) was used to probe topology, conformational changes and initial substratecarrier interactions of Na+-glucose co-transporter (SGLT1) in living cells on a single-molecule level. By scanning SGLT1-transfected Chinese hamster ovary (CHO) cells with AFM tips carrying an epitope-specific antibody directed against the extramembranous C-terminal loop 13, significant recognition events could be detected. Specificity was confirmed by the absence of events in nontransfected CHO cells and by the use of free antigen and free antibody superfusion. Thus, contrary to computer predictions on SGLT1 topology, loop 13 seems to be part of the extracellular surface of the transporter. Binding probability of the antibody decreased upon addition of phlorizin, a specific inhibitor of SGLT1, suggesting a considerable conformational change of loop 13 when the inhibitor occludes the sugar translocation pathway. Using an AFM tip carrying 1-thio-D-glucose, direct evidence could be obtained that in the presence of Na+ a sugarbinding site appears on the transporter surface. The binding site accepts the sugar residue of the glucoside phlorizin, free D-glucose, and D-galactose, but not free Lglucose and probably represents the first of several selectivity filters of the transporter. This work demonstrates the potential of AFM to study the presence and dynamics of plasma membrane transporters in intact cells on the single molecule level.

Simultaneous topography and recognition imaging (TREC), 2] a recent development in dynamic force microscopy, has proven to be a powerful technique in biophysical research. In contrast to the common force mapping mode, the slow imaging speed and the low lateral resolution are overcome by oscillating a functionalized tip close to its resonance frequency during the scan across the surface. In these studies, the topographical imaging of receptor molecules is combined with molecular recognition by their cognate ligands bound to the atomic force microscope (AFM) tip via a distensible tether. The binding sites are evident from the reduction in the oscillation amplitude, as a result of specific recognition during the lateral scan. The receptors are recognized by the ligand on the scanning tip with a lateral resolution of a few nanometers, yielding a topographic image and a separate map of recognition sites from a single scan. Because this new technique opens a broad range of biological applications, a stable and easy-to-use setup is of key interest for new users. Using well-characterized, high-affinity biological binding partners, such as avidin–biotin, for TREC imaging enables a better understanding of the key factors for optimizing the scanning parameters. The robust and well-described avidin–biotin interaction offers an ideal pair of binding partners. Avidin–biotin is 1) robust and reliable, 2) well-known in terms of binding properties, 3) easy to prepare with commercially available components. Moreover, 4) avidin can simply be adsorbed to mica, and mica is an ideal support for AFM imaging. Finally, 5) the biotin–PEG (poly(ethylene glycol)) tether is commercially available and can be attached to amino-functionalized AFM tips in one step. Single avidin molecules can easily be immobilized onto mica via electrostatic adsorption by virtue of their positive net charge at neutral pH. Using different concentrations of avidin in the adsorption buffer, the surface coverage of avidin on mica can be adjusted easily. In addition, a biotin residue was coupled to the AFM tip via a distensible PEG chain. More specifically, biotin was covalently tethered to the amino-functionalized tip in a single coupling step, using “biotin-PEG-NHS” which consists of a PEG chain with a biotin on one end and an amino-reactive N-hydroxysuccinimide ester function (NHS group) on the other. With this configuration, two independent maps were simultaneously acquired, that is, a topography image of the immobilized avidin molecules and a lateral map of the corresponding recognition sites, both recorded at experimental times comparable to normal AFM imaging. Before being applied to TREC, the tips were examined for a functional biotin residue by force spectroscopy experiments on a mica surface densely covered with avidin molecules. Force spectroscopy with an oscillating tip offers an important tool for adjusting the scanning parameters in TREC microscopy. In simultaneously recorded amplitude–distance and force–distance cycles (Figure 1), the oscillation amplitude and the cantilever bending force, respectively, were investigated.

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2′-aminoethyl β-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.

Combination of high-resolution atomic force microscope topography imaging with single molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Here simultaneous topography and recognition imaging (TREC) was applied to gently fixed microvascular endothelial cells from mouse myocardium (MyEnd) to identify binding sites of vascular endothelial (VE)-cadherin, known to play a crucial role in calcium-dependent, homophilic cell-to-cell adhesion. TREC images were acquired with magnetically oscillating atomic-force microscope tips functionalized with a recombinant VE-cadherin-Fc cis-dimer. The recognition images revealed single molecular binding sites and prominent, irregularly shaped dark spots (domains) with sizes ranging from 10 to 100nm. These domains arose from a decrease of the oscillation amplitude during specific binding between active VE-cadherin cis-dimers. The VE-cadherin clusters were subsequently assigned to topography features. TREC represents an exquisite method to quickly obtain the local distribution of receptors on cellular surface with an unprecedented lateral resolution of 5nm.

