Optical diffraction for measurements of nano-mechanical bending

Micromechanical transducers such as cantilevers for AFM often rely on optical readout methods that require illumination of a specific region of the microstructure. Here we explore and exploit the diffraction effects that have been previously neglected when modeling cantilever bending measurement techniques. The illumination of a cantilever end causes an asymmetric diffraction pattern at the photodetector that significantly affects the calibration of the signal in the popular optical beam deflection technique (OBDT). Conditions for optimized linear signals that avoid detection artifacts conflict with small numerical aperture illumination and narrow cantilevers which are softer and therefore more sensitive. Embracing diffraction patterns as a physical measurable allows a richer detection technique that decouples measurements of tilt and curvature and simultaneously relaxes the requirements on the alignment of illumination and detector. We show analytical results, numerical simulations and physiologically relevant experimental data demonstrating the usefulness of these diffraction features. We offer experimental design guidelines and identify and quantify possible sources of systematic error of up to 10% in OBDT. We demonstrate a new nanometre resolution detection method that can replace OBDT, where Frauenhofer and Bragg diffraction effects from finite sized and patterned cantilevers are exploited. Such effects are readily generalized to arrays, and allow transmission detection of mechanical curvature, enabling in-line instruments. In particular, a cantilever with a periodic array of slots produces Bragg peaks which can be analyzed to deduce the cantilever curvature. We highlight the comparative advantages over OBDT by detecting molecular activity of antibiotic Vancomycin, with an RMS noise equivalent to less than $2.5 \mu M$ (1.5 nm), as example of possible multi-maker bio-assays.Comment: 9 pages, 8 figure

Nanomechanical sensors: Measuring a response in blood

Nanomechanical cantilevers can determine the concentration of active drugs in human serum

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 solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity(1-5). The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions. Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures(1,6,7). We developed a new model(1) which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly, revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections

Pharmacokinetic Characterisation and Comparison of Bioavailability of Intranasal Fentanyl, Transmucosal, and Intravenous Administration through a Three-Way Crossover Study in 24 Healthy Volunteers

Background. For more than 60 years, the synthetic opioid fentanyl has been widely used in anaesthesia and analgesia. While the intravenous formulation is primarily used for general anaesthesia and intensive care settings, the drug’s high lipophilic properties also allow various noninvasive routes of administration. Published data suggest that intranasal administration is also attractive for use as intranasal patient-controlled analgesia (PCA). A newly developed intranasal fentanyl formulation containing 47 μg fentanyl, intravenous fentanyl, and oral transmucosal fentanyl citrate were characterised, and bioavailability was compared to assess the suitability of the intranasal formulation for an intranasal PCA product. Methods. 27 healthy volunteers were enrolled in a single-centre, open-label, randomised (order of treatments), single-dose study in a three-period crossover design. The pharmacokinetics of one intranasal puff of fentanyl formulation (47 μg, 140 mL per puff), one short intravenous infusion of 50 μg fentanyl, and one lozenge with an integrated applicator (200 μg fentanyl) were studied, and bioavailability was calculated. Blood samples were collected over 12 hours, and plasma concentrations of fentanyl were determined by HPLC with MS/MS detection. Results. 24 volunteers completed the study. The geometric mean of AUC0-tlast was the highest with oral transmucosal administration (1106 h  pg/ml, CV% = 32.86), followed by intravenous (672 h  pg/ml, CV% = 32.18) and intranasal administration (515 h  pg/ml, CV% = 30.10). Cmax was 886 pg/ml (CV% = 59.38) for intravenous, 338 pg/ml (CV% = 45.61) for intranasal, and 310 pg/ml (CV% = 29.58) for oral transmucosal administration. tmax was shortest for intravenous administration (0.06 h, SD = 0.056), followed by intranasal (0.21 h, SD = 0.078) and oral transmucosal administration (1.20 h, SD = 0.763). Dose-adjusted absolute bioavailability was determined to be 74.70% for the intranasal formulation and 41.25% for the oral transmucosal product. In total, 38 adverse events (AEs) occurred. Fourteen AEs were potentially related to the investigational items. No serious AE occurred. Conclusion. Pharmacokinetic parameters and bioavailability of the investigated intranasal fentanyl indicated suitability for its intended use as an intranasal PCA option

Modified cantilever arrays improve sensitivity and reproducibility of nanomechanical sensing in living cells

