No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Biomolecular Stiffness Detection Based on Positive Frequency Shift of CMOS Compatible Gigahertz Solidly Mounted Resonators
Abstract
In this work, gigahertz solidly mounted resonators (SMRs) (2.5 GHz) were designed and fabricated to construct a novel particle-resonator system to achieve the biomolecular stiffness sensing in real time. The positive frequency shift of the system was used to estimate the stiffness of biomolecules connecting between the SMR and attached particles. The working principle was revealed by the mathematical analysis of the general block-spring model of the system. Further interpretations about the mechanism of such elastic interaction from the perspective of acoustic resonant modes of SMRs were given by finite element method. Biotin-streptavidin, antibody and antigen binding system were used as model molecular linkers to study the frequency shift varied with different particle diameters and particle densities. Different linker stiffness was realized by adjusting the concentrations of antigens connected with particles which form specific binding with antibodies immobilized on the SMR. The results fairly agree with the simulation results demonstrating the proposed particle-resonator system as an effective method to realize the real-time biomolecular stiffness detection.
... Among the various detection technologies, mass-sensitive sensors based on piezoelectric resonators have attracted considerable attention for biomolecule detection during the last few decades due to their high selectivity and sensitivity. 10,11 In generally, these piezoelectric resonator sensors can be divided into two categories (determined by the transmission paths of acoustic wave resonance) surface acoustic wave (SAW) and bulk acoustic wave (BAW) sensors. However, the SAW sensors cannot achieve a higher sensitivity due to the restriction on dimension of the delay line for the interdigital transducer (IDT) and the resonant frequency (from 30 MHz to 1 GHz). ...
... However, the SAW sensors cannot achieve a higher sensitivity due to the restriction on dimension of the delay line for the interdigital transducer (IDT) and the resonant frequency (from 30 MHz to 1 GHz). 12,13 In addition, the quartz crystal microbalance (QCM) employed in BAW sensors has a mass detection limit in the order of nanograms determined from their low operation frequency (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) due to the bulk quartz substrate thickness. Thus BAW sensors do not have adequate sensitivity for the detection of small molecules in low concentrations. ...
We report the fabrication of a solidly mounted resonator (SMR) that can also function as a sensor for biological molecules. The SMR, consisting of a Au electrode, aluminum nitride (AlN) piezoelectric thin film and Bragg acoustic reflector, was fabricated on a Si substrate by radio frequency (RF) magnetron sputtering. The Bragg acoustic reflector, made entirely of metal, has small internal stress and good heat conduction. Human immunoglobulin G (IgG) antibody was immobilized on the modified (by self-assembled monolayer method) Au electrode surface of the SMR and goat anti-human IgG antigen was captured through the specificity of bond between the antibody and antigen on the electrode surface. We found a linear relationship between the resonant frequency shift and the concentration of goat anti-human IgG antigen for concentrations smaller than 0.4 mg ml-1 and a relatively constant frequency shift for concentrations greater than 0.5 mg ml-1. A series of interference experiments can prove that the selectivity of the sensor is satisfactory. Our findings suggest that the SMR sensor is an attractive alternative for biomolecule detection.
... The more detailed information can be found in Figure S1 and our previous studies. 35,37 Experimental Setup. After fabrication, the device was attached to an evaluation board. ...
Diffusion limitations and nonspecific surface absorption are great challenges for developing micro-/nanoscale affinity biosensors. There are very limited approaches that can solve these issues at the same time. Here, an acoustic streaming approach enabled by a gigahertz (GHz) resonator is presented to promote mass transfer of analytes through the jet mode and biofouling removal through the shear mode, which can be switched by tuning the deviation angle, α, between the resonator and the sensor. Simulations show that the jet mode (α ≤ 0) drives the analytes in the fluid toward the sensing surface, overcomes the diffusion limitation, and enhances the binding; while the shear mode (0 < α < π/4) provides a scouring action to remove the biofouling from the sensor. Experimental studies were performed by integrating this GHz resonator with optoelectronic sensing systems, where a 34-fold enhancement for the initial binding rate was obtained. Featuring high efficiency, controllability, and versatility, we believe that this GHz acoustic streaming approach holds promise for many kinds of biosensing systems as well as lab-on-chip systems.
Viral particles bind to receptors through multivalent protein interactions. Such high avidity interactions on sensor surfaces are less studied. In this work, three polyelectrolytes that can form biosensing surfaces with different interfacial characteristics in probe density and spatial arrangement were designed. Quartz crystal microbalance, interferometry and atomic force microscopy were used to study their surface density and binding behaviors with proteins and virus particles. A multivalent adsorption kinetic model was developed to estimate the number of bonds from the viral particles bound to the polyelectrolyte surfaces. Experimental results show that the heterogeneous 3D surface with jagged forest-like structure enhances the virus capture ability by maximizing the multivalent interactions. As a proof of concept, specific coronavirus detection was achieved in spiked swab samples. These results indicate the importance of both probe density and their spatial arrangement on the sensing performance, which could be used as a guideline for rational biosensing surface design.
We have developed a hybrid device that fuses the techniques of acoustofluidic localization and resonator-based mass sensing of particles in liquid-phase for in-situ analysis in a
droplet. An elliptic plate resonator (EPR) was used in this work to verify the capability of acoustofluidic manipulation on a resonator chip. Particles were focused on the EPR by the acoustic tweezers to increase the effectiveness of detection. The EPR has a reasonably high liquid-phase quality factor (
) and low motional resistance (
) to allow the detection of focused particles in liquid. Moreover, the resonator chip can be detached from the acoustic tweezing device without damage. This allows replacement of just the chip when required. A potential application of this work is organizing and sensing cells on-chip using the concepts herein.
This work shows a differential rotational thermal piezoresistive resonator (DR-TPR) design, fabrication, and characterization. The resonator uses silicon on insulator (SOI) microelectromechanical systems (SOI MEMS) technology. The sensing of carboxyl-polystyrene beads requires estimating the variation in the frequency of the resonator. It can serve as an alternative enabling technology to detect small molecules having wide implementations in clinical and industrial applications. The resonator uses a negative frequency shift to find the change in the mass due to the chemical absorption of the carboxyl-polystyrene beads on the DR-TPR surface. The method of using DR-TPR can attain label-free detection for many future biomarkers. The use of carboxyl-polystyrene beads to showcase the potential of DR-TPR as a mass sensor for future chemical/biosensing applications is the highlight of the paper. It includes the characterization in different concentrations of immobilization on the resonator surface. The work shows the operation of the resonator in an ionic buffer solution which is crucial for its future applications. The Q value of 80 with differential feedthrough cancellation is measured in phosphate-buffered saline (PBS) using DR-TPR.
Detection sensitivity is one of the essential indices for nano-resonator-based sensing. Multiple mechanisms, such as the counteractive effects of mass and stiffness, have been revealed to contribute to the detection sensitivity, making the sensitivity analysis a complex and under-explored issue. In this study, we develop an analytical frequency model and quantitatively reveal how the detection sensitivity relies on the complex interactions between the nano-resonator-based sensor and the biochemical adsorbate for the measurement of elasticity and density. We further specify the detection limits and approaches to enhance the detection sensitivity for mechanical properties of biochemical adsorbate in nano-resonator-based label-free measurements. Results indicate that the detection sensitivity to elasticity/density could be exponentially enhanced by decreasing the elasticity/density ratio between the adsorbate and the nano-resonator until it reaches the limit. Besides, it is effective to maximize the detection sensitivity through the deposition of the biochemical adsorbate at specific regions combined with the optimization of its geometry according to the mechanical property to be measured. The maximum detection sensitivity to the elasticity relies on the distribution and geometry of the adsorbate more than that of the density. This study provides significant insights into the identification of biochemical nano-entities and helps to enhance the accuracy and capability of the nano-resonator-based sensors in mechanical measurements.
Here, we present a high performance uncooled near-infrared (NIR) detector comprising of a giga hertz (GHz) solidly mounted resonator (SMR) and gold nanorods (GNRs) arrays. By coupling the localized surface plasmon resonances of GNRs, the resonator system exhibits optimized optical response to vis-NIR region. Both simulation and experiments demonstrate the hybrid GNRs-SMR exhibit significantly enhanced optical responsive sensitivity of NIR, the tunable aspect ratios (AR) of GNRs enable resonator respond sensitively to selected light. Specially, taking advantage of the acoustofluidic effect of SMR, the GNRs can be controllably and precisely modified on the microchip surface in an ultra-short time, which addresses one of the most fundamental challenges in the localized functionalization of micro/nano scale surface. The presented work opens new directions in development of novel miniaturized, tunable NIR detector.
In this report, a solidly mounted resonator (SMR), consisting of an Au electrode, Mg-doped ZnO (MgXZn1−XO) piezoelectric film and Bragg acoustic reflector, was fabricated on a Si substrate by radio frequency (RF) magnetron sputtering. As a key processing step for the SMR, MgXZn1−XO films with high c-axis orientation were fabricated and the crystalline structure, surface morphology and roughness of the films were investigated. The surface morphology, optical transmittance and shape control of MgXZn1−XO films were investigated by the chemical wet-etching method with various etchants. The profiles and line patterns of MgXZn1−XO films etched with FeCl3·6H2O solutions are satisfactory and fully meet the industrial requirements. The Bragg acoustic reflector, made entirely of metal, has small internal stress and good heat conduction. An SMR based on a MgXZn1−XO piezoelectric film shows a resonant frequency of 2.402 GHz, and the keff², QS and QP of the SMR are 3.07%, 415 and 546, respectively.
Polyethyleneimine-modified single-wall carbon nanotubes were self-assembled as a sensitive coating for the micro-scale film bulk acoustic formaldehyde sensor. The film bulk acoustic resonator has a high working frequency (∼4.5 GHz) and can detect the ultra-small mass changes in the sensitive layer induced by the absorption of formaldehyde molecules. The proposed sensor demonstrated a reversible and linear response towards formaldehyde over the range of 50–400 ppb, with a limit of detection of 24 ppb. Both the response and recovery times were less than 1 min. The frequency response was improved with increasing coverage of carbon nanotubes on the resonator surface due to the larger surface area and adsorption space. The polyethyleneimine-modified single-wall carbon nanotubes provided an excellent selectivity towards formaldehyde over interfering organic vapors. The results show that the film bulk acoustic sensor is a promising candidate for the portable and convenient detection of indoor air pollution with high sensitivity.
A micro-scale gas sensor based on mass-sensitive film bulk acoustic resonator is demonstrated for the detection of trace formaldehyde at room temperature. The composites mixed with multiwalled carbon nanotubes and polyethyleneimine (MWNTs-PEI) were coated on the resonator surface as the sensitive layer to specifically absorb formaldehyde molecules using a facile spray process. The influence of spraying processes on the formaldehyde sensing properties were investigated. Different response behaviors were determined by both the chemical absorption between formaldehyde molecules and the amine functional groups on PEI and the increase of absorption surface came from the nanostructure. The combination of high frequency of the film bulk acoustic resonator (∼4.3 GHz) and the specific absorbability of MWNTs-PEI composites provided a high sensitivity in the detections of trace formaldehyde. The obtained ultra-low limit of detection was as low as 60 ppb with linear response, quick response/recovery time, good reproducibility and selectivity. The proposed sensor shows potential as a portable and convenient gas-sensing system for monitoring the low-level concentration of indoor air pollution.
Colloidal spheres attached to a quartz crystal microbalance (QCM) produce the so-called “coupled resonances”. They are resonators of their own, characterized by a particle resonance frequency, a resonance bandwidth, and a modal mass. When the frequency of the main resonator comes close to the frequency of the coupled resonance, the bandwidth goes through a maximum. A coupled resonance can be viewed as an absorption line in acoustic shear-wave spectroscopy. The known concepts from spectroscopy apply. This includes the mode assignment problem, selection rules, and the oscillator strength. In this work, the mode assignment problem was be addressed with Finite Element calculations. These reveal that a rigid sphere in contact with a QCM displays two modes of vibration, termed “slipping” and “rocking”. In the slipping mode, the sphere rotates about its center; it exerts a tangential force onto the resonator surface at the point of contact. In the rocking mode, the sphere rotates about the point of contact; it exerts a torque onto the substrate. In liquids, both axes of rotation are slightly displaced from their ideal positions. Characteristic for spectroscopy, the two modes do not couple to the mechanical excitation equally well. The degree of coupling is quantified by an oscillator strength. Because the rocking mode mostly exerts a torque (rather than a tangential force), its coupling to the resonator's tangential motion is weak; the oscillator strength consequently is small. Recent experiments on surface-adsorbed colloidal spheres can be explained by the mode of vibration being of the rocking type.
A novel signal amplification strategy of quartz crystal microbalance (QCM) based on catalytic gas generation was developed to construct an ultrasensitive immunosensor for the detection of proteins (immunoglobulin G, IgG, used as a model). Catalase modified immunoparticle was prepared to form a sandwich-type immunocomplex with the IgG and anti-IgG antibody that was immobilized on the QCM sensor. The amount of the immunoparticles on the sensor surface was thus controlled by the IgG concentration. Then H2O2 was added and catalyzed by catalase for oxygen generation. The generated oxygen replaced some of liquid on the sensor surface, leading to the change in the shear modulus of the immunocomplex layer and the apparent viscosity and density of the liquid layer. Due to ultrasensitive response of QCM to these changes, a significant frequency shift related to the IgG concentration was achieved. The different parameters, including flow cell structure, operation temperature, immunoparticle concentration, and H2O2 concentration were optimized to achieve steady and efficient frequency shifts. Under the optimal conditions, the proposed gas-phase amplified QCM sensor could achieve up to 72 times improvement of detection sensitivity compared to the label-free sensor as a control, in the concentration range of 0.1-3.0 µg mL-1. The detection limit was also reduced from 236 ng mL-1 to 51.0 ng mL-1 at the 3Sblank level.
Sensor performance of a dielectric filled silicon bulk acoustic resonator type label-free biosensor is verified with biotin-streptavidin binding interactions as a model system. The mass sensor is a micromachined silicon square plate with a dielectric filled capacitive excitation mechanism. The resonance frequency of the biotin modified resonator decreased 315 ppm when exposed to streptavidin solution for 15 min with a concentration of 10-7 M, corresponding to an added mass of 3.43 ng on the resonator surface. An additional control is added by exposing a bovine serum albumin (BSA)-covered device to streptavidin in the absence of the attached biotin. No resonance frequency shift was observed in the control experiment, which confirms the specificity of the detection. The sensor-to-sensor variability is also measured to be 4.3%. Consequently, the developed sensor can be used to observe in biotin-streptavidin interaction without the use of labelling or molecular tags. In addition, biosensor can be used in a variety of different immunoassay tests.
Force spectroscopies have emerged as a powerful and unprecedented tool to study and manipulate biomolecules directly at a molecular level. Usually, protein and DNA behavior under force is described within the framework of the worm-like chain (WLC) model for polymer elasticity. Although it has been surprisingly successful for the interpretation of experimental data, especially at high forces, the WLC model lacks structural and dynamical molecular details associated with protein relaxation under force that are key to the understanding of how force affects protein flexibility and reactivity. We use molecular dynamics simulations of ubiquitin to provide a deeper understanding of protein relaxation under force. We find that the WLC model successfully describes the simulations of ubiquitin, especially at higher forces, and we show how protein flexibility and persistence length, probed in the force regime of the experiments, are related to how specific classes of backbone dihedral angles respond to applied force. Although the WLC model is an average, backbone model, we show how the protein side chains affect the persistence length. Finally, we find that the diffusion coefficient of the protein's end-to-end distance is on the order of 10 nm/s, is position and side-chain dependent, but is independent of the length and independent of the applied force, in contrast with other descriptions.