The measuring tip of a force microscope can be converted into a monomolecular sensorif one or few "ligand" molecules are attached to the apex of the tip while maintainingligand function. Functionalized tips are used to study fine details of receptor-ligand interactionby force spectroscopy or to map cognate "receptor" molecules on the sample surface. Thereceptor (or target) molecules can be present on the surface of a biological specimen; alternatively,soluble target molecules must be immobilized on ultraflat supports. This review describes the methodsof tip functionalization, as well as target molecule immobilization. Silicon nitride tips, siliconchips, and mica have usually been functionalized in three steps: (1) aminofunctionalization,(2) crosslinker attachment, and (3) ligand/receptor coupling, whereby numerous crosslinkersare available to couple widely different ligand molecules. Gold-covered tips and/or supports haveusually been coated with a self-assembled monolayer, on top of which the ligand/receptor moleculehas been coupled either directly or via a crosslinker molecule. Apart from these general strategies,many simplified methods have been used for tip and/or support functionalization, even single-stepmethods such as adsorption or chemisorption being very efficient under suitable circumstances. Allmethods are described with the same explicitness and critical parameters are discussed. In conclusion,this review should help to find suitable methods for specific problems of tip and support functionalization.

Tannerella forsythia ATCC 43037 (Tf) is a Gram-negative oral anaerobe which is one of the major contributors to periodontal disease in humans [1]. It possesses a glycosylated S-layer consisting of two regularly arrayed subunits as outermost cell envelope layer. The S-layer of Tf was shown to be a virulence factor, capable of delaying the bacterium's recognition by the innate immune system [2] and mediating adhesion and invasion of host cells [3]. In this study, we used scanning probe microscopy to probe the S-layer surface of live Tf cells by investigating the nano subunit structure so as to characterize the surface structure and the interactions of Tf S-layer proteins. High-resolution imaging of living Tf wild-type cells has shown a periodic square lattice structure with about 10 nm dimension, while on the surface of an S-layer single mutant no periodic structure was visible. In addition, single-molecule force spectroscopy using a TfsA S-layer protein specific antibody immobilized onto AFM tips revealed specific interaction forces with the S-layer structure on living Tf wild-type cells. Our data contribute to the elucidation of a surface structure on living bacteria and to the understanding of their subunit arrangement with nano-meter resolution.[1] S. Holt, and J. Ebersole Periodontology 2000 38 (2000) 72.[2] G. Sekot, G. Posch, P. Messner, M. Matejka, X. Rausch-Fan, O. Andrukhov, and C. Schaffer, J. Dent, Res 90 (2011) 109[3] J. Sakakibara, K. Nagano, and Y. Murakami, Microbiology 153 (2007) 866.

Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

Human rhinoviruses are the causative agents of the common cold. The serotypes belonging to the minor receptor group attach to members of the low-density lipoprotein receptor family and enter the host cell via receptor-mediated endocytosis. Receptor binding, the very first step in infection, was characterized by force spectroscopy measurements at the single molecule level. We demonstrate how kinetic on- and off-rate constants can be derived from such experiments carried out with the atomic force microscope.