Mechanical signaling involved in molecular interactions lies at the heart of materials science and biological systems, but the mechanisms involved are poorly understood. Here we use nanomechanical sensors and intact human cells to provide unique insights into the signaling pathways of connectivity networks, which deliver the ability to probe cells to produce biologically relevant, quantifiable and reproducible signals. We quantify the mechanical signals from malignant cancer cells, with 10 cells per ml in 1000-fold excess of non-neoplastic human epithelial cells. Moreover, we demonstrate that a direct link between cells and molecules creates a continuous connectivity which acts like a percolating network to propagate mechanical forces over both short and long length-scales. The findings provide mechanistic insights into how cancer cells interact with one another and with their microenvironments, enabling them to invade the surrounding tissues. Further, with this system it is possible to understand how cancer clusters are able to co-ordinate their migration through narrow blood capillaries

Modified Cantilever Arrays Improve Sensitivity and Reproducibility of Nanomechanical Sensing in Living Cells

Mechanical signaling involved in molecular interactions lies at the heart of materials science and biological systems, but the mechanisms involved are poorly understood. Here we use nanomechanical sensors and intact human cells to provide unique insights into the signaling pathways of connectivity networks, which deliver the ability to probe cells to produce biologically relevant, quantifiable and reproducible signals. We quantify the mechanical signals from malignant cancer cells, with 10 cells per ml in 1000-fold excess of non-neoplastic human epithelial cells. Moreover, we demonstrate that a direct link between cells and molecules creates a continuous connectivity which acts like a percolating network to propagate mechanical forces over both short and long length-scales. The findings provide mechanistic insights into how cancer cells interact with one another and with their microenvironments, enabling them to invade the surrounding tissues. Further, with this system it is possible to understand how cancer clusters are able to co-ordinate their migration through narrow blood capillaries

Decoupling competing surface binding kinetics and reconfiguration of receptor footprint for ultrasensitive stress assays

Cantilever arrays have been used to monitor biochemical interactions and their associated stress. However, it is often necessary to passivate the underside of the cantilever to prevent unwanted ligand adsorption, and this process requires tedious optimization. Here, we show a way to immobilize membrane receptors on nanomechanical cantilevers so that they can function without passivating the underlying surface. Using equilibrium theory, we quantitatively describe the mechanical responses of vancomycin, human immunodeficiency virus type 1 antigens and coagulation factor VIII captured on the cantilever in the presence of competing stresses from the top and bottom cantilever surfaces. We show that the area per receptor molecule on the cantilever surface influences ligand-receptor binding and plays an important role on stress. Our results offer a new way to sense biomolecules and will aid in the creation of ultrasensitive biosensors

Surface mediated cooperative interactions of drugs enhance mechanical forces for antibiotic action

The alarming increase of pathogenic bacteria that are resistant to multiple antibiotics is now recognized as a major health issue fuelling demand for new drugs. Bacterial resistance is often caused by molecular changes at the bacterial surface, which alter the nature of specific drug-target interactions. Here, we identify a novel mechanism by which drug-target interactions in resistant bacteria can be enhanced. We examined the surface forces generated by four antibiotics; vancomycin, ristomycin, chloroeremomycin and oritavancin against drug-susceptible and drug-resistant targets on a cantilever and demonstrated significant differences in mechanical response when drug-resistant targets are challenged with different antibiotics although no significant differences were observed when using susceptible targets. Remarkably, the binding affinity for oritavancin against drug-resistant targets (70 nM) was found to be 11,000 times stronger than for vancomycin (800 μM), a powerful antibiotic used as the last resort treatment for streptococcal and staphylococcal bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Using an exactly solvable model, which takes into account the solvent and membrane effects, we demonstrate that drug-target interactions are strengthened by pronounced polyvalent interactions catalyzed by the surface itself. These findings further enhance our understanding of antibiotic mode of action and will enable development of more effective therapies.We thank the EPSRC Interdisciplinary Research Centre in Nanotechnology (Cambridge, UCL, Bristol (GR/R45680/01), the EPSRC Grand Challenge in Nanotechnology for Healthcare (EP/G0620064/1), I-sense EPSRC IRC in Early Warning Sensing Systems for Infectious Diseases (EP/G062064/1), the EPSRC Speculative Engineering Program (EP/D50925/1), Royal Society (RS), Medicine Company Inc., USA, NHMRC Australia Fellowship (AF511105), UCL Graduate School Scholarship, UCL COMPLEX, Bio Nano Consulting (BNC), European Union FP7 Project VSMMART Nano (managed by BNC) and NHS Trusts Biomedical Research Centre (BRC) for funding

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

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 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 affected 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 impact on our understanding of antibiotic drug action in bacteria.Comment: Comments: This paper consists of the main article (6 pages, 5 figures) plus Supplemental Material (6 pages, 3 figures). More details are available at http://www.london-nano.co

Experimental and Computational Characterization of Biological Liquid Crystals: A Review of Single-Molecule Bioassays

Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM) have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins