High Dynamic Range Nanowire Resonators.

Dynamic range quantifies the linear operation regime available in nanomechanical resonators. Nonlinearities dominate the response of flexural beams in the limit of very high aspect ratio and very small diameter, which leads to expectation of low dynamic range for nanowire resonators in general. However, the highest achievable dynamic range for nanowire resonators with practical dimensions remains to be determined. We report dynamic range measurements on singly clamped silicon nanowire resonators reaching remarkably high values of up to 90 dB obtained with a simple harmonic actuation scheme. We explain these measurements by a comprehensive theoretical examination of dynamic range in singly clamped flexural beams including the effect of tapering, a usual feature of semiconductor nanowires. Our analysis reveals the nanowire characteristics required for broad linear operation, and given the relationship between dynamic range and mass sensing performance, it also enables analytical determination of mass detection limits, reaching atomic-scale resolution for feasible nanowires.

Nanomechanical Molecular Mass Sensing Using Suspended Microchannel Resonators.

In this work we study the different phenomena taking place when a hydrostatic pressure is applied in the inner fluid of a suspended microchannel resonator. Additionally to pressure-induced stiffness terms, we have theoretically predicted and experimentally demonstrated that the pressure also induces mass effects which depend on both the applied pressure and the fluid properties. We have used these phenomena to characterize the frequency response of the device as a function of the fluid compressibility and molecular masses of different fluids ranging from liquids to gases. The proposed device in this work can measure the mass density of an unknown liquid sample with a resolution of 0.7 µg/mL and perform gas mixtures characterization by measuring its average molecular mass with a resolution of 0.01 atomic mass units.

Optical Transduction for Vertical Nanowire Resonators.

We describe an optical transduction mechanism to measure the flexural mode vibrations of vertically aligned nanowires on a flat substrate with high sensitivity, linearity, and ease of implementation. We demonstrate that the light reflected from the substrate when a laser beam strikes it parallel to the nanowires is modulated proportionally to their vibration, so that measuring such modulation provides a highly efficient resonance readout. This mechanism is applicable to single nanowires or arrays without specific requirements regarding their geometry or array pattern, and no fabrication process besides the nanowire generation is required. We show how to optimize the performance of this mechanism by characterizing the split flexural modes of vertical silicon nanowires in their full dynamic range and up to the fifth mode order. The presented transduction approach is relevant for any application of nanowire resonators, particularly for integrating nanomechanical sensing in functional substrates based on vertical nanowires for biological applications.

Optomechanical devices for deep plasma cancer proteomics.

Most of the cancer deaths could be avoided by early detection of the tumor when it is confined to its primary site and it has not metastasized. To this aim, one of the most promising strategies is the discovery and detection of protein biomarkers shed by the young tumor to the bloodstream. Proteomic technologies, mainly mass spectrometry and multiplexed immunoassays, have rapidly developed during last years with improved limits of detection and multiplexing capability. Unfortunately, these developments together major investments and large international efforts have not resulted into new useful protein biomarkers. Here, we analyze the potential and limitations of current proteomic technologies for detecting protein biomarkers released into circulation by the tumor. We find that these technologies can hardly probe the deepest region of the plasma proteome, at concentrations below the pg/mL level, where protein biomarkers for early cancer detection may exist. This clearly indicates the need of incorporating novel ultrasensitive techniques to the proteomic tool-box that can cover the inaccessible regions of the plasma proteome. We here propose biological detectors based on nanomechanical systems for discovery and detection of cancer protein biomarkers in plasma. We review the modes of operation of these devices, putting our focus on recent developments on nanomechanical sandwich immunoassays and nanomechanical spectrometry. The first technique enables reproducible immunodetection of proteins at concentrations well below the pg/mL level, with a limit of detection on the verge of 10 ag/mL. This technology can potentially detect low abundance tumor-associated proteins in plasma at the very early stages of the tumor. The second technique enables the identification of individual intact proteins by two physical coordinates, the mass and stiffness, instead of the mass-to-charge ratio of the protein constituents. This technology enormously simplifies the identification of proteins and it can provide useful information on interactions and posttranslational modifications, that otherwise is lost in mass spectrometry.

Coherent Optical Transduction of Suspended Microcapillary Resonators for Multi-Parameter Sensing Applications.

Characterization of micro and nanoparticle mass has become increasingly relevant in a wide range of fields, from materials science to drug development. The real-time analysis of complex mixtures in liquids demands very high mass sensitivity and high throughput. One of the most promising approaches for real-time measurements in liquid, with an excellent mass sensitivity, is the use of suspended microchannel resonators, where a carrier liquid containing the analytes flows through a nanomechanical resonator while tracking its resonance frequency shift. To this end, an extremely sensitive mechanical displacement technique is necessary. Here, we have developed an optomechanical transduction technique to enhance the mechanical displacement sensitivity of optically transparent hollow nanomechanical resonators. The capillaries have been fabricated by using a thermal stretching technique, which allows to accurately control the final dimensions of the device. We have experimentally demonstrated the light coupling into the fused silica capillary walls and how the evanescent light coming out from the silica interferes with the surrounding electromagnetic field distribution, a standing wave sustained by the incident laser and the reflected power from the substrate, modulating the reflectivity. The enhancement of the displacement sensitivity due to this interferometric modulation (two orders of magnitude better than compared with previous accomplishments) has been theoretically predicted and experimentally demonstrated.

Nanomechanical Plasmon Spectroscopy of Single Gold Nanoparticles.

We experimentally demonstrate the effect of the localized surface plasmon resonance (LSPR) of a single gold nanoparticle (AuNP) of 100 nm in diameter on the mechanical resonance frequency of a free-standing silicon nitride membrane by means of optomechanical transduction. We discover that a key effect to explain the coupling in these systems is the extinction cross section enhancement due to the excitation of the LSPR at selected wavelengths. In order to validate this coupling, we have developed a fixed wavelength interferometric readout system with an integrated tunable laser source, which allows us to perform the first experimental demonstration of nanomechanical spectroscopy of deposited AuNPs onto the membrane, discerning in between single particles and dimers by the mechanical frequency shift. We have also introduced three-axis mechanical scanners with nanometer-scale resolution in our experimental setup to selectively study single nanoparticles or small clusters. Whereas the single particles are polarization-insensitive, the gold dimers have a clearly defined polarization angle dependency as expected by theory. Finally, we found an unexpected long-distance (∼200 nm) coupling of the LSPR of separated AuNPs coming out from the guided light by the silicon nitride membrane.

Direct Detection of OXA-48 Carbapenemase Gene in Lysate Samples through Changes in Mechanical Properties of DNA Monolayers upon Hybridization.

Carbapenem-resistant Enterobacteriaceae have recently become an important cause of morbidity and mortality due to healthcare-associated infections. Most commonly used diagnostic methods are incompatible with fast and accurate directed therapy. We report here the direct identification of the blaOXA48 gene, which codes for the carbapenemase OXA-48, in lysate samples from Klebsiella pneumoniae. The method is PCR-free and label-free. It is based on the measurement of changes in the stiffness of DNA self-assembled monolayers anchored to microcantilevers that occur as a consequence of the hybridization. The stiffness of the DNA layer is measured through changes of the sensor resonance frequency upon hybridization and at varying relative humidity.

Spatially Multiplexed Micro-Spectrophotometry in Bright Field Mode for Thin Film Characterization.

Thickness characterization of thin films is of primary importance in a variety of nanotechnology applications, either in the semiconductor industry, quality control in nanofabrication processes or engineering of nanoelectromechanical systems (NEMS) because small thickness variability can strongly compromise the device performance. Here, we present an alternative optical method in bright field mode called Spatially Multiplexed Micro-Spectrophotometry that allows rapid and non-destructive characterization of thin films over areas of mm² and with 1 µm of lateral resolution. We demonstrate an accuracy of 0.1% in the thickness characterization through measurements performed on four microcantilevers that expand an area of 1.8 mm² in one minute of analysis time. The measured thickness variation in the range of few tens of nm translates into a mechanical variability that produces an error of up to 2% in the response of the studied devices when they are used to measure surface stress variations.

Label-free DNA-based detection of Mycobacterium tuberculosis and rifampicin resistance through hydration induced stress in microcantilevers.

We have developed a label-free assay for the genomic detection of Mycobacterium tuberculosis and rifampicin resistance. The method relies on the quantification of the hydration induced stress on microcantilever biosensors functionalized with oligonucleotide probes, before and after hybridization with specific targets. We have found a limit of detection of 10 fg/mL for PCR amplified products of 122 bp. Furthermore, the technique can successfully target genomic DNA (gDNA) fragments of length >500 bp, and it can successfully discriminate single mismatches. We have used both loci IS6110 and rpoB as targets to detect the mycobacteria and the rifampicin resistance from gDNA directly extracted from bacterial culture and without PCR amplification. We have been able to detect 2 pg/mL target concentration in samples with an excess of interfering DNA and in a total analysis time of 1 h and 30 min. The detection limit found demonstrates the capability to develop direct assays without the need for long culture steps or PCR amplification. The methodology can be easily translated to different microbial targets, and it is suitable for further development of miniaturized devices and multiplexed detection.

Highly sensitive measurement of liquid density in air using suspended microcapillary resonators.

We report the use of commercially available glass microcapillaries as micromechanical resonators for real-time monitoring of the mass density of a liquid that flows through the capillary. The vibration of a suspended region of the microcapillary is optically detected by measuring the forward scattering of a laser beam. The resonance frequency of the liquid filled microcapillary is measured for liquid binary mixtures of ethanol in water, glycerol in water and Triton in ethanol. The method achieves a detection limit in an air environment of 50 µg/mL that is only five times higher than that obtained with state-of-the-art suspended microchannel resonators encapsulated in vacuum. The method opens the door to novel advances for miniaturized total analysis systems based on microcapillaries with the add-on of mechanical transduction for sensing the rheological properties of the analyzed fluids without the need for vacuum encapsulation of the resonators.

Hydration induced stress on DNA monolayers grafted on microcantilevers.

Surface tethered single-stranded DNA films are relevant biorecognition layers for oligonucleotide sequence identification. Also, hydration induced effects on these films have proven useful for the nanomechanical detection of DNA hybridization. Here, we apply nanomechanical sensors and atomic force microscopy to characterize in air and upon varying relative humidity conditions the swelling and deswelling of grafted single stranded and double stranded DNA films. The combination of these techniques validates a two-step hybridization process, where complementary strands first bind to the surface tethered single stranded DNA probes and then slowly proceed to a fully zipped configuration. Our results also demonstrate that, despite the slow hybridization kinetics observed for grafted DNA onto microcantilever surfaces, ex situ sequence identification does not require hybridization times typically longer than 1 h, while quantification is a major challenge.

Biosensors based on nanomechanical systems.

The advances in micro- and nanofabrication technologies enable the preparation of increasingly smaller mechanical transducers capable of detecting the forces, motion, mechanical properties and masses that emerge in biomolecular interactions and fundamental biological processes. Thus, biosensors based on nanomechanical systems have gained considerable relevance in the last decade. This review provides insight into the mechanical phenomena that occur in suspended mechanical structures when either biological adsorption or interactions take place on their surface. This review guides the reader through the parameters that change as a consequence of biomolecular adsorption: mass, surface stress, effective Young's modulus and viscoelasticity. The mathematical background needed to correctly interpret the output signals from nanomechanical biosensors is also outlined here. Other practical issues reviewed are the immobilization of biomolecular receptors on the surface of nanomechanical systems and methods to attain that in large arrays of sensors. We then describe some relevant realizations of biosensor devices based on nanomechanical systems that harness some of the mechanical effects cited above. We finally discuss the intrinsic detection limits of the devices and the limitation that arises from non-specific adsorption.

Simultaneous Optical and Mechanical Sensing Based on Optomechanical Resonators.

Optical and mechanical resonators have each been abundantly employed in sensing applications, albeit following separate development. Here we show that bringing together optical and mechanical resonances in a unique sensing device significantly improves the sensor performance. To that purpose, we employ nanoscale optomechanical disk resonators that simultaneously support high quality optical and mechanical modes localized in tiny volumes, which provide extraordinary sensitivities. We perform environmental sensing, but the conclusions of our work extend to other sensing applications. First, we determine optical and mechanical responsivities to temperature and relative humidity changes. Second, by characterizing mechanical and optical frequency stabilities, we determine the corresponding limits of detection. Mechanical modes appear more sensitive to relative humidity changes, while optical modes appear more sensitive to temperature ones, reaching, respectively, 0.05% and 0.6 mK of independent resolution. We then prove that simultaneous optical and mechanical monitoring enables disentangling both effects and demonstrates 0.1% and 1 mK resolution, even considering that both parameters may change at the same time. Finally, we highlight the importance of actively tracking the optical mode when optomechanical sensor devices. Not doing so enforces tedious independent calibration, influences the device sensitivity during the experiment, and shortens the sensing range. The present work hence clarifies the requirements for the optimal operation of optomechanical sensors, which will be of importance for chemical and biological sensing.

Tackling reproducibility in microcantilever biosensors: a statistical approach for sensitive and specific end-point detection of immunoreactions.

In the last decade, microcantilever biosensors have shown enormous potential for highly sensitive label-free detection of nucleic acid and proteins. Despite the enormous advances, the promise of applications of this technology in the biomedical field has been frustrated because of its low reproducibility. Here we tackle the reproducibility issue in microcantilever biosensors and provide the guidelines to minimize the deviations in the biosensor response between different assays. We use as a model system the label-free end-point detection of horseradish peroxidase. We choose the end-point detection mode because of its suitability for implementation in the clinical field that requires simplicity and point-of-care capability. Our study comprises the analysis of 1012 cantilevers with different antibody surface densities, two blocking strategies based on polyethylene-glycol (PEG) and bovine serum albumin (BSA) and stringent controls. The study reveals that the performance of the assay critically depends on both antibody surface density and blocking strategies. We find that the optimal conditions involve antibody surface densities near but below saturation and blocking with PEG. We find that the surface stress induced by the antibody-antigen binding is significantly correlated with the