The measuring tip of an atomic force microscope (AFM) can be upgraded to a specific biosensor by attaching one or a few biomolecules to the apex of the tip. The biofunctionalized tip is then used to map cognate target molecules on a sample surface or to study biophysical parameters of interaction with the target molecules. The functionality of tip-bound sensor molecules is greatly enhanced if they are linked via a thin, flexible polymer chain. In a typical scheme of tip functionalization, reactive groups are first generated on the tip surface, a bifunctional cross-linker is then attached with one of its two reactive ends, and finally the probe molecule of interest is coupled to the free end of the cross-linker. Unfortunately, the most popular functional group generated on the tip surface is the amino group, while at the same time, the only useful coupling functions of many biomolecules (such as antibodies) are also NH(2) groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH(2) groups on the tip surface. In the present study, an uncompromising solution to this problem was found with the help of a new cross-linker ("acetal-PEG-NHS") which possesses one activated carboxyl group and one acetal-protected benzaldehyde function. The activated carboxyl ensures rapid unilateral attachment to the amino-functionalized tip, and only then is the terminal acetal group converted into the amino-reactive benzaldehyde function by mild treatment (1% citric acid, 1-10 min) which does not harm the AFM tip. As an exception, AFM tips with magnetic coating become demagnetized in 1% citric acid. This problem was solved by deprotecting the acetal group before coupling the PEG linker to the AFM tip. Bivalent binding of the corresponding linker ("aldehyde-PEG-NHS") to adjacent NH(2) groups on the tip was largely suppressed by high linker concentrations. In this way, magnetic AFM tips could be functionalized with an ethylene diamine derivative of ATP which showed specific interaction with mitochondrial uncoupling protein 1 (UCP1) that had been purified and reconstituted in a mica-supported planar lipid bilayer.

Human rhinoviruses are the causative agents of the common cold. The serotypes belonging to the minor receptor group attach to members of the low-density lipoprotein receptor family and enter the host cell via receptor-mediated endocytosis. Receptor binding, the very first step in infection, was characterized by force spectroscopy measurements at the single molecule level. We demonstrate how kinetic on- and off-rate constants can be derived from such experiments carried out with the atomic force microscope.

Vascular endothelial (VE)-cadherin is predominantly responsible for the mechanical linkage between endothelial cells, where VE-cadherin molecules are clustered and linked through their cytoplasmic domain to the actin-based cytoskeleton. Clustering and linkage of VE-cadherin to actin filaments is a dynamic process and changes according to the functional state of the cells. Here nano-mapping of VE-cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium (MyEnd). The recognition maps revealed prominent ‘dark’ spots (domains or clusters) with the sizes from 10 to 250 nm. These spots arose from a decrease of oscillation amplitude during specific binding between VE-cadherin cis-dimers. They were assigned to characteristic structures of the topography images. After treatment with nocodazole so as to depolymerize microtubules, VE-cadherin domains with a typical ellipsoidal form were still found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE-cadherin is linked to actin filaments. Compared to other conventional techniques such as immunochemistry or single molecule optical microscopy, TREC represents an alternative method to quickly obtain the local distribution of receptors on cell surface with an unprecedented lateral resolution of several nanometers. Copyright © 2010 John Wiley & Sons, Ltd.

Atomic force microscopy (AFM) has developed into a key technique for the investigation of biological samples. In contrast to other microscopy methods, high lateral resolution down to the nanometer scale and measurements under physiological conditions are possible. Additionally, the piconewton force sensitivity allows accurate data collection for single-molecule interactions. This chapter describes the combination of high-resolution imaging and single-molecule interaction measurements. In the so-called topography and recognition imaging (TREC) mode, the scanning AFM tip is upgraded into a molecular sensor by anchoring a ligand to the tip. Enhanced electronics, including a recently developed feedback loop, allow measurement of the sample topography while simultaneously mapping ligand-binding sites. This results in topography images recorded alongside with recognition images, thereby allowing accurate allocation of the binding sites with a lateral resolution of one to a few nanometers. TREC has been successfully used for recognition imaging on isolated proteins, native and artificial membranes, and cells.

Functional nanoarrays were fabricated using the chimeric bacterial cell surface layer (S-layer) protein rSbpA fused with the affinity tag Strep-tagII and characterized using various atomic force microscopy (AFM) techniques in aqueous environment. The accessibility of Strep-tagII was verified by single-molecule force spectroscopy studies employing Strep-Tactin as specific ligand. Simultaneous topography and recognition imaging (TREC) of the nanoarray yielded high resolution maps of the Strep-tagll binding sites with a positional accuracy of 1.5 nm. The nanoarrays were used as template for constructing highly ordered molecular binding blocks.

The nanoscale exploration of microbes using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Here, we show that single-molecule force spectroscopy is a valuable tool for the localization and conformational analysis of individual polysaccharides on live bacteria. We focus on the clinically important probiotic bacterium Lactobacillus rhamnosus GG, demonstrating the power of AFM to reveal the coexistence of polysaccharide chains of different nature on the cell surface. Applicable to a wide variety of cells, this single molecule method offers exciting prospects for analyzing the heterogeneity and diversity of macromolecules constituting cell membranes and cell walls. ispartof: ACS Nano vol:2 issue:9 pages:1921-1929 ispartof: location:United States status: published

The mineralocorticoid hormone aldosterone acts on target cells of kidney, colon, and the cardiovascular system through genomic and nongenomic pathways. Although the classical intracellular mineralocorticoid receptor plays a key role in mediating both pathways, it is unclear whether there are specific aldosterone receptors located on the cell surface. To search for such sites in vascular endothelium, we used an atomic force microscope (AFM) which measures unbinding forces based on single molecular recognition between an aldosterone-loaded AFM tip and the cell membrane. Aldosterone was tethered covalently via linker molecules to an AFM tip. Human endothelial cells (EA.hy926) were grown in culture and studied in buffer at 37 degrees C. Using the aldosterone-functionalized AFM tip as a mechanical nanoscale indenter, unbinding forces could be measured at randomly chosen sites of the plasma membrane. Sites with strong interactions between AFM tip and cell surface could be identified exhibiting unbinding forces of about 65 pN. The binding probability between the aldosterone-loaded tip and the cell surface at selected membrane sites was 53 +/- 7.2%. Addition of an excess supply of aldosterone to the bath solution blocked the binding of the aldosterone-loaded tip to the cell surface. The binding probability was reduced to 8.0 +/- 1.8% when an excess supply of aldosterone was added to the bath. However, it was not influenced by the addition of spironolactone or dexamethasone. We conclude that aldosterone receptor sites exist on the cell surface of vascular endothelial cells distinct from the classical mineralocorticoid receptors and insensitive to glucocorticoids. Binding of aldosterone to these receptors initiates an intracellular signaling cascade that precedes the classical genomic response and most likely participates in the control of vascular resistance.

The measuring tip of a force microscope can be converted into a monomolecular sensorif one or few “ligand” molecules are attached to the apex of the tip while maintainingligand function. Functionalized tips are used to study fine details of receptor–ligand interactionby force spectroscopy or to map cognate “receptor” molecules on the sample surface. Thereceptor (or target) molecules can be present on the surface of a biological specimen; alternatively,soluble target molecules must be immobilized on ultraflat supports. This review describes the methodsof tip functionalization, as well as target molecule immobilization. Silicon nitride tips, siliconchips, and mica have usually been functionalized in three steps: (1) aminofunctionalization,(2) crosslinker attachment, and (3) ligand/receptor coupling, whereby numerous crosslinkersare available to couple widely different ligand molecules. Gold-covered tips and/or supports haveusually been coated with a self-assembled monolayer, on top of which the ligand/receptor moleculehas been coupled either directly or via a crosslinker molecule. Apart from these general strategies,many simplified methods have been used for tip and/or support functionalization, even single-stepmethods such as adsorption or chemisorption being very efficient under suitable circumstances. Allmethods are described with the same explicitness and critical parameters are discussed. In conclusion,this review should help to find suitable methods for specific problems of tip and support functionalization.

The inhibition of the human ether-à-go-go-related (hERG) K+ channels is the major cause of long QT syndromes inducing fatal cardiac arrhythmias. Ergtoxin 1 (ErgTx1) belongs to scorpion-toxins, which are K+ channel-blockers, and binds to hERG channel with 1:1 stoichiometry and high affinity (Kd approximately 10 nM). Nevertheless, patch-clamp recordings recently demonstrated that ErgTx1 does not establish complete blockade of hERG currents, even at high ErgTx1 concentrations. Such phenomenon is supposed to be consistent with highly dynamic conformational changes of the outer pore domain of hERG. In this study, simultaneous topography and recognition imaging (TREC) on hERG HEK 293 cells was used to visualize binding sites on the extracellular part of hERG channel (on S1-S2 region) for Anti-Kv11.1 (hERG-extracellular-antibody). The recognition maps of hERG channels contained recognition spots, haphazardly distributed and organized in clusters. Recognition images after the addition of ErgTx1 at high concentrations ( approximately 1 microM) revealed subsequent partial disappearance of clusters, indicating that ErgTx1 was bound to the S1-S2 region. These results were supported by AFM force spectroscopy data, showing for the first time that voltage sensing domain (S1-S4) of hERG K+ channel might be one of the multiple binding sites of ErgTx1.

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.