Simplified Aeroelastic Model for Fluid Structure Interaction between Microcantilever Sensors and Fluid Surroundings

Fluid-structural coupling occurs when microcantilever sensors vibrate in a fluid. Due to the complexity of the mechanical characteristics of microcantilevers and lack of high-precision microscopic mechanical testing instruments, effective methods for studying the fluid-structural coupling of microcantilevers are lacking, especially for non-rectangular microcantilevers. Here, we report fluid-structure interactions (FSI) of the cable-membrane structure via a macroscopic study. The simplified aeroelastic model was introduced into the microscopic field to establish a fluid-structure coupling vibration model for microcantilever sensors. We used the finite element method to solve the coupled FSI system. Based on the simplified aeroelastic model, simulation analysis of the effects of the air environment on the vibration of the commonly used rectangular microcantilever was also performed. The obtained results are consistent with the literature. The proposed model can also be applied to the auxiliary design of rectangular and non-rectangular sensors used in fluid environments.     

Microcantilevers modified by specific peptide for selective detection of trimethylamine

We report a biosensor based on a microcantilever that is modified by a specific peptide for highly selective detection of trimethylamine (TMA). The assay is based on binding-induced bending of the peptide functionalized microcantilevers. The sensor is selectively responsive to TMA. The amplitude of microcantilever bending at equilibrium is a function of the concentration of TMA with a dynamic range from 8 ppm to 800 ppm. The detection limit is approximately 8 ppm. There is a good intra-sensor and an acceptable inter-sensor reproducibility as evidenced by the standard deviation of 5% and 15%, respectively.     

Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase

We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.     

Detection of heavy metal ions using protein-functionalized microcantilever sensors

Microcantilevers functionalized with metal-binding protein, AgNt84-6, are demonstrated to be sensors for the detection of heavy metal ions like Hg(2+) and Zn(2+). AgNt84-6, a protein that has the ability to bind multiple atoms of Ni(2+), Zn(2+), Co(2+), Cu(2+), Cd(2+) and Hg(2+) was attached to the gold-coated side of silicon nitride cantilevers via linker groups. Upon exposure to 0.1 mM HgCl(2) and 0.1 mM ZnCl(2) solutions, the microcantilevers underwent bending corresponding to an expanding gold side. Exposure to a 0.1 mM solution of MnCl(2) solution did not result in a similar bending indicating a weak or no interaction of Mn(2+) ions with the AgNt84-6 protein. The microcantilever bending data were consistent with data from electrophoresis carried out on SDS-PAGE gels containing metal ions that showed protein interaction with Zn(2+) ions but not with Mn(2+) ions. Thus, we demonstrate that microcantilever bending can be used to discriminate between metal ions that bind and do not bind to AgNt84-6 protein in real time.     

A three-dimensional model of fluid-structural interactions for quantifying the contractile force for cardiomyocytes on hybrid biopolymer microcantilever

Quantitatively analysis of the contractility of cardiomyocytes is important for understanding the mechanism of heart failure as well as the molecular alterations in diseased heart cells. This paper presents a realistic computational model, which considers the three-dimensional fluid-structural interactions (FSI), to quantify the contractile force of cardiomyocytes on hybrid biopolymer microcantilevers. Prior to this study, only static modeling of the microscale cellular force has been reported. This study modeled the dynamics of cardiomyocytes on microcantilevers in a medium using the FSI. This realistic model was compared with static finite element modeling (FEM) analysis and the Stoney's equation-based analytical solution, and was validated by the deflections of the microcantilevers in the experimental results. Using harmonic response analysis in FSI modeling, the motion of a hybrid biopolymer microcantilever in the medium was identified as a second-order system and the influence of the dynamics of cardiomyocytes could be evaluated quantitatively.     

Peptide receptor-based selective dinitrotoluene detection using a microcantilever sensor

We reported that peptide could be utilized as receptor molecule in the gas phase for application in micro/nano sensors by using a specific peptide that recognizes 2,4-dinitrotoluene at room temperature and in an atmospheric environment and measuring changes in the resonant frequency of the peptide immobilized microcantilevers. By using these peptides as receptors on a microcantilever sensor, we were able to experimentally detect 2,4-dinitrotoluene (DNT) vapor at concentrations as low as parts per billion (ppb) in the gas phase. While resonant frequency changes after binding between 2,4-DNT and the specific peptide receptor that was immobilized on microcantilevers were observed, the resonant frequency of DNT nonspecific peptide immobilized microcantilever did not change when exposed to 2,4-DNT vapor. The limit of detection (LOD) was calculated to be 431 ppt of limit of detection is numerically expected by experimental based on an equation that describes the relationship between the noise-equivalent analyte concentration. These results indicate that the peptide receptors hold great promise for use in the development of an artificial olfactory system and electronic nose based on micro/nanotechnology for monitoring various chemical vapors in the gas phase such as explosive mixtures of chemicals and/or volatile organic compounds.     

In situ detection of calcium ions with chemically modified microcantilevers

We report a novel technique of micromechanical detection of trace amounts of calcium ions by using microcantilevers modified with ion-selective self-assembled monolayers (SAMs). The SAM-modified microcantilevers undergo bending due to selective adsorption of calcium ions. Experiments conducted under flow conditions show that the modified cantilevers respond sensitively to calcium ions (Ca(2+)); a Ca(2+) concentration of 10(-9) M can be detected with this technique. Other cations, such as Na(+) and K(+), do not have any effect on the deflection of these cantilevers. We demonstrate two different kinds of SAMs having selectivity for calcium ions.     

Bioassay of prostate-specific antigen (PSA) using microcantilevers

Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.     

Highly specific label-free protein detection from lysed cells using internally referenced microcantilever sensors

We report the investigation of label-free protein detection directly from lysed cells using microcantilever sensors. The integration of an internally referenced microcantilever sensor combined with peptide aptamer technology enables scalable and label-free detection of proteins from a complex biological environment (e.g. cell lysate). The internally referenced microcantilever sensor was found to be effective in minimizing both the effects of thermal drift and non-specific binding interactions with the backside of the cantilever, thereby allowing protein detection in a complex biological background. Highly specific peptide aptamers are used to modify the cantilever surface to specifically detect less than 80nM CDK2 protein from yeast cell lysate. This binding of CDK2 on the microcantilever generates a tensile surface stress of average magnitude equal to 70+/-22mN/m. Similar experiments conducted with quartz crystal microbalance (QCM) technology are consistent with the response observed using microcantilever sensors.     

Study of the near-neutral pH-sensitivity of chitosan/gelatin hydrogels by turbidimetry and microcantilever deflection

The fundamental properties and pH-sensitivity of chitosan/gelating hydrogels were investigated using spectroscopic and microelectro mechanical (MEMS) measurement approaches. Turbidimetric titration revealed that there were electrostatic attractive interactions between tripolyphosphate (TPP), chitosan, and gelatin in the acidic pH range, depending on their degree of ionization. The pH-sensitive swelling behavior of the hydrogels was investigated by monitoring the deflection of hydrogel-coated microcantilevers, which exhibited a sensitive and repeatable response to solution pH. The deflection of the microcantilever increased as the pH decreased, and the response speed of the system exhibited a nearly linear relationship with pH. The effects of the pH and concentration of TPP solution, as well as the ratio of chitosan to gelatin in gel precursor solutions, on the pH sensitivity of the hydrogels were also investigated. It was found that the swelling of the hydrogel is mainly a result of chain relaxation of chitosan-TPP complexes caused by protonation of free amino groups in chitosan, which depends on the crosslinking density set during the formation of the network. An increase in initial crosslink density induced a decrease in swelling and pH sensitivity. It can be concluded from this study that pH-sensitive chitosan gel properties can be tuned by preparatory conditions and inclusion of gelatin. Furthermore, microcantilevers can be used as a platform for gaining increased understanding of environmentally sensitive polymers.     

Mechanisms Controlling Cell Size and Shape during Isotropic Cell Spreading

Cell motility is important for many developmental and physiological processes. Motility arises from interactions between physical forces at the cell surface membrane and the biochemical reactions that control the actin cytoskeleton. To computationally analyze how these factors interact, we built a three-dimensional stochastic model of the experimentally observed isotropic spreading phase of mammalian fibroblasts. The multiscale model is composed at the microscopic levels of three actin filament remodeling reactions that occur stochastically in space and time, and these reactions are regulated by the membrane forces due to membrane surface resistance (load) and bending energy. The macroscopic output of the model (isotropic spreading of the whole cell) occurs due to the movement of the leading edge, resulting solely from membrane force-constrained biochemical reactions. Numerical simulations indicate that our model qualitatively captures the experimentally observed isotropic cell-spreading behavior. The model predicts that increasing the capping protein concentration will lead to a proportional decrease in the spread radius of the cell. This prediction was experimentally confirmed with the use of Cytochalasin D, which caps growing actin filaments. Similarly, the predicted effect of actin monomer concentration was experimentally verified by using Latrunculin A. Parameter variation analyses indicate that membrane physical forces control cell shape during spreading, whereas the biochemical reactions underlying actin cytoskeleton dynamics control cell size (i.e., the rate of spreading). Thus, during cell spreading, a balance between the biochemical and biophysical properties determines the cell size and shape. These mechanistic insights can provide a format for understanding how force and chemical signals together modulate cellular regulatory networks to control cell motility.     

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 model¹ 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 (K_d) 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.     

Delivery of nascent polypeptides to the mitochondrial surface

Thousands of polypeptides with diverse biochemical properties, some of which are extremely hydrophobic, are targeted from cytoplasmic ribosomes to the surface of mitochondria. Localised synthesis, as well as transient interactions with a wide array of molecular chaperones and other cytoplasmic factors, can promote productive interaction of mitochondrial proteins with the TOM complex to initiate protein import into mitochondria.     

Study of binding between protein A and immunoglobulin G using a surface tension probe

Molecular interactions and binding are one of the most important and fundamental properties in the study of biochemical and biomedical systems. The understanding of such interactions and binding among biomolecules forms the basis for the design and processing of many biotechnological applications, such as bioseparation and immunoadsorption. In this study, we present a novel method to probe molecular interactions and binding based on surface tension measurement. This method complements conventional techniques, which are largely based on optical, spectroscopic, fluorescence polarization, chromatographic or atomic force microscopy measurements, by being definite in determining molecular binding ratio and flexible in sample preparation. Both dynamic and equilibrium (or quasi-equilibrium) information on molecular binding can be obtained through dynamic and equilibrium surface tension measurements. For an important pair of biological ligand and ligate, Protein A and immunoglobulin G (IgG), the existence of molecular interactions and the binding ratio of 1:2 have been determined unequivocally with the proposed surface tension method. These results are confirmed/supported by a mass balance calculation and spectrophotometry experiment. In addition, adsorption isotherms for Protein A and IgG separately at the air/water interface have been established with the dynamic surface tension measurements. The results show that the Langmuir isotherm equation can describe the adsorption data satisfactorily for both Protein A and IgG solutions.     

Flexoelectric origin of nanomechanic deflection in DNA-microcantilever system

The membrane theory is used to study the recently observed nanomechanical bending of cantilevers, which have processed biomolecular adsorption or biochemical reactions. To be different from entropy-controlling bending mechanism discussed before, we propose that the flexoelectric effect induces cantilever bending. With the introduction of flexoelectric spontaneous curvature, the relation between the bending and biopolymer character is constructed by a simple analytical formula. The cantilever motion induced by adsorption of single-strand DNA and DNA hybridization reaction is quantified analytically and our results show good agreement with experiments.     

Surface plasmon resonance sensors in cell biology: basics and application

Optical sensors based on the excitation of surface plasmons, referred to as surface plasmon resonance (SPR) sensors, have become a central analytical tool for characterizing and quantifying a wide variety of macromolecular interactions, like receptor–ligand contacts. Besides this classical field of application, in the last 15 years, the development of SPR sensors aiming for the detection and analysis of ligand/cell or host/pathogen interactions, cell/cell contacts, and cellular reactions gained considerable momentum. The number of publications reporting about applications of SPR sensors implementing vital prokaryotic or eukaryotic cells as biorecognition elements for medical diagnostics, environmental monitoring, or biological safety is steadily growing. This review gives a short introduction to the technique of surface plasmon resonance and the parameters that are important for its application in the field of vital cell sensors. Furthermore, the publications concerning the application of such sensors in the analysis of cellular interactions and cellular reactions to extra- and intracellular stimuli are summarized.     

Investigation of biotin-streptavidin binding interactions using microcantilever sensors

We report the investigation of biotin-streptavidin binding interactions using microcantilever sensors. A symmetric cantilever construction is employed to minimize the effects of thermal drift and the control of surface chemistry on the backside of the cantilever is demonstrated to reduce the effects of non-specific binding interactions on the cantilever. Three structurally different biotin modified cantilever surfaces are used as a model system to study the binding interaction with streptavidin. The cantilever response to the binding of streptavidin on these biotin sensing monolayers is compared. The lowest detection limit of streptavidin using biotin-HPDP is found to be between 1 and 10nM limited by the optical measurement setup. Surface characterization using quartz crystal microbalance (QCM) and high-resolution atomic force microscope (AFM) is used to benchmark the cantilever sensor response. In addition, the QCM and AFM studies reveal that the surface density of bound streptavidin on biotin modified surfaces was low, thereby implying that effects other than steric hindrance are responsible for defining cantilever response.     

Fluidics-resolved estimation of protein adsorption kinetics in a biomicrofluidic system

Protein adsorption on surfaces is a complex phenomenon that is described by the balance of convective/diffusive transport of the protein species to the surface and its adsorption/desorption at the surface. The extent of binding depends on a variety of factors such as protein/surface interactions, availability of binding sites, localized concentrations of protein near biomaterial surfaces and flow characteristics of the protein in that region. Factors such as time-varying flows, complex device geometries, presence of multiple competitive species, or possible denaturing of proteins when they attach to the surface make it extremely difficult to quantitatively analyze protein interactions with surfaces. Adsorption/desorption rate constants are often inferred using simplistic models which neglect mass transport and have limited use across different microfluidic systems and flow protocols. In this work, we have developed and demonstrated a fluidics-resolved model that evaluates protein adsorption, accounting for both the fluidic transport and the biochemical kinetics in complex biomicrofluidic devices. The model is valid for both flow and static conditions. An automated procedure was also developed to extract the "intrinsic" mass-transport-independent adsorption kinetic rate constants from experimental data using a least squares optimization method. The automated data extraction methodology is applied to two proteins (alkaline phosphatase and glucose oxidase) that have been brought into contact with poly(etheretherketone) and Teflon capillaries. The applicability of the procedure in analyzing flow and adsorption in complex microfluidic structures is also demonstrated.     

Surface plasmon resonance protein sensor using Vroman effect

We report a new surface plasmon resonance (SPR) protein sensor using the Vroman effect for real-time, sensitive and selective detection of protein. The sensor relies on the competitive nature of protein adsorption onto the surface, directly depending upon protein's molecular weight. The sensor uses SPR for highly sensitive biomolecular interactions detection and the Vroman effect for highly selective detection. By using the Vroman effect we bypass having to rely on bio-receptors and their attachment to transducers, a process known to be complex and time-consuming. The protein sensor is microfabricated to perform real-time protein detection using four different proteins including aprotinin (0.65kDa), lysozyme (14.7kDa), streptavidine (53kDa), and isolectin (114kDa) on three different surfaces, namely a bare-gold surface and two others modified by OH- and COOH-terminated self-assembled monolayer (SAM). The real-time adsorption and displacement of the proteins are observed by SPR and evaluated using an atomic force microscope (AFM). The sensor can distinguish proteins of at least 14.05kDa in molecular weight and demonstrate a very low false positive rate. The protein detector can be integrated with microfluidic systems to provide extremely sensitive and selective analytical capability.     

In situ, in-liquid, all-electrical detection of Salmonella typhimurium using lead titanate zirconate/gold-coated glass cantilevers at any dipping depth

Most biosensing techniques are indirect, slow, and require labeling. Even though silicon-based microcantilever sensors are sensitive and label-free, they are not suitable for in-liquid detection. More recently lead zirconate titanate (PZT) thin-film-based microcantilevers are shown to be sensitive and in situ. However, they require microfabrication and must be electrically insulated. In this study, we show that highly sensitive, in situ, Salmonella typhimurium detection can be achieved at 90% relative humidity using a lead zirconate titanate (PZT)/gold-coated glass cantilever 0.7 mm long with a non-piezoelectric 2.7 mm long gold-coated glass tip by partially dipping the gold-coated glass tip in the suspension at any depth without electrically insulating the PZT. In particular, we showed that at 90% relative humidity and with a dipping depth larger than 0.8 mm the PZT/gold-coated glass cantilever showed virtually no background resonance frequency up-shift due to water evaporation and exhibited a mass detection sensitivity of Δm/Δf = −5 × 10⁻¹¹ g/Hz. The concentration sensitivities of this PZT/gold-coated glass cantilever were 1 × 10³ and 500 cells/ml in 2 ml of liquid with a 1 and 1.5 mm dipping depth, respectively, both more than two orders of magnitude lower than the infectious dose and more than one order of magnitude lower than the detection limit of a commercial Raptor sensor.