Single DNA molecules were chemically attached by one end to a glass surface and by their other end to a magnetic bead. Equilibrium
positions of the beads were observed in an optical microscope while the beads were acted on by known magnetic and hydrodynamic
forces. Extension versus force curves were obtained for individual DNA molecules at three different salt concentrations with
forces between 10(-14) and 10(-11) newtons. Deviations from the force curves predicted by the freely jointed chain model suggest
that DNA has significant local curvature in solution. Ethidium bromide and 4',6-diamidino-2-phenylindole had little effect
on the elastic response of the molecules, but their extent of intercalation was directly measured. Conversely, the effect
of bend-inducing cis-diamminedichloroplatinum (II) was large and supports the hypothesis of natural curvature in DNA.
Interaction forces between single strands of DNA were measured with the atomic force microscope by a procedure in which DNA
oligonucleotides were covalently attached to a spherical probe and surface. Adhesive forces measured between complementary
20-base strands fell into three distinct distributions centered at 1.52, 1.11, and 0.83 nano-newtons, which are associated
with the rupture of the interchain interaction between a single pair of molecules involving 20, 16, and 12 base pairs, respectively.
When a third long DNA molecule was coupled between complementary surfaces, both intra- and interchain forces were observed.
The intrachain interaction resulting from the molecule's elasticity manifested itself as a long-range cohesive force.
Single linear DNA molecules were bound at multiple sites at one extremity to a treated glass cover slip and at the other to
a magnetic bead. The DNA was therefore torsionally constrained. A magnetic field was used to rotate the beads and thus to
coil and pull the DNA. The stretching force was determined by analysis of the Brownian fluctuations of the bead. Here, the
elastic behavior of individual λ DNA molecules over- and underwound by up to 500 turns was studied. A sharp transition was
discovered from a low to a high extension state at a force of ∼0.45 piconewtons for underwound molecules and at a force of
∼3 piconewtons for overwound ones. These transitions, probably reflecting the formation of alternative structures in stretched
coiled DNA molecules, might be relevant for DNA transcription and replication.
Single molecules of double-stranded DNA (dsDNA) were stretched with force-measuring laser tweezers. Under a longitudinal stress
of ∼65 piconewtons (pN), dsDNA molecules in aqueous buffer undergo a highly cooperative transition into a stable form with
5.8 angstroms rise per base pair, that is, 70% longer than B-form dsDNA. When the stress was relaxed below 65 pN, the molecules
rapidly and reversibly contracted to their normal contour lengths. This transition was affected by changes in the ionic strength
of the medium and the water activity or by cross-linking of the two strands of dsDNA. Individual molecules of single-stranded
DNA were also stretched giving a persistence length of 7.5 angstroms and a stretch modulus of 800 pN. The overstretched form
may play a significant role in the energetics of DNA recombination.
Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were approximately 25 nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain.
The basic features of DNA were elucidated during the half-century following the discovery of the double helix. But it is only during the past decade that researchers have been able to manipulate single molecules of DNA to make direct measurements of its mechanical properties. These studies have illuminated the nature of interactions between DNA and proteins, the constraints within which the cellular machinery operates, and the forces created by DNA-dependent motors.
duce active movement against load (for example, "molecular motors" such as myosin, which drives muscle contraction). A number of "molecular machines" (for instance, DNA-processing enzymes) are now accessible to single-molecule observation and manipulation. Among other obvious targets were more passive molecules (such as biopolymers) that respond to an applied force in various ways. This field was initially developed by work on DNA, but more recently, it has been extended to structural biopolymers. In the past 6 years, observations of molecular motors and biopolymers have included the following: stepwise motion of single kinesin molecules along a microtubule track, displacement of an actin filament by a single myosin molecule, forces and transcriptional pauses associated with RNA polymerase activity, nonlinear elasticity of single polymers, reversible unfolding of single-protein domains by applied force, and discrete rotations of a single F 1 subunit of the F 0<F
Surface immobilized bacteriophages (phages) are increasingly used as biorecognition elements on bacterial biosensors (e.g., on acoustical, electrical, or optical platforms). The phage surface density is a critical factor determining a sensor’s bacterial binding efficiencies; in fact, phage surface densities that are too low or too high can result in significantly reduced bacterial binding capacities. Identifying an optimum phage surface density is thus crucial when exploiting the bacteriophages’ bacterial capture capabilities in biosensing applications. Herein, we investigated surface immobilization of the Pseudomonas aeruginosa specific E79 (tailed) phage and the Salmonella Typhimurium specific PRD1 (non-tailed) phage and their subsequent bacterial capture abilities using quartz crystal microbalance with dissipation monitoring (QCM-D). The QCM-D was used in two experimental set-ups, (i) a conventional set-up, and (ii) a combined setup with ellipsometry. Both setups were exploited to link the phages’ immobilization behaviors to their bacterium capture efficiency. While E79 displayed characteristic optima in both the mechanical (QCM-D) and the optical (ellipsometry) data that coincided with its specific bacterial capture optimum, no optima were observed during PRD1 immobilization. The characteristic optima suggests that the E79 phage undergoes a surface rearrangement event that changes the hydration state of the phage film, thereby impairing the E79 phage’s ability to capture bacteria. However, the absence of such optima during deposition of the non-tailed PRD1 phage suggests that other mechanisms may also lead to reduced bacterial capture by surface immobilized bacteriophages.
Titin, a giant filamentous polypeptide, is believed to play a fundamental role in maintaining sarcomeric structural integrity and developing what is known as passive force in muscle. Measurements of the force required to stretch a single molecule revealed that titin behaves as a highly nonlinear entropic spring. The molecule unfolds in a high-force transition beginning at 20 to 30 piconewtons and refolds in a low-force transition at ∼2.5 piconewtons. A fraction of the molecule (5 to 40 percent) remains permanently unfolded, behaving as a wormlike chain with a persistence length (a measure of the chain’s bending rigidity) of 20 angstroms. Force hysteresis arises from a difference between the unfolding and refolding kinetics of the molecule relative to the stretch and release rates in the experiments, respectively. Scaling the molecular data up to sarcomeric dimensions reproduced many features of the passive force versus extension curve of muscle fibers.
Single-molecule atomic force microscopy (AFM) was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle. Individual titin molecules were repeatedly stretched, and the applied force was recorded as a function of the elongation. At large extensions, the restoring force exhibited a sawtoothlike pattern, with a periodicity that varied between 25 and 28 nanometers. Measurements of recombinant titin immunoglobulin segments of two different lengths exhibited the same pattern and allowed attribution of the discontinuities to the unfolding of individual immunoglobulin domains. The forces required to unfold individual domains ranged from 150 to 300 piconewtons and depended on the pulling speed. Upon relaxation, refolding of immunoglobulin domains was observed.
A new surface functionalization scheme for nano-Bio field effect transistors (FETs) using biocompatible polyelectrolyte thin films (PET) is developed. PET assemblies on Si nanowires (Si-NWs) are driven by electrostatic interactions between the positively charged polymer backbone and negatively charged Si/SiO2 surface. Such assemblies can be directly coated from PET aqueous solutions and result in a uniform nanoscale thin film, which is more stable compared to the conventional amine silanization. Short oligo-ethylene glycol chains are grafted on the PETs to prevent nonspecific protein binding. Moreover, the reactive groups of the polymer chains can be further functionalized to other chemical groups in specific stoichiometry for biomolecules detection. Therefore, it opens a new strategy to precisely control the functional group densities on various biosensor surfaces at the molecular level. In addition, such assemblies of the polymers together with the bound analytes can be removed with the pH stimulation resulting in regeneration of a bare sensor surface without compromising the integrity and performance of the Si-NWs. Thus, it is believed that the developed PET coating and sensing systems on Si-NW FETs represent a versatile, promising approach for regenerative biosensors which can be applied to other biosensors and will benefit real device applications, enhancing sensor lifetime, reliability, and repeatability.
We use an AT‐cut quartz resonator with metal electrodes as the substrate. The material properties of the quartz are well characterized, and the resonant frequency can be determined accurately (1 part in 108 or better). When a particle was placed on the electrode, the resonant frequency increased, contrary to mass loading theory. If the particle‐resonator system is modeled mechanically as a coupled oscillator system, indeed the resonant frequency should increase. The increase is determined by the bonding force constant between particle and substrate, which we calculate. Experimental data on the autohesion force constants of Au spheres on etched Au electrodes in air is presented.
a b s t r a c t The detection of the biotin–streptavidin interaction is presented based on surface stress changes of func-tionalized Si membranes. During the biomolecular interaction, the surface stress of the functionalized membranes alters resulting in their deflection and finally in capacitance variations. The biological inter-action on the sensors surface was verified after each experiment using fluorescent microscopy. Successful detection of 21 nM of streptavidin using 3.55 mM biotin probes is presented.
Wird eine Fremdschicht auf eine zu Dickenscherungsschwingungen angeregte Schwingquarzplatte aufgebracht, so ndert sich die Eigenfrequenz der Platte infolge Vergrerung der schwingenden Masse. Da die Frequenznderung eines Schwingquarzes sehr genau vermessen werden kann, ergibt sich daraus eine sehr empfindliche Methode zur Wgung dnner Schichten.Massenbelegung der Fremdschicht und Frequenznderung sind einander proportional. Die Proportionalittskonstante lt sich aus der Eigenfrequenz des Schwingquarzes berechnen, so da eine empirische Eichung bei der Schichtwgung mit Schwingquarzen entfllt.Die Genauigkeit des Schichtwgeverfahrens ist in erster Linie durch die Temperaturabhngigkeit der Quarzeigenfrequenz begrenzt und betrgt bei 1 C zugelassener Temperaturschwankung etwa 4 10–9 g cm–2. Das entspricht einer mittleren Dicke von 0,4 bei der Dichte =1 g cm–3.Das Verfahren wurde auch zur direkten Wgung einer Masse ausgenutzt (Mikrowgung). Dabei lie sich eine Genauigkeit von 10–10g erreichen.
In a quartz crystal microbalance, particles adhering to a sensor crystal are perturbed around their equilibrium positions via thickness-shear vibrations at the crystal's fundamental frequency and overtones. The amount of adsorbed molecular mass is measured as a shift in resonance frequency. In inertial loading, frequency shifts are negative and proportional to the adsorbed mass, in contrast with "elastic loading", where particles adhere via small contact points. Elastic loading in air yields positive frequency shifts according to a coupled resonance model. We explore here the novel application of a coupled resonance model for colloidal particle adhesion in a liquid phase theoretically and demonstrate its applicability experimentally. Particles with different radii and in the absence and presence of ligand-receptor binding showed evidence of coupled resonance. By plotting the frequency shifts versus the quartz crystal microbalance with dissipation overtone number, frequencies of zero-crossing could be inferred, indicative of adhesive bond stiffness. As a novelty of the model, it points to a circular relation between bandwidth versus frequency shift, with radii indicative of bond stiffness. The model indicates that bond stiffness for bare silica particles adhering on a crystal surface is determined by attractive Lifshitz-van der Waals and ionic-strength-dependent, repulsive electrostatic forces. In the presence of ligand-receptor interactions, softer interfaces develop that yield stiffer bonds due to increased contact areas. In analogy with molecular vibrations, the radii of adhering particles strongly affect the resonance frequencies, while bond stiffness depends on environmental parameters to a larger degree than for molecular adsorption.
We report investigations on the use of film bulk acoustic resonators for the label-free, multiplexed biosensing of DNA and proteins. The used acoustic resonators were operated in shear mode at about 800 MHz. From the measured changes of frequency and in dissipation, the mass and the viscoelasticity of biomolecular films formed at the top electrode of the device could be derived, respectively. A mass sensitivity of ∼2 kHz cm2/ng and a minimum detectable mass of ∼1 ng/cm2 were achieved. To demonstrate the highly sensitive detection of the time evolution of protein adsorption, the adsorption kinetics and recrystallisation of bacterial surface layer proteins on gold surfaces were investigated.
Zinc oxide based film bulk acoustic resonator as mass sensor was fabricated by multi-target magnetron sputtering under optimized deposition condition. Each layer of the device was well crystallized and highly textural observed by transmission electron microscopy and X-ray diffraction measurement. Through piezoelectric test, the device vibrated with significant distance. The influence of top electrode on resonant frequency and the bio-specimen of mass loading effect were investigated. Data show that the device has qualified properties as mass biosensor, with a resonant frequency of 3–4 GHz and a high sensitivity of 8–10 kHz cm2/ng.
A shear mode AlN solidly mounted resonator (SMR) microfluidic sensor system was fabricated and characterized. The AlN SMR fabrication process is fully IC compatible and uses reactive sputtering to deposit piezoelectric AlN thin films with a non-zero mean inclination of the c-axis, which allows in-liquid operation through the excitation of the shear mode. Silicone encapsulation bonded on top of the Si sensor chip includes a microfluidic system to transport the analyte and confine the flow to the active area of the sensor chip. The sensor operation in air, water, glycerol and acetone was characterized. The resonator had a resonance frequency of around 1.2 GHz and a Q value in water of around 100. Results concerning the stability and resolution are also presented. The results indicate a potential of highly sensitive low-cost microfluidic sensor systems for applications in, e.g. point-of-care testing.
Micro/nanobeads with different materials (magnetic, silica and polymer) and different sizes (diameters from 30nm to 970nm) were investigated for their use as amplifiers in a quartz crystal microbalance (QCM) immunosensor for more sensitive detection of Escherichia coli O157:H7. The micro/nanobeads were conjugated with anti-E. coli antibodies. E. coli O157:H7 cells were first captured by the first antibody immobilized on the electrode surface, and then micro/nanobeads labeled secondary antibodies attached to the cells, and finally the complexes of antibody-E. coli-antibody modified beads were formed. The results showed that antibody-labeled beads lead to signal amplification in both the change in frequency (ΔF) and the change in resistance (ΔR). Since the penetration depth of the oscillation-induced shear-waves for a ∼8MHz crystal is limited to 200nm, the interpretation of how the signal is amplified by the adsorbed particles was represented in terms of the coupled-oscillator theory. The amplification is not sensed in terms of increase in mass on the sensor surface. Amplification is sensed as a change in bacterial resonance frequency when the spheres adsorb to the bacteria. The change in the values of ΔF caused by different micro/nanobeads (amplifiers) attaching on target bacterial cells is indicative of the ratio between the resonance frequency of the absorbed bacterial-particle complex (ω(s)), and the resonance frequency of the crystal (ω).
Forces play a pivotal role in life, and the response of live systems to forces requires molecules and molecular interactions with adequate properties to counteract both in a passive and also, if needed, in an active, dynamic manner. However, at the level of individual molecules these forces are so minute, that the development of sophisticated experiments to measure and control them was required. With the maturation of these techniques, particularly the AFM-based single-molecule force spectroscopy into commercial instruments, the scope has widened considerably and more and more studies shed light onto the different aspects of biomolecular mechanics. Many surprises turned up and more are waiting for us.
The giant muscle protein titin, also called connectin, is responsible for the elasticity of relaxed striated muscle, as well as acting as the molecular scaffold for thick-filament formation. The titin molecule consists largely of tandem domains of the immunoglobulin and fibronectin-III types, together with specialized binding regions and a putative elastic region, the PEVK domain. We have done mechanical experiments on single molecules of titin to determine their visco-elastic properties, using an optical-tweezers technique. On a fast (0.1s) timescale titin is elastic and force-extension data can be fitted with standard random-coil polymer models, showing that there are two main sources of elasticity: one deriving from the entropy of straightening the molecule; the other consistent with extension of the polypeptide chain in the PEVK region. On a slower timescale and above a certain force threshold, the molecule displays stress-relaxation, which occurs in rapid steps of a few piconewtons, corresponding to yielding of internal structures by about 20 nm. This stress-relaxation probably derives from unfolding of immunoglobulin and fibronectin domains.
We have used optical tweezers to study the elasticity of red cell membranes; force was applied to a bead attached to a permeabilized spherical ghost and the force-extension relation was obtained from the response of a second bead bound at a diametrically opposite position. Interruption of the skeletal network by dissociation of spectrin tetramers or extraction of the actin junctions engendered a fourfold reduction in stiffness at low applied force, but only a twofold change at larger extensions. Proteolytic scission of the ankyrin, which links the membrane skeleton to the integral membrane protein, band 3, induced a similar effect. The modified, unlike the native membranes, showed plastic relaxation under a prolonged stretch. Flaccid giant liposomes showed no measurable elasticity. Our observations indicate that the elastic character is at least as much a consequence of the attachment of spectrin as of a continuous membrane-bound network, and they offer a rationale for formation of elliptocytes in genetic conditions associated with membrane-skeletal perturbations. The theory of Parker and Winlove for elastic deformation of axisymmetric shells (accompanying paper) allows us to determine the function BH(2) for the spherical saponin-permeabilized ghost membranes (where B is the bending modulus and H the shear modulus); taking the literature value of 2 x 10(-19) Nm for B, H then emerges as 2 x 10(-6) Nm(-1). This is an order of magnitude higher than the value reported for intact cells from micropipette aspiration. Reasons for the difference are discussed.
During the past decade, physical techniques such as optical tweezers and atomic force microscopy were used to study the mechanical properties of DNA at the single-molecule level. Knowledge of DNA's stretching and twisting properties now permits these single-molecule techniques to be used in the study of biological processes such as DNA replication and transcription.
We report biophysical experiments performed on the bacterium Listeria monocytogenes, a model system to study actin-based motility. Using optical tweezers and electrophoresis experiments, we find that the bacterium is firmly attached to its tail, and we demonstrate that the tail responds as an elastic gel when deformed. We have measured its elastic modulus at a value of 10(3)-10(4) Pa, which is 10 times higher than the rigidity of the eukaryotic cytoplasm. These results demonstrate that the bacterium and its tail form a very robust system, consistent with the steadyness of the motion observed in vivo. We propose an elastic model for the propulsion mechanism which takes into account the connection and thus the interaction between the actin filaments. It provides a generic description of the various aspects of actin-tail based movements.
Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.
Coiled-coils occur in a variety of proteins involved in mechanical and structural tasks in the cell. Their mechanical properties are important in various contexts ranging from hair elasticity to synaptic fusion. Beyond their importance in biology, coiled-coils have also attracted interest as programmable protein sequences for the design of novel hydrogels and materials. We have studied the elastic properties of the myosin coiled-coil at the single molecule level. The coiled-coil undergoes a massive structural transition at forces between 20 and 25 pN where the coil extends to about 2.5 times its original length. Unlike all other proteins investigated mechanically so far, this transition is reversible on a timescale of less than a second, making the coiled-coil a truly elastic protein.
Atomic force microscopy allows three-dimensional imaging and measurements of unstained and uncoated biological samples in air or fluid. Using this technology it offers resolution on the nanometer scale and detection of temporal changes in the mechanical properties, i.e. surface stiffness or elasticity in live cells and membranes. Various biological processes including ligand-receptor interactions, reorganization, and restructuring of the cytoskeleton associated with cell motility that are governed by intermolecular forces and their mode of detection will be discussed.
The adhesion of lactic acid bacteria to the intestinal epithelium is one of the most important factors determining probiotic ability of a bacterial strain. Studying bacterial adhesion requires knowledge of the structure and properties of the bacterial surface, which can be studied by atomic force microscopy under native conditions. The observation of the surface topography of bacteria from the species Lactobacillus crispatus, L. helveticus and L. johnsonii shows major differences between bacteria having a crystalline-like protein layer as part of the cell wall and those without such layers. Force volume images calculated into elasticity and adhesion force maps of different bacterial strains show that L. crispatus and L. helveticus have a surface with a homogeneous stiffness with no adhesion events. This is most likely caused by the S-layer, which completely covers the surface of the bacteria. We infer that the absence of adhesion peaks is caused by the semi-crystalline character of such protein layers, in agreement with the results obtained from electron microscopy. Analysis of a number of L. johnsonii strains shows that these bacteria have surface properties which strongly differ from the L. crispatus and L. helveticus strains. For L. johnsonii DMS20533 and L. johnsonii ATCC33200 high adhesion forces are observed, which can be related to a surface rich in polysaccharides. L. johnsonii ATCC332 has lower adhesion forces compared to the other two and, furthermore, the surface topography shows depressions. We suppose that this strain has a surface pattern consisting of crystalline-like proteins alternating with polysaccharide-rich domains. The wide variety in surface properties of lactobacilli could well have wide-ranging implications for food processing and for health benefits.
A novel integrated bio-sensor technology based on thin-film bulk acoustic wave resonators on silicon is presented and the feasibility of detecting DNA and protein molecules proofed. The detection principle of these sensors is label-free and relies on a resonance frequency shift caused by mass loading of an acoustic resonator, a principle very well known from quartz crystal micro balances. Integrated ZnO bulk acoustic wave resonators with resonance frequencies around 2 GHz have been fabricated, employing an acoustic mirror for isolation from the silicon substrate. DNA oligos have been thiol-coupled to the gold electrode by on-wafer dispensing. In a further step, samples have either been hybridised or alternatively a protein has been coupled to the receptor. The measurement results show the new bio-sensor being capable of both, detecting proteins as well as the DNA hybridisation without using a label. Due to the substantially higher oscillation frequency, these sensors already show much higher sensitivity and resolution comparable to quartz crystal micro balances. The potential for these sensors and sensors arrays as well as technological challenges will be discussed in detail.
We show how the water content (and effective density) of thin adsorbed films composed of biomolecules can be determined using combined quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) analysis. In particular, these techniques, combined with theoretical treatment using a Voigt-based viscoelastic model, were applied to analyze the state of surface immobilized single stranded biotin-modified probe DNA (b-DNA) coupled via streptavidin to a biotin-doped supported phospholipid bilayer (b-SPB)). From a proper analysis, it is demonstrated how changes in effective thickness, delta(f), and the viscoelastic components (shear viscosity, eta(f), and shear elasticity, mu(f))) can be obtained during both DNA immobilization and hybridization with single stranded fully complementary target DNA. In particular, it is demonstrated how this type of analysis can be used to control the state of streptavidin arrangement for improved measurements of DNA hybridization kinetics. The latter is demonstrated by identifying a surface-coverage dependent viscoelastic behavior of immobilized b-DNA, which is shown to influence the hybridization efficiency.
For a thin viscoelastic film deposited on a quartz crystal microbalance in a liquid environment, the change in dissipation induced by the presence of a film is proportional to the film's elastic compliance, Jf'. This surprising result is a consequence of the fact that the film is "clamped" by a viscous fluid.
Particulate interactions are dominated by aspects such as surface topography, exposed chemical moieties, environmental conditions, and thermodynamic properties such as surface free energy (gamma). The absolute value and relative magnitude of surface energies of a drug and excipients within a formulation can significantly influence manufacture, processing, and use. This study utilizes and compares the potentially complementary analytical techniques of atomic force microscopy (AFM) and inverse gas chromatography (IGC) in the quantitative determination of the surface energy of drug (budesonide) particles (micronized and unmilled) relevant to inhaled delivery. In addition, the study investigates with AFM another important parameter in determining material interactions, the local mechanical properties of the drug.
AFM was used to acquire force of adhesion (Fadh) and related work of adhesion (WA) and surface energy values between individual mironized drug particles and also model substrates (graphite and mica). In addition, AFM probes were used to interrogate the surface energy of unmilled drug particles. Measurement with AFM probes also yielded localized measurements of Young's modulus for the unmilled drug. IGC was also used to probe the surface characteristics of the bulk drug material.
The average values for surface energies acquired from budesonide micronized particle interactions with graphite, mica, and drug particles of the same substance were found to range from 35 to 175, 5 to 40, and 10 to 32 mJ m(-2), respectively. The unmilled material displayed a range of values of 39-88 mJ m(-2) with an average of 60 mJ m(-2). The IGC result for the surface energy of the micronized material was 68.47 +/- 1.60 mJ m(-2). The variability in surface energy from AFM, a feature particularly apparent for the micronized material was attributed to two factors, intrinsic material variations within a single particle and assumptions present within the contact mechanics model used. Here we provide a detailed description of these factors to go some way to rationalize the results. The Young's modulus of the unmilled drug was determined to be approximately 10 GPa.
The range of determined surface energies between the AFM measurement on graphite, mica, and the drug is proposed to reflect the different chemistries displayed by the drug at the single particle level. The maximum values of these ranges can be related to the sites most likely to be involved in adhesion. AFM and IGC yield surface energy estimates in approximate agreement, but clearly are interrogating surfaces in different fashions. This raises questions as to the nature of the measurement being made by these approaches and to the most appropriate time to use these methods in terms of a direct relation to formulation design, manufacture, and drug delivery. Finally, we demonstrate a novel method for assessing the Young's modulus of a drug from a single particle.
The hydration-dehydration process of an adsorbed human serum albumin film has been studied using atomic force microscopy (AFM) and a quartz crystal microbalance (QCM). All measurements were performed with identically prepared protein films deposited on highly hydrophilic substrates. Both techniques are shown to be suitable for following in situ the kinetics of protein hydration, and for providing quantitative values of the adsorbed adlayer mass. The results obtained by the two methods have been compared and combined to study changes of physical properties of the films in terms of viscosity, shear, Young's modulus, density and film thickness. These properties were found to be reversible during hydration-dehydration cycles.