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Magnetophoretic lab on a chip technologies are rapidly evolving into integrated systems for the identification of biomarkers and cells with ultra-high sensitivity. We demonstrate the highly efficient detection of the Human herpes simplex virus type 1 (HSV) UL27 gene through the programmed assembly of superparamagnetic (SPM) nanoparticles based on oligonucleotide hybridization. The state of assembly of the SPM nanoparticles was determined by optical signature of the synchronized motion on the beads on a micromagnetic array (MMA). This technique has been used to identify

Magnetic nanoparticles are promising new tools for therapeutic applications, such as magnetic nanoparticle hyperthermia therapy and targeted drug delivery. Recent in vitro studies have demonstrated that a force application with magnetic tweezers can also affect cell fate, suggesting a therapeutic potential for magnetically modulated mechanical stimulation. The magnetic properties of nanoparticles that induce physical responses and the subtle responses that result from mechanically induced membrane damage and/or intracellular signaling are evaluated. Magnetic particles with various physical, geometric, and magnetic properties and specific functionalization can now be used to apply mechanical force to specific regions of cells, which permit the modulation of cellular behavior through the use of spatially and time controlled magnetic fields. On one hand, mechanochemical stimulation has been used to direct the outgrowth on neuronal growth cones, indicating a therapeutic potential for neural repair. On the other hand, it has been used to kill cancer cells that preferentially express specific receptors. Advances made in the synthesis and characterization of magnetic nanomaterials and a better understanding of cellular mechanotransduction mechanisms may support the translation of mechanochemical stimulation into the clinic as an emerging therapeutic approach. European Commission - European Regional Development Fund Science Foundation Ireland Programme for Research in Third-Level Institutions Marie Curie Intra-European Fellowship

Due to their homophilic and heterophilic binding properties, cell adhesion molecules (CAMs) such as integrin, cadherin and the immunoglobulin superfamily CAMs are of primary importance in cell-cell and cell-substrate interactions, signalling pathways and other crucial biological processes. We study the molecular structures and conformational dynamics of the two fibronectin type III (Fn-III) extracellular domains of the Aplysia californica CAM (apCAM) protein, by constructing and probing an atomically-detailed structural model based on apCAM's homology with other CAMs. The stability and dynamic properties of the Fn-III domains, individually and in tandem, are probed and analysed using all-atom explicit-solvent molecular dynamics (MD) simulations and normal mode analysis of their corresponding elastic network models. The refined structural model of the Fn-III tandem of apCAM reveals a specific pattern of amino acid interactions that controls the stability of the β-sheet rich structure and could affect apCAM's response to physical or chemical changes of its environment. It also exposes the important role of several specific charged residues in modulating the structural properties of the linker segment connecting the two Fn-III domains, as well as of the inter-domain interface.

Candida albicans is the lead fungal pathogen of nosocomial bloodstream infections worldwide and has mortality rates of 43%. Nanoparticles have been identified as a means to improve medical outcomes for Candida infections, enabling sample concentration, serving as contrast agents for in vivo imaging, and delivering therapeutics. However, little is known about how nanoparticles interact with the fungal cell wall. In this report we used laser scanning confocal microscopy to examine the interaction of fluorescent polystyrene nanoparticles of specific surface chemistry and diameter with C. albicans and mutant strains deficient in various C. albicans surface proteins. Carboxylate-functionalized nanoparticles adsorbed mainly to the hyphae of wild-type C. albicans. The dissociative binding constant of the nanoparticles was ∼150, ∼30 and ∼2.5 pM for 40, 100 nm and 200 nm diameter particles, respectively. A significant reduction in particle binding was observed with a Δals3 strain compared to wild-type strains, identifying the Als3 adhesin as the main mediator of this nanoparticle adhesion. In the absence of Als3, nanoparticles bound to germ tubes and yeast cells in a pattern resembling the localization of Als1, indicating Als1 also plays a role. Nanoparticle surface charge was shown to influence binding – positively charged amine-functionalized nanoparticles failed to bind to the hyphal cell wall. Binding of carboxylate-functionalized nanoparticles was observed in the presence of serum, though interactions were reduced. These observations show that Als3 and Als1 are important targets for nanoparticle-mediated diagnostics and therapeutics, and provide direction for optimal diameter and surface characteristics of nanoparticles that bind to the fungal cell wall. Science Foundation Ireland Whitaker International Program of the IEE

