Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

University College London

The unique ability of living systems to translate biochemical reactions into mechanical work has inspired the design of synthetic DNA motors which generate nanoscale motion via controlled conformational change. However, while Nature has evolved intricate mechanisms to convert molecular shape change into specific micrometer-scale mechanical cellular responses, the integration of artificial DNA motors with mechanical devices presents a major challenge. Here we report the direct integration between an ensemble of DNA motors and an array of microfabricated silicon cantilevers. The forces exerted by the precise duplex to nonclassical i-motif conformational change were probed via differential measurements using an in-situ reference cantilever coated with a nonspecific sequence of DNA. Fueled by the addition of protons, the open to close stroke of the motor induced 32 +/- 3 mN/m compressive surface stress, which corresponds to a single motor force of approximately 11 pN/m, an order of magnitude larger than previous classical hybridization studies. Furthermore, the surface-tethered conformational change was found to be highly reversible, in contrast to classical DNA motors which typically suffer rapid system poisoning. The direction and amplitude of motor-induced cantilever motion was tuneable via control of buffer pH and ionic strength, indicating that electrostatic forces play an important role in stress generation. Hybrid devices which directly harness the multiple accessible conformational states of dynamic oligonucleotides and aptamers, translating biochemical energy into micromechanical work, present a radical new approach to the construction of "smart" nanoscale machinery and mechano-biosensors.

DNA molecular motor driven micromechanical cantilever arrays.

Surface charge patterns generated by atomic force microscopy-based charge writing were used to pattern amyloid-like peptide fibrils on a solid substrate. Fibrils of the short peptide TTR105-115 were encapsulated inside water droplets of a water-in-perfluorocarbon oil emulsion and retained their rod morphology. They were observed to deposit selectively with a lateral resolution of approximately 1 microm onto negatively charged patterns on a polymethyl-methacrylate substrate.

Patterning amyloid peptide fibrils by AFM charge writing.

Synthetic, amyloid-like peptide fibrils have recently attracted interest as a novel, potentially biocompatible material for applications in biotechnology and tissue-engineering. In this paper, we report atomic force microscopy (AFM) studies of the morphology and mechanical stability of fibrils self-assembled in vitro from the short peptide TTR(105-115), which serves as a model system for amyloid fibrils. It forms predominantly straight rods of approximately 1 microm in length and of diameters between 7 nm and 12 nm. We found polymorphism, with some fibrils exhibiting an unstructured morphology and others showing a regular, longitudinal surface pattern of 90 nm periodicity. Contact mode AFM-imaging in air was utilised to perform mechanical tests of individual fibrils on the nanometer scale with a defined, vertical force in the nN-range applied by the AFM-tip. Above 100 nN, all fibrils showed a permanent, mechanical deformation whereas below 40 nN, fibrils remained unaffected. Tapping-mode AFM-imaging in water led to fibril decomposition within 1.5 h whereas tapping-mode imaging in air left fibrils intact. Additional investigations by circular-dichroism spectroscopy showed that dispersed fibrils were structurally stable in aqueous solution between pH 3 and pH 8, and in sodium phosphate buffer of concentration between 50 mM and 1 M.

Morphology and mechanical stability of amyloid-like peptide fibrils.

Free-standing cantilevers, which directly translate specific biochemical reactions into micromechanical motion, have recently attracted much attention as label-free biosensors and micro/nano robotic devices. To exploit this mechanochemical sensing technology, it is essential to develop a fundamental understanding of the origins of surface stress. Here we report a detailed study into the molecular basis of stress generation in aqueous environments focusing on the pH titration of model mercaptohexadecanoic acid self-assembled monolayers (SAMs), using in situ reference cantilevers coated with nonionizable hexadecanethiol SAMs. Semiautomated data analysis and a statistical model were developed to quantify cyclic deprotonation/protonation reactions on multiple arrays. In-plane force titrations were found to have the sensitivity to detect ionic hydrogen bond formation between protonated and nonprotonated carboxylic acid groups in the proximity of the surface pK1/2, which generated a mean tensile differential surface stress of +1.2 +/- 0.3 mN/m at pH 6.0, corresponding to 1 pN attractive force between two adjacent MHA molecules. Conversely, the magnitude of compressive differential surface stress was found to increase progressively with pH >/= 7.0, reaching a maximum of -14.5 +/- 0.5 mN/m at pH 9.0, attributed to enhanced electrostatic repulsion between deprotonated carboxylic acid groups. However, striking differences were observed in the micromechanical responses to different ionic strength and ion species present in the aqueous environment, highlighting the critical role of counter- and co-ions on surface stress. Our findings provide fundamental insights into the molecular mechanisms of in-plane mechanochemistry, which may be exploited for biosensing and nanoactuation applications.

Investigating the molecular mechanisms of in-plane mechanochemistry on cantilever arrays.

The alarming growth of the antibiotic-resistant superbugs methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) is driving the development of new technologies to investigate antibiotics and their modes of action. We report the label-free detection of vancomycin binding to bacterial cell wall precursor analogues (mucopeptides) on cantilever arrays, with 10 nM sensitivity and at clinically relevant concentrations in blood serum. Differential measurements have quantified binding constants for vancomycin-sensitive and vancomycin-resistant mucopeptide analogues. Moreover, by systematically modifying the mucopeptide density we gain new insights into the origin of surface stress. We propose that stress is a product of a local chemical binding factor and a geometrical factor describing the mechanical connectivity of regions activated by local binding in terms of a percolation process. Our findings place BioMEMS devices in a new class of percolative systems. The percolation concept will underpin the design of devices and coatings to significantly lower the drug detection limit and may also have an impact on our understanding of antibiotic drug action in bacteria.

Nanomechanical detection of antibiotic-mucopeptide binding in a model for superbug drug resistance.

Biologically inspired cantilever systems which transform biochemical reactions into nanomechanical motion have attracted attention for label-free biosensing and nanorobotic applications. Here, we take advantage of chemically programmable proton-driven reactions to actuate both the direction and amplitude of nanomechanical cantilever motion in aqueous environments, corresponding to femto-Newton single molecule surface stress. By altering the end groups of self-assembled coatings, we deconvolute the dominant role of surface charge over hydrophilic/hydrophobic interactions and attribute reference cantilever signals to the silicon underside of the cantilever. These findings and underlying concepts will lead to the next generation of massively parallel intelligent nanomechanical systems triggered by self-assembled reactions.

Chemically programmed nanomechanical motion of multiple cantilever arrays.

Because physical form and function are intimately linked, mechanisms that maintain cell shape and size within strict limits are likely to be important for a wide variety of biological processes. However, while intrinsic controls have been found to contribute to the relatively well-defined shape of bacteria and yeast cells, the extent to which individual cells from a multicellular animal control their plastic form remains unclear. Here, using micropatterned lines to limit cell extension to one dimension, we show that cells spread to a characteristic steady-state length that is independent of cell size, pattern width, and cortical actin. Instead, homeostatic length control on lines depends on a population of dynamic microtubules that lead during cell extension, and that are aligned along the long cell axis as the result of interactions of microtubule plus ends with the lateral cell cortex. Similarly, during the development of the zebrafish neural tube, elongated neuroepithelial cells maintain a relatively well-defined length that is independent of cell size but dependent upon oriented microtubules. A simple, quantitative model of cellular extension driven by microtubules recapitulates cell elongation on lines, the steady-state distribution of microtubules, and cell length homeostasis, and predicts the effects of microtubule inhibitors on cell length. Together this experimental and theoretical analysis suggests that microtubule dynamics impose unexpected limits on cell geometry that enable cells to regulate their length. Since cells are the building blocks and architects of tissue morphogenesis, such intrinsically defined limits may be important for development and homeostasis in multicellular organisms.

A polarised population of dynamic microtubules mediates homeostatic length control in animal cells.

Polycrystalline gold films coated with thiol-based self-assembled monolayers (SAM) form the basis of a wide range of nanomechanical sensor platforms. The detection of adsorbates with such devices relies on the transmission of mechanical forces, which is mediated by chemically derived stress at the organic-inorganic interface. Here, we show that the structure of a single 300-nm-diameter facetted gold nanocrystal, measured with coherent X-ray diffraction, changes profoundly after the adsorption of one of the simplest SAM-forming organic molecules. On self-assembly of propane thiol, the crystal's flat facets contract radially inwards relative to its spherical regions. Finite-element modelling indicates that this geometry change requires large stresses that are comparable to those observed in cantilever measurements. The large magnitude and slow kinetics of the contraction can be explained by an intermixed gold-sulphur layer that has recently been identified crystallographically. Our results illustrate the importance of crystal edges and grain boundaries in interface chemistry and have broad implications for the application of thiol-based SAMs, ranging from nanomechanical sensors to coating technologies.

Differential stress induced by thiol adsorption on facetted nanocrystals.

Micromechanic resonators provide a small-volume and potentially high-throughput method to determine rheological properties of fluids. Here we explore the accuracy in measuring mass density and viscosity of ethanol-water and glycerol-water model solutions, using a simple and easily implemented model to deduce the hydrodynamic effects on resonating cantilevers of various length-to-width aspect ratios. We next show that these measurements can be extended to determine the alcohol percentage of both model solutions and commercial beverages such as beer, wine and liquor. This demonstrates how micromechanical resonators can be used for quality control of every-day drinks.

Using micromechanical resonators to measure rheological properties and alcohol content of model solutions and commercial beverages.

The alarming rise in drug-resistant hospital 'superbugs' and the associated increase in fatalities is driving the development of technologies to search for new antibiotics and improve disease diagnostics. One of the most successful drug targets is the bacterial cell wall, an evolutionary feature of virtually all prokaryotes and vital for their survival by providing mechanical strength. The recent discovery of bacterial cytoskeletal proteins analogous to the key force-bearing machinery in eukaryotes also provides new opportunities for drug discovery, but little is known about their mechanical role in bacteria. In the present short article, I review recent developments in the field of nanotechnology to investigate the mechanical mechanisms of action of potent antibiotics on cell wall and cytoskeletal targets with unprecedented spatial, temporal and force resolution and the development of a new generation of nanomechanical devices to detect pathogens for point-of-care diagnostics.

Rachel A. McKendry

London Centre for Nanotechnology and Departments of Medicine

University College London

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

Rachel A. McKendry

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University College London

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

London Centre for Nanotechnology and Departments of Medicine