Nanomechanics of the formation of DNA self-assembled monolayers and hybridization on microcantilevers

Biomolecular interactions over the surface of a microcantilever can produce its bending motion via changes of the surface stress, which is referred to nanomechanical response. Here, we have studied the interaction forces responsible for the bending motion during the formation of a self-assembled monolayer of thiolated 27-mer single-stranded DNA on the gold-coated side of a microcantilever and during the subsequent hybridization with the complementary nucleic acid. The immobilization of the single-stranded DNA probe gives a mean surface stress of 25 mN/m and a mean bending of 23 nm for microcantilevers with a length and thickness of about 200 microm and 0.8 microm, respectively. The hybridization with the complementary sequence could not be inferred from the nanomechanical response. The nanomechanical response was compared with data from well-established techniques such as surface plasmon resonance and radiolabeling, to determine the surface coverage and study the intermolecular forces between neighboring DNA molecules anchored to the microcantilever surface. From both techniques, an immobilization surface density of 3 x 10(12) molecules/cm(2) and a hybridization efficiency of 40% were determined. More importantly, label-free hybridization was clearly detected in the same conditions with a conventional sensor based on surface plasmon resonance. The results imply that the nanomechanical signal during the immobilization process arises mainly from the covalent attachment to the gold surface, and the interchain interactions between neighboring DNA molecules are weak, producing an undetectable surface stress. We conclude that detection of nucleic acid hybridization with nanomechanical sensors requires reference cantilevers to remove nonspecific signals, more sensitive microcantilever geometries, and immobilization chemistries specially addressed to enhance the surface stress variations.     

Mechano-transduction of DNA hybridization and dopamine oxidation through electrodeposited chitosan network

While microcantilevers offer exciting opportunities for mechano-detection, they often suffer from limitations in either sensitivity or selectivity. To address these limitations, we electrodeposited a chitosan film onto a cantilever surface and mechano-transduced detection events through the chitosan network. Our first demonstration was the detection of nucleic acid hybridization. In this instance, we electrodeposited the chitosan film onto the cantilever, biofunctionalized the film with oligonucleotide probe, and detected target DNA hybridization by cantilever bending in solution (static mode) or resonant frequency shifts in air (dynamic mode). In both detection modes, we observed a two-order of magnitude increase in sensitivity compared to values reported in literature for DNA immobilized on self-assembled monolayers. In our second demonstration, we coupled electrochemical and mechanical modes to selectively detect the neurotransmitter dopamine. A chitosan-coated cantilever was biased to electrochemically oxidize dopamine solution. Dopamine's oxidation products react with the chitosan film and create a tensile stress of approximately 1.7 MPa, causing substantial cantilever bending. A control experiment was performed with ascorbic acid solution. It was shown that the electrochemical oxidation of ascorbic acid does not lead to reactions with chitosan and does not change cantilever bending. These results suggest that chitosan can confer increased sensitivity and selectivity to microcantilever sensors.     

Sensing lipid bilayer formation and expansion with a microfabricated cantilever array

We show that cantilever array sensors can sense the formation of supported phospholipid bilayers on their surface and that they can monitor changes in mechanical properties of lipid bilayers. Supported lipid bilayers were formed on top of microfabricated cantilevers by vesicle fusion. The formation of bilayers led to a bending of the cantilevers of 70-590 nm comparable to a surface stress of 27-224 mN/m. Physisorption of bilayers of DOPC and other bilayers on the silicon oxide surface of cantilevers led to a tensile bending of about 70 nm whereas formation of chemisorbed bilayers of mixed thiolated (DPPTE) and non-thiolated lipids (DOPC) on the gold side of cantilevers led to a compressive bending of nearly 600 nm which depended on the ratio of DPPTE to DOPC. First results on bending of bilayer-covered cantilevers due to their interaction with the pore-forming peptide melittin are shown. The results demonstrate that cantilever sensors with immobilized bilayers can be used as model systems to investigate mechanical properties of cellular membranes and may be used for screening of membrane processes involving modification, lateral expansion, or contraction of membranes.     

Laser‐micromachined cellulose acetate adhesive tape as a low‐cost smart material

An off‐the‐shelf, moisture‐responsive, acetate‐backed adhesive tape is investigated as a commercially available smart material for fabricating low‐cost, multifunctional, humidity‐responsive millimeter‐scale structures. Laser ablation is used for cutting and thinning‐down the tape to enhance its response. Water‐submerged cantilevers show a radius of curvature of 3 mm or lower (for laser‐thinned cantilevers). Additionally, their humidity response is a function of the angle between the longitudinal axis of the cantilever and polymer orientation. A cut angled at 80° with respect to this orientation results in a tip rotation of up to 25°, enabling the formation of bending cantilevers with twisting behavior. The tape cantilevers are further functionalized with magnetic nanoparticles and used to create four‐finger grippers that close underwater within minutes and can sample 100 µL of liquid. A cyclic humidity monitor is also fabricated using a tape strip that walks unidirectionally on a ratchet‐shaped surface upon exposure to humidity variations. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2013 , 51, 1263–1267     

AlN on nanocrystalline diamond piezoelectric cantilevers for sensors/actuators

Micro-cantilevers can be used as both sensors and actuators. In this work, the design, fabrication and characterization of piezoelectrically driven nano-crystalline diamond (NCD) cantilevers are reported Diamond films were grown on silicon (100) substrates by microwave plasma enhanced chemical vapor deposition (MW-PECVD). Cantilevers are coated by DC pulsed piezoelectric with AlN films that is sandwiched between two metallic electrodes. The thicknesses of AlN and diamond layers are 1μm and 700nm, respectively. The influence on the electromechanical response of cantilevers length was studied. The motion of the electrically driven cantilevers is performed by measuring the evolution of the electrical impedance at the resonant frequencies that varies between 10 kHz and 130 kHz for the resonant mode.     

Polymer microcantilever biochemical sensors with integrated polymer composites for electrical detection

Micro fabricated sensors based on nanomechanical motion with piezoresistive electrical readout have become a promising biochemical sensing tool. The conventional microcantilever materials are mostly silicon-based. The sensitivity of the sensor depends on Young's modulus of the structural material, thickness of the cantilever as well as on the gauge factor of the piezoresistor. UV patternable polymers such as SU-8 have a very low Young's modulus compared to the silicon-based materials. Polymer cantilevers with a piezoresistive material having a large gauge factor and a lower Young's modulus are therefore highly suited for sensing applications. In this work, a spin coatable and photopatternable mixture of carbon black (CB) and SU-8, with proper dispersion characteristics, has been demonstrated as a piezoresistive thin film for polymer microcantilevers. Results on percolation experiments of SU-8/CB composite and fabrication of piezoresistive SU-8 microcantilevers using this composite are presented. With our controlled dispersion experiments, we could get a uniform piezoresistive thin film of thickness less than 1.2 μm and resistivity of 2.7 Ω cm using 10 wt% of CB in SU-8. The overall thickness of the SU-8/composite/SU-8 is approximately 3 μm. We further present results on the electromechanical characterization and biofunctionalization of the cantilever structures for biochemical sensing applications. These cantilevers show a deflection sensitivity of 0.55 ppm/nm. Since the surface stress sensitivity is 4.1 × 10⁻³ [N/m]⁻¹, these cantilevers can well be used for detection of protein markers for pathological applications.     

Static deflection measurements of cantilever arrays reveal polymer film expansion and contraction

An optical static method of detection is used to interpret surface stress induced bending related to cantilevers coated on one side with poly(vinyl alcohol), poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate), and poly(vinyl chloride-co-vinyl acetate-co-2-hydroxypropyl acrylate), or respectively, PVA, PVB, and PVC, and exposed to various solvent vapors. Results indicate that the adsorption and surface interactions of the different solvent vapors that cause polymer swelling and shrinking lead to rearrangements, which have been shown to change the elastic properties of the polymer film, and subsequently, the spring constant of the polymer coated cantilever. Static deflection measurements allow the direction of cantilever bending to be determined, which adds a new dimension of usefulness for surface functionalized cantilevers as transducers in the development of novel microelectromechanical systems (MEMS).     

Analysis of DNA hybridization regarding the conformation of molecular layer with piezoelectric microcantilevers

Lead Zirconate Titanate (PZT)-embedded microcantilevers were fabricated with dimensions of 30 × 90 × 3 μm(3) (width × length × thickness). A thicker PZT layer improved the actuation and enabled long-term data acquisition in common aqueous buffers with a frequency resolution of 20 Hz. A quantitative assay was conducted in the range of 1-20 μM and the resonant frequency was found to increase with the concentration of target DNAs and the probe DNAs were almost saturated at 20 μM. Back-filling with ethyleneglycol-modified alkanethiol was shown to facilitate the hybridization efficiency and stabilize the surface reaction, resulting in a signal enhancement of 40%. We report for the first time how secondary structures in oligonucleotide monolayer change the surface property of a dynamic mode microcantilever and subsequently affect its oscillating behavior. Using fabricated microcantilevers, the real time changes in resonant frequency upon hybridization were measured by utilizing different probe and target sets. The results revealed that the microcantilevers experienced a resonant frequency upshift during the hybridization with complementary DNAs if a dimer structure was present between DNA probes. A resonant frequency downshift was observed for DNA probes that did not contain any complex secondary structures. In addition, the results demonstrate the potential of using these microcantilevers to extract structural information of oligonucleotides.     

'Living cantilever arrays' for characterization of mass of single live cells in fluids

The size of a cell is a fundamental physiological property and is closely regulated by various environmental and genetic factors. Optical or confocal microscopy can be used to measure the dimensions of adherent cells, and Coulter counter or flow cytometry (forward scattering light intensity) can be used to estimate the volume of single cells in a flow. Although these methods could be used to obtain the mass of single live cells, no method suitable for directly measuring the mass of single adherent cells without detaching them from the surface is currently available. We report the design, fabrication, and testing of 'living cantilever arrays', an approach to measure the mass of single adherent live cells in fluid using silicon cantilever mass sensor. HeLa cells were injected into microfluidic channels with a linear array of functionalized silicon cantilevers and the cells were subsequently captured on the cantilevers with positive dielectrophoresis. The captured cells were then cultured on the cantilevers in a microfluidic environment and the resonant frequencies of the cantilevers were measured. The mass of a single HeLa cell was extracted from the resonance frequency shift of the cantilever and was found to be close to the mass value calculated from the cell density from the literature and the cell volume obtained from confocal microscopy. This approach can provide a new method for mass measurement of a single adherent cell in its physiological condition in a non-invasive manner, as well as optical observations of the same cell. We believe this technology would be very valuable for single cell time-course studies of adherent live cells.     

In-situ quantitative analysis of a prostate-specific antigen (PSA) using a nanomechanical PZT cantilever

We report on a novel technique of resonant frequency shift measurement based on a nanomechanical cantilever with a PZT actuating layer for label-free detection of a prostate-specific antigen (PSA) in a liquid environment. The nanomechanical PZT thin film cantilever is composed of SiO(2)/Ta/Pt/PZT/Pt/SiO(2) on a SiN(x) supporting layer for simultaneous self-exciting and sensing; it was fabricated using a standard MEMS (micro electromechanical system) process. The specific binding characteristics of the PSA antigen to its antibody, which is immobilized with Calixcrown self-assembled monolayers (SAMs) on a gold surface deposited on a cantilever, are determined to a high sensitivity. For the bioassay in a liquid environment, a liquid test cell with a 20 microl volume reaction chamber has been fabricated, using a bonding technique between poly(dimethyl siloxane) (PDMS) bilayers. An observed trend of resonant frequency change with respect to time could be explained by the binding kinetics due to the Langmuir isotherm and diffusion and by the effects of a small volume reaction chamber. In the saturated regimes, the resonant frequency of the cantilever increased with increase of the PSA concentration in the reaction chamber, showing that the trend of the resonance frequency change was similar to that of the fluorescence results. The saturated resonance frequency shift of the cantilever was proportional to the PSA antigen concentration of analyte solution.     

Microcantilevers as gas‐phase sensing platforms: Simplification and optimization of the production of polymer coated porous‐silicon‐over‐silicon microcantilevers

The asymmetric roughening of silicon microcantilevers using different vapor stain‐etching conditions is studied with the aim of optimizing face selective coating of microcantilevers by polymers through simple dipping. The effect of roughening is studied by following the time‐dependent guest‐induced bending of silicone microcantilevers coated with a poly‐4‐vinylpyridine sensing layer. A correlation between the surface roughness of the microcantilevers and their time‐dependent guest‐induced bending is gained from combining high resolution scanning electron microscopy studies of the surface of the microcantilevers as well as their cross‐sections with time‐dependent guest‐induced microcantilever bending. The purpose of the present work is to lay the foundations for a small and relatively simple gas‐phase sensing tool based on a microcantilever platform capable of offering wide range sensing capabilities. © 2013 Wiley Periodicals, 2014 , 52, 141–146     

Towards a modular,versatile and portable sensor system for measurements in gaseous environments based on microcantilevers

Microfabricated silicon cantilever sensor arrays represent a powerful platform for sensing applications in physics, chemistry, material science, biology and medicine. The sensor response is mechanical bending due to absorption of molecules. In gaseous environment, polymer-coated microcantilevers are used as electronic nose for characterization of vapors, resulting in cantilever bending due to polymer swelling upon exposure. Medical applications involve fast characterization of exhaled patient's breath samples for detection of diseases, based on the presence of certain chemicals in breath. We present a portable, compact, modular microcantilever setup, which uses a micropump for aspiration and a bluetooth interface for remote data acquisition.     

Noncontact dielectric friction

Dielectric fluctuations are shown to be the dominant source of noncontact friction in high-sensitivity scanning probe microscopy of dielectric materials. Recent measurements have directly determined the friction acting on custom-fabricated single-crystal silicon cantilevers whose capacitively charged tips are located 3-200 nm above thin films of poly(methyl methacrylate), poly(vinyl acetate), and polystyrene. Differences in measured friction among these polymers are explained here by relating electric field fluctuations at the cantilever tip to dielectric relaxation of the polymer.     

Microdrops on atomic force microscope cantilevers: evaporation of water and spring constant calibration

The evaporation of water drops with radii approximately 20 microm was investigated experimentally by depositing them onto atomic force microscope (AFM) cantilevers and measuring the deflection versus time. Because of the surface tension of the liquid, the Laplace pressure inside the drop, and the change of interfacial stress at the solid-liquid interface, the cantilever is deflected by typically a few hundred nanometers. The experimental results are in accordance with an analytic theory developed. The evaporation process could be monitored with high accuracy even at the last stage of evaporation because (1) cantilever deflections can be measured with nanometer resolution and (2) the time resolution, given by the inverse of the resonance frequency of the cantilever of approximately 0.3 ms, is much faster than the typical evaporation time of 1 s. Experimental results indicate that evaporation of the last thin layer of water is significantly slower than the rest of the drop, which can be due to surface forces. This drop-on-cantilever system can also be used to analyze the drop impact dynamics on a surface and to determine the spring constant of cantilevers.     

Use of biospecific reactions for the design of high-sensitivity biosensors based on nanomechanical cantilever systems

The study of nanomechanical cantilever systems is among the priority directions in the progression of nanotechnologies. Principally new ways of designing biosensors based on nanocantilever transducers are investigated, the effect of orientation of receptor immunoglobulin molecules in the sensor layer on the formation of lateral strain during complementary binding is examined for the first time, and unique techniques for creation of selective receptor transducers based on cantilevers are developed. The unique data of this study make it possible to state that, owing to the presence of 13-thiotridecane-1,1,2-triol molecules in the probe DNA layer, the lateral strain tends to increase during hybridization of complementary molecules. Theoretical predictions and experimental data are compared, and the effect of the formation of lateral strain in polymer layers on the bending of the cantilever transducer is revealed. The nature of lateral strain arising in films of biopolymers (proteins and DNA) during complementary binding (formation of the immune complex for protein molecules and hybridization for DNA) is ascertained.     

Detection of Pb2+ using a hydrogel swelling microcantilever sensor.

Hydrogels containing benzo-18-crown-6 were used to modify microcantilevers for measurements of the concentration of Pb2+ in aqueous solutions. These microcantilevers undergo bending deflection upon exposure to solutions containing various Pb2+ concentrations as the result of a swelling of the hydrogels. It was found that a concentration of 10(-6) M Pb2+ can be detected using this technology. Other cations, such as Na+, have no effect on the deflection of this cantilever. The cation K+, which also complexes with benzo-18-crown-6, could interfere with Pb2+ detection, but only at high concentrations (> 10(-4) M).     

Hamaker constants in integrated circuit metalization

A new method for determining Hamaker constants was examined for materials of interest in integrated circuit manufacture. An ultra-high vacuum atomic force microscope and an atomic force microscope operated in a nitrogen environment were used to measure the interaction forces between metals, dielectrics, and barriers used during the metalization portion of integrated circuit manufacturing. The materials studied included copper, silver, titanium nitride, silicon dioxide, poly(tetrafluoroethylene), and parylene-N. Spheres coated with a material of interest were mounted on AFM cantilevers and brought into contact with substrates of interest. The interaction force was measured as the cantilever approached the substrate but before the two surfaces came into contact, and also when the particle was pulled out of contact with the substrate. The Hamaker constant calculation from the contact measurement is based on an adhesion model that quantifies the contribution of geometrical, morphological and mechanical properties of materials to the measured adhesion force. Hamaker constants determined with this new approach were compared with values found by using the Derjaguin approximation for a sphere to describe the interaction force as the cantilever approaches the surface. Both approaches produced similar values for most of the systems studied, with variations of less than 10%.     

Detection of Bacillus subtilis spores using peptide-functionalized cantilever arrays

We move beyond antibody-antigen binding systems and demonstrate that short peptide ligands can be used to efficiently capture Bacillus subtilis (a simulant of Bacillus anthracis) spores in liquids. On an eight-cantilever array chip, four cantilevers were coated with binding peptide (NHFLPKV-GGGC) and the other four were coated with control peptide (LFNKHVP-GGGC) for reagentless detection of whole B. subtilis spores in liquids. The peptide-ligand-functionalized microcantilever chip was mounted onto a fluid cell filled with a B. subtilis spore suspension for approximately 40 min; a 40 nm net differential deflection was observed. Fifth-mode resonant frequency measurements were also performed before and after dipping microcantilever arrays into a static B. subtilis solution showing a substantial decrease in frequency for binding-peptide-coated microcantilevers as compared to that for control peptide cantilevers. Further confirmation was obtained by subsequent examination of the microcantilever arrays under a dark-field microscope. Applications of this technology will serve as a platform for the detection of pathogenic organisms including biowarfare agents.     

Phase transitions between the rotator phases of paraffin investigated using silicon microcantilevers

Nanogram amounts of paraffin were coated onto a silicon cantilever, and the resonance frequency and deflection of the cantilever were measured as a function of temperature. Changes in the cantilever resonance frequency were used to determine the temperatures at which phase transitions between the rotator phases of tricosane, tetracosane, and pentacosane occurred. The phase transition measured using the cantilever was found to be more apparent than that obtained using conventional methods. The thermal hysteresis in the resonance frequency of a tetracosane-coated cantilever differed from that of the tricosane- and pentacosane-coated cantilevers, which was attributed to the even-odd effect on the crystal structures of paraffin. The even-odd effect was also observed in the temperature dependent deflection measurements. Further, the overshoot at the transition R(V) → crystal in the deflection measurement was observed and attributed to the steep increase in the modulus of paraffin during the transition.     

Low-energy electron scattering on deuterated nanocrystalline diamond films-a model system for understanding the interplay between density-of-states, excitation mechanisms and surface versus lattice contributions

Electron energy loss spectrum, elastic reflectivity and selected vibrational excitation functions were measured by High Resolution Electron Energy Loss Spectroscopy (HREELS) for deuterated nanocrystalline dc GD CVD diamond films. The electron elastic reflectivity is strongly enhanced at about 13 eV, as a consequence of the second absolute band gap of diamond preserved up to the surface for D-nano-crystallites. The pure bending modes δ(CD(x)) at 88 meV and 107 meV are dominantly excited through the impact mechanism and their vibration excitation functions mimic the electron elastic reflectivity curve. Pure diamond phonon mode ν(CC) can be probed through the resolved fundamental loss located at 152 meV and through the multiple loss located at 300 meV. In addition to the well-known 8 eV resonance, two supplementary resonances located at 4.5 eV and 11.5 eV were identified and clearly resolved for the first time. A comprehensive set of data is now available on low-energy electron scattering at hydride terminated polycrystalline diamond films grown either by HF (microcrystalline) or dc GD (nanocrystalline) chemical vapour deposition. The careful comparison of the vibrational excitation functions for hydrogen/deuterium termination stretching modes ν(sp(3)-CH(x)) and ν(sp(3)-CD(x)), for hydrogen termination bending modes δ(CH(x)) mixed with diamond lattice modes ν(CC), for deuterium termination bending modes δ(CD(x)), and for multiple loss 2ν(CC) demonstrates the close interplay between three characteristics: (i) the density-of-states of the substrate, (ii) the vibrational excitation mechanisms (dipolar and/or impact scattering including resonant scattering) and (iii) the surface versus lattice character of the excited vibrational modes. This work shows clearly that excitation function measurement provides a powerful and sensitive tool to clarify loss attributions, involved excitation mechanisms, and surface versus lattice characters of the excited vibrational modes.