Multifunctional nanoparticles that actively target specific cells are promising tools for cancer diagnosis and therapy. In this article we review the synthesis and surface chemistry of Fe–Au nanorods and their characterization using microscopy. The diameter of the rods used in this study was selected to be 150–200 nm so that they did not enter the cells. The 80 nm-long Au tips of the nanorods were functionalized with heregulin (HRG), and the micron-long Fe portion was coated with a poly(ethylene glycol) monolayer to minimize non-specific interactions. Nanorods functionalized with HRG were found to preferentially bind to MCF7 cells that express high levels of the receptor tyrosine-protein kinase ErbB2/3. Magnetic tweezers measurements were used to characterize the kinetic properties of the bond between the HRG on the rods and ErbB2/3 on the surface of the cells. The strong magnetization of Fe–Au nanorods makes them excellent candidates for in-vitro and in-vivo imaging, and magnetic therapeutic applications targeting cancer cells in circulation. Science Foundation Ireland Erasmus Mundus Gulf Countries Postdoctoral Fellowship Marie Curie Intra-European Fellowship

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) play a crucial role in cell-cell interactions during nervous system development and function. The Aplysia CAM (apCAM), an invertebrate IgCAM, shares structural and functional similarities with vertebrate NCAM and therefore has been considered as the Aplysia homolog of NCAM. Despite these similarities, the binding properties of apCAM have not been investigated thus far. Using magnetic tweezers, we applied physiologically relevant, constant forces to apCAM-coated magnetic particles interacting with apCAM-coated model surfaces and characterized the kinetics of bond rupture. The average bond lifetime decreased with increasing external force, as predicted by theoretical considerations. Mathematical simulations suggest that the apCAM homophilic interaction is mediated by two distinct bonds, one involving all five immunoglobulin (Ig)-like domains in an antiparallel alignment and the other involving only two Ig domains. In summary, this study provides biophysical evidence that apCAM undergoes homophilic interactions, and that magnetic tweezers-based, force-clamp measurements provide a rapid and reliable method for characterizing relatively weak CAM interactions. European Commission Science Foundation Ireland National Institutes of Health

Aplysia californica neurons comprise a powerful model system for quantitative analysis of cellular and biophysical properties that are essential for neuronal development and function. The Aplysia cell adhesion molecule (apCAM), a member of the immunoglobulin superfamily of cell adhesion molecules, is present in the growth cone plasma membrane and involved in neurite growth, synapse formation, and synaptic plasticity. apCAM has been considered to be the Aplysia homolog of the vertebrate neural cell adhesion molecule (NCAM); however, whether apCAM exhibits similar binding properties and neuronal functions has not been fully established because of the lack of detailed binding data for the extracellular portion of apCAM. In this work, we used the atomic force microscope to perform single-molecule force spectroscopy of the extracellular region of apCAM and show for the first time (to our knowledge) that apCAM, like NCAM, is indeed a homophilic cell adhesion molecule. Furthermore, like NCAM, apCAM exhibits two distinct bonds in the trans configuration, although the kinetic and structural parameters of the apCAM bonds are quite different from those of NCAM. In summary, these single-molecule analyses further indicate that apCAM and NCAM are species homologs likely performing similar functions.

Nanoparticles (NPs) are now used in numerous technologies and serve as carriers for several new classes of therapeutics. Studies of the distribution of NPs in vivo demonstrate that they can be transported through biological barriers and are concentrated in specific tissues. Here, transport behavior, and final destination of polystyrene NPs are reported in primary mouse cortical neurons and SH-SY5Y cells, cultured in two-compartmental microfluidic devices. In both cell types, negative polystyrene NPs (PS(-)) smaller than 100 nm are taken up by the axons, undergo axonal retrograde transport, and accumulate in the somata. Examination of NP transport reveals different transport mechanisms depending on the cell type, particle charge, and particle internalization by the lysosomes. In cortical neurons, PS(-) inside lysosomes and 40 nm positive polystyrene NPs undergo slow axonal transport, whereas PS(-) outside lysosomes undergo fast axonal transport. Inhibition of dynein in cortical neurons decreases the transport velocity and cause a dose-dependent reduction in the number of accumulated PS(-), suggesting that the fast axonal transport is dynein mediated. These results show that the axonal retrograde transport of NPs depends on the endosomal pathway taken and establishes a means for screening nanoparticle-based therapeutics for diseases that involve neurons.

