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In this work we show how hydrodynamic forces can be used to locally trap molecules in a supported lipid bilayer (SLB). The method uses the hydrodynamic drag forces arising from a flow through a conical pipette with a tip radius of 1-1.5 µm, placed approximately 1 µm above the investigated SLB. This results in a localized forcefield that acts on molecules protruding from the SLB, yielding a hydrodynamic trap with a size approximately given by the size of the pipette tip. We demonstrate this concept by trapping the protein streptavidin, bound to biotin receptors in the SLB. It is also shown how static and kinetic information about the intermolecular interactions in the lipid bilayer can be obtained by relating how the magnitude of the hydrodynamic forces affects the accumulation of protein molecules in the trap.
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Membrana Dobles de Lípidos , Colorantes Fluorescentes/química , CinéticaRESUMEN
We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions.
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Células Cultivadas/ultraestructura , Microscopía de Sonda de Barrido/instrumentación , Microscopía de Sonda de Barrido/métodos , Nanotecnología/instrumentación , Nanotecnología/métodos , Animales , Conductividad Eléctrica , Diseño de Equipo , Análisis de Falla de Equipo , Aumento de la Imagen/instrumentación , Aumento de la Imagen/métodos , Iones , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The acoustic response of conventional mechanical oscillators, such as a piezoelectric crystal, is predominantly harmonic at modest amplitudes. However, here, we observe from the electrical response that significant motional anharmonicity is introduced in the presence of attached analyte. Experiments were conducted with streptavidin-coated polystyrene microbeads of various sizes attached to a quartz crystal resonator via specific and nonspecific molecular tethers in liquid. Quantitative analysis reveals that the deviation of odd Fourier harmonics of the response caused by introduction of microbeads as a function of oscillation amplitude presents a unique signature of the molecular tether. Hence, the described anharmonic detection technique (ADT) based on this function allows screening of biomolecules and provides an additional level of selectivity in receptor-based detection that is often associated with nonspecific interactions. We also propose methods to extract mechanical force-extension characteristics of the molecular tether and activation energy using this technique.
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Cuarzo/química , Acústica , Microesferas , Modelos Moleculares , Física/métodos , Poliestirenos/química , Resistencia al Corte , Estreptavidina/química , Propiedades de SuperficieRESUMEN
The challenges with frequency-based acoustic detection systems in sensitive, selective, and reliable quantitative estimation of surface-bound analyte are well-known. These systems are traditionally used in their linear incarnations; i.e., the measurement frequency is the same as the driving frequency. However, it was found in this work that interactions of adsorbents with sensor surface show significant anharmonicity even at low drive amplitudes. In particular, using streptavidin-coated polystyrene microbeads on an oscillating quartz surface in air, it has been demonstrated through modeling and experiments that the anharmonic signal from microparticle to surface interaction is significantly higher relative to that from bare quartz and orders of magnitude higher than relative shifts in resonant frequency. The signal is proportional to the number of microparticles and holds a well-defined functional relationship with the amplitude of oscillation, distinct to the nature of interaction with the surface for a given analyte. This approach, thus, can be used for ultrasensitive and quantitative detection of surface adsorbents and characterization of different kinds of surface interactions, distinguishing specific from nonspecific adsorbents. The modeling also reveals a direct functional relationship between the measured anharmonic signal and the interaction potential of the adsorbent with the surface.
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Small molecule detection is of wide interest in clinical and industrial applications. However, its accessibility is still limited as miniaturisation and system integration is challenged in reliability, costs and complexity. Here we combined a 14.3 MHz quartz crystal resonator (QCR), actuated and analysed using a fixed frequency drive (FFD) method, with a nanomolecular imprinted polymer for label-free, realtime detection of N-hexanoyl-L-homoserine lactone (199 Da), a gram-negative bacterial infection biomarker. The lowest concentration detected (1 µM) without any optimisation was comparable with that of a BIAcore SPR system, an expensive laboratory gold standard, with significant enhancement in sensitivity and specificity beyond the state-of-the-art QCR. The analytical formula-based FFD method can potentially allow a multiplexed "QCR-on-chip" technology, bringing a paradigm shift in speed, accessibility and affordability of small molecule detection.
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Técnicas Biosensibles , Polímeros Impresos Molecularmente , Nanotecnología , Tecnicas de Microbalanza del Cristal de Cuarzo , Impresión Molecular , Sensibilidad y Especificidad , Técnicas de Síntesis en Fase SólidaRESUMEN
Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.
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Células/citología , Animales , Fenómenos Biomecánicos , Calibración , Supervivencia Celular , Células Epiteliales/citología , Eritrocitos/citología , Modelos Biológicos , Miocitos Cardíacos/citología , Neuronas/citología , Presión , Reproducibilidad de los ResultadosRESUMEN
We have developed a sensitive and economical method to directly detect bacteria, based on the interaction between the bacteria and specific antibodies attached to an oscillating surface. By monotonously increasing the amplitude of oscillation of a quartz crystal microbalance (QCM) coated with the antibody, the QCM can be used to sensitively detect the acoustic noise produced when the interactions between the bacteria and the surface were broken. We term this process rupture event scanning (REVS). The method is quantitative over at least 6 orders of magnitude and can detect as few as 10 bacteria. We demonstrate here that this approach allows one to arrange separation of bacteria and follow the process completion on the basis of the acoustic signal. Detection is not significantly affected by non-specific binding of sample contaminants and thus can be achieved both in buffer and in serum.
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Técnicas Biosensibles/métodos , Escherichia coli/aislamiento & purificación , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Staphylococcus aureus/aislamiento & purificación , Anticuerpos/metabolismo , Sitios de Unión de Anticuerpos , Tampones (Química) , Escherichia coli/metabolismo , Microscopía Electrónica de Rastreo/métodos , Sensibilidad y Especificidad , Staphylococcus aureus/metabolismoRESUMEN
Receptor-based detection of pathogens often suffers from non-specific interactions, and as most detection techniques cannot distinguish between affinities of interactions, false positive responses remain a plaguing reality. Here, we report an anharmonic acoustic based method of detection that addresses the inherent weakness of current ligand dependant assays. Spores of Bacillus subtilis (Bacillus anthracis simulant) were immobilized on a thickness-shear mode AT-cut quartz crystal functionalized with anti-spore antibody and the sensor was driven by a pure sinusoidal oscillation at increasing amplitude. Biomolecular interaction forces between the coupled spores and the accelerating surface caused a nonlinear modulation of the acoustic response of the crystal. In particular, the deviation in the third harmonic of the transduced electrical response versus oscillation amplitude of the sensor (signal) was found to be significant. Signals from the specifically-bound spores were clearly distinguishable in shape from those of the physisorbed streptavidin-coated polystyrene microbeads. The analytical model presented here enables estimation of the biomolecular interaction forces from the measured response. Thus, probing biomolecular interaction forces using the described technique can quantitatively detect pathogens and distinguish specific from non-specific interactions, with potential applicability to rapid point-of-care detection. This also serves as a potential tool for rapid force-spectroscopy, affinity-based biomolecular screening and mapping of molecular interaction networks.
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Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Esporas Bacterianas/aislamiento & purificación , Acústica , Anticuerpos Antibacterianos , Anticuerpos Inmovilizados , Bacillus subtilis/inmunología , Bacillus subtilis/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/estadística & datos numéricos , Técnicas Biosensibles/métodos , Técnicas Biosensibles/estadística & datos numéricos , Microscopía de Fuerza Atómica , Microesferas , Sistemas de Atención de Punto , Poliestirenos , Tecnicas de Microbalanza del Cristal de Cuarzo/estadística & datos numéricos , Esporas Bacterianas/inmunología , EstreptavidinaRESUMEN
A complete system for the simultaneous monitoring of multiple cantilever sensors from different sensor arrays has been developed and tested for gas- and liquid-phase applications. The cantilever sensors are operated in static-deflection mode and the readout is achieved with phase-shifting interferometric microscopy (PSIM). In contrast to existing cantilever-sensor readout methods, PSIM is not dependent on alignment and allows the monitoring of the entire displacement profiles of all cantilevers within the field of view, using just one light source. To complement the PSIM readout, we have developed a sample cell, which can hold multiple cantilever-array chips, allows for very fast and reproducible sensor-chip replacement, has very low sample-volume requirements, and allows for individual or common addressing of all chips in the sample cell. We demonstrate the functionality of our microcantilever sensor system with a setup that can monitor eight cantilevers from four different sensor chips simultaneously.