We present a microfluidic chip that generates linear concentration gradients of multiple solutes that are orthogonally-aligned to each other. The kinetics of gradient formation was characterized using a fluorescent tracer matching the molecular weight of small inhibitory drugs. Live-cell signalling and motility experiments were conducted to demonstrate the potential uses and advantages of the device. A431 epidermoid carcinoma cells, where EGF induces apoptosis in a concentration-dependent manner, were simultaneously exposed to gradients of MEK inhibitor and EGF receptor (EGFR) inhibitor. By monitoring live caspase activation in the entire chip, we were able to quickly assess the combinatorial interaction between MEK and EGFR pathways, which otherwise would require costly and time consuming titration experiments. We also characterized the motility and morphology of MDA-MB-231 breast cancer cells exposed to orthogonal gradients of EGF and EGFR inhibitor. The microfluidic chip not only permitted the quantitative analysis of a population of cells exposed to drug combinations, but also enabled the morphological characterization of individual cells. In summary, our microfluidic device, capable of establishing concentration gradients of multiple compounds over a group of cells, facilitates and accelerates in vitro cell biology experiments, such as those required for cell-based drug combination assays. Higher Education Authority Science Foundation Ireland Marie Curie Intra-European Fellowship AXA Research Fund Doctoral Fellowship Erasmus scholarship

Multifunctional nanoparticles that actively target-specific tissues are studied for cancer diagnosis and treatment. Magnetically and optically active particles are of particular interest because they enable multiple imaging modalities and physically modulated therapies, such as magnetic hyperthermia. Fe–Au nanorods are synthesized that have a long iron segment, coated with polyethylene glycol, and a short gold tip functionalized with heregulin (HRG), a known ligand of ErbB family of receptors. HRG–nanorods preferentially target MCF7 cells relative to MDA-MB-231 cells, as demonstrated in a novel microfluidics device. Targeting rates of these classical breast cancer cells correlate with their differential expression of ErbB2/3 receptors. HRG–nanorod binding stimulates the extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in MCF7 cells. The increase in ERK phosphorylation is linked to 'active zones,' dynamic regions in the cell periphery, which exhibit higher rates of particle binding than the rest of the cell. Periodically stretching cells using magnetic tweezers further activates ERK, which leads to cell death in cells co-treated with B-Raf inhibitors, through ERK hyperactivation. Although to a lesser extent, cell death is also achieved through magnetic hyperthermia. These results demonstrate nanoscale targeting and localized mechanochemical treatment of specific cancer cell lines based on their receptor expression using multifunctional nanoparticles. Science Foundation Ireland Nanoremedies Programme Programme for Research in Third-Level Institutions European Regional Development Fund Erasmus Mundus Gulf Countries Postdoctoral Fellowship Marie Curie Intra-European Fellowship

A new method has been developed for measuring the binding affinity of phage displayed peptides and a target protein using magnetic particles. The specific interaction between the phage displayed peptides and the target protein was subject to a force generated by the magnetic particle. The binding affinity was obtained by analyzing the force–bond lifetime.

Growth cones, dynamic structures at axon tips, integrate chemical and physical stimuli and translate them into coordinated axon behaviour, e.g., elongation or turning. External force application to growth cones directs and enhances axon elongation in vitro; however, direct mechanical stimulation is rarely combined with chemotactic stimulation. We describe a microfluidic device that exposes isolated cortical axons to gradients of diffusing and substrate-bound molecules, and permits the simultaneous application of piconewton (pN) forces to multiple individual growth cones via magnetic tweezers. Axons treated with Y-27632, a RhoA kinase inhibitor, were successfully towed against Semaphorin 3A gradients, which repel untreated axons, with less than 12 pN acting on a small number of neural cell adhesion molecules. Treatment with Y-27632 or monastrol, a kinesin-5 inhibitor, promoted axon towing on substrates coated with chondroitin sulfate proteoglycans, potent axon repellents. Thus, modulating key molecular pathways that regulate contractile stress generation in axons counteracts the effects of repellent molecules and promotes tension-induced growth. The demonstration of parallel towing of axons towards inhibitory environments with minute forces suggests that mechanochemical stimulation may be a promising therapeutic approach for the repair of the damaged central nervous system, where regenerating axons face repellent factors over-expressed in the glial scar.

A technique has been developed to apply force to the antibody-antigen complex in a solid-phase immunoassay. Force was applied to the immunochemical complex by labeling the secondary antibody with a magnetically susceptible, micrometer-size particle and placing the assay chamber in a magnetic field of defined magnitude and orientation. The force was strong enough to displace weakly bound particles but was not strong enough to rupture the immunochemical complex. The number of particles bound to the surface after applying the differentiation force was related to the analyte concentration, thus an optical detection scheme was developed for counting the number of particles on the surface. The sensitivity of the force differentiation assay was demonstrated to be one to two orders of magnitude higher than conventional solid-phase immunoassay techniques for model protein, virus, and bacterial analytes, with 99% specificity. The enhanced sensitivity of this assay appears to result from lowering the assay background through the identification of weakly adhesive, nonspecific interactions. (C) 2000 Academic Press.

The end-to-end stretching of a duplex DNA oligonucleotide has been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) simulations and atomic force microscopy (AFM) experiments. Near quantitative agreement between the calculations and experiments was obtained for both the extension length and forces associated with strand separation. The PMF calculations show that the oligonucleotide extends without a significant energetic barrier from a length shorter than A-DNA to a length 2.4 times the contour length of B-DNA at which the barrier to strand separation is encountered. Calculated forces associated with the barrier are 0.09 +/- 0.03 nN, based on assumptions concerning tip and thermal-activated barrier crossing contributions to the forces. Direct AFM measurements show the oligonucleotide strands separating at 2.6 +/- 0.8 contour lengths with a force of 0.13 +/- 0.05 nN. Analysis of the energies from the MD simulations during extension reveals compensation between increases in the DNA-self energy and decreases in the DNA-solvent interaction energy, allowing for the barrierless extension of DNA beyond the canonical B form. The barrier to strand separation occurs when unfavorable DNA interstrand repulsion cannot be compensated for by favorable DNA-solvent interactions. The present combination of single molecule theoretical and experimental approaches produces a comprehensive picture of the free energy surface of biological macromolecular structural transitions.

Magnetic tweezers (MTW) enable highly accurate forces to be transduced to molecules to study mechanotransduction at the molecular or cellular level. We review recent MTW studies in single molecule and cell biophysics that demonstrate the flexibility of this technique. We also discuss technical advances in the method on several fronts, i.e., from novel approaches for the measurement of torque to multiplexed biophysical assays. Finally, we describe multi-component nanorods with enhanced optical and magnetic properties and discuss their potential as future MTW probes. European Commission - European Regional Development Fund Higher Education Authority Science Foundation Ireland Marie Curie Intra-European Fellowship

Molecular recognition forms the basis for assembly and regulation in living organisms. We have used the atomic force microscope (AFM) to study the interaction of a model receptor streptavidin with its ligand biotin under physiological conditions. Surfaces functionalized with biotin and streptavidin exhibited adhesive forces 3-8 times greater than the nonspecific interactions observed between blocked streptavidin and biotinylated surfaces. The magnitude and distribution of the observed adhesive forces suggest they result from individual streptavidin-biotin interactions. This technique provides a means to directly study molecular recognition interactions at the molecular level. Nature has developed the unique ability for molecular recognition through the use of multiple noncovalent bonds (Le., van der Waals, hydrogen, ionic, and hydrophobic interactions) which possess a high degree of spatial and orientational specificity. Molecular recognition plays a central role in cellular behavior1 and the immunological response,2 and has also become the basis for a wide range of bioanalytical technique^.^ Although the structure and binding properties of molecular recognition systems can be measured, the forces involved in intermolecular interactions remain largely unknown. Molecular recognition interactions have been characterized through observations of the behavior of cells on which ligands and receptors naturally occur or have been atta~hed.~?~ Although these studies have largely been qualitative, the application of micropipet techniques to the study of cellular interactions5 has provided the meansfor micromanipulation with a force sensitivity of N. The surface force apparatus has also recently been used to characterize quantitatively the surface forces between model molecular recognition systems.6 The control of surface properties and the very small forces and distances involved in intermolecular interactions continue to limit the characterization of discrete molecular recognition interactions. The AFM has several properties that make it an ideal tool for measuring intermolecular forces: theoretical force sensitivity on the order of N, displacement sensitivity of 0.01 nm, contact areas as small as 10 nm2, and ability to operate under physiological condition^.^ In fact, the measurement of interactions as small as asingle hydrogen bond has recently been reported.8 The bond energies for specific molecular interactions fall between those typical * To whom correspondence should be addressed.

Neuronal growth cones are motile structures located at the end of axons that translate extracellular guidance information into directional movements. Despite the important role of growth cones in neuronal development and regeneration, relatively little is known about the topography and mechanical properties of distinct subcellular growth cone regions under live conditions. In this study, we used the AFM to study the P domain, T zone, and C domain of live Aplysia growth cones. The average height of these regions was calculated from contact mode AFM images to be 183 ± 33, 690 ± 274, and 1322 ± 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nano-indentation measurements indicate that the elastic moduli of the C domain and T zone ruffling region ranged between 3–7 and 7–23 kPa, respectively. The range of the measured elastic modulus of the P domain was 10–40 kPa. High resolution images of the P domain suggest its relatively high elastic modulus results from a dense meshwork of actin filaments in lamellipodia and from actin bundles in the filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering.

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three-dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force-distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F-actin bundles are stiffer than surrounding F-actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions.

During the past decade, the atomic force microscope (AFM) has become a key technique in biochemistry and biophysics to characterize supported lipid films, as testified by the continuous growth in the number of papers published in the field. The unique capabilities of AFM are: (i) capacity to probe, in real time and in aqueous environment, the surface structure of lipid films; (ii) ability to directly measure physical properties at high spatial resolution; (iii) possibility to modify the film structure and biophysical processes in a controlled way. Such experiments, published up to June 2000, are the focus of the present review. First, we provide a general introduction on the preparation and characterization of supported lipid films as well as on the principles of AFM. The section ‘Structural properties’ focuses on the various applications of AFM for characterizing the structure of supported lipid films: visualization of molecular structure, formation of structural defects, effect of external agents, formation of supported films, organization of phase-separated films (coexistence region, mixed films) and, finally, the use of supported lipid bilayers for anchoring biomolecules such as DNA, enzymes and crystalline protein arrays. The section ‘Physical properties’ introduces the principles of force measurements by AFM, interpretation of these measurements and their recent application to supported lipid films and related structures. Finally, we highlight the major achievements brought by the technique and some of the current limitations.

This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy (AFM) images of phase-separated Langmuir-Blodgett bilayers with imaging load. Force curves collected on pure monolayers of both distearoylphosphatidylethanolamine (DSPE) and monogalactosylethanolamine (MGDG) and dioleoylethanolamine (DOPE) deposited at similar surface pressures onto a monolayer of DSPE show an abrupt breakthrough event at a repeatable, material-dependent force. The breakthrough force for DSPE and MGDG is sizable, whereas the breakthrough force for DOPE is too small to measure accurately. Contact-mode AFM images on 1:1 mixed monolayers of DSPE/DOPE and MGDG/DOPE have a high topographic contrast at loads between the breakthrough force of each phase, and a low topographic contrast at loads above the breakthrough force of both phases. Frictional contrast is inverted and magnified at loads above the breakthrough force of both phases. These results emphasize the important role that surface forces and mechanics can play in imaging multicomponent biomembranes with AFM.

We are developing a biosensor that will measure, at the level of single molecules, the forces that bind DNA-DNA, antibody-antigen, or ligand-receptor pairs together. The Bead Array Counter (BARC) will use these interaction forces to hold magnetic microbeads to a solid substrate. Microfabricated magnetoresistive transducers on the substrate will indicate whether or not the beads are removed when pulled by magnetic forces. By adapting magnetoresistive computer memory technology, it may be possible to fabricate millions of transducers on a chip and detect or screen thousands of analytes. The multi-analyte capability of this portable sensor would be ideal for on-site testing, while the potential to directly gauge intermolecular interaction strengths suggests drug discovery applications.

Regeneration of central nervous system (CNS) axons into hostile environments such as the glial scar that forms after the spinal cord injury is a crucial medical problem. Successful therapies will combine pharmaceutical interventions with physical techniques that control the mechanical environment along the axon path. Axon growth cones are highly motile regions that constantly probe the surrounding tissue and integrate external chemotactic stimuli into motility decisions such as elongation, retraction or turning. While a range of chemical guidance molecules and subsequent signaling pathways are well established, the effect of mechanics on axon elongation is largely unknown. We describe a novel in vitro system that enables direct force application to large number of CNS growth cones that are fluidically isolated from their cell bodies and exposed to controlled gradients of diffusing guidance cues. Growth cones were preferentially targeted by anti-neuronal cell adhesion molecule (NCAM) magnetic particles. Particles exhibited low non-specific interaction with axon shafts and resulting in a massively parallel magnetic tweezers platform. Results suggested that pulling with

STM is one of the most promising techniques available for visualizing the fine details of biomolecular structure. It has been used to map the surface topography of inorganic materials in atomic dimensions, and thus has the resolving power not only to determine the conformation of small molecules but to distinguish site-specific features within a molecule. That level of detail is of critical importance in understanding the relationship between form and function in biological systems. The size, shape, and accessibility of molecular structures can be determined much more accurately by STM than by electron microscopy since no staining, shadowing or labeling with heavy metals is required, and there is no exposure to damaging radiation by electrons. Crystallography and most other physical techniques do not give information about individual molecules.We have obtained striking images of DNA and RNA, using calf thymus DNA and two synthetic polynucleotides, poly(dG-me5dC)·poly(dG-me5dC) and poly(rA)·poly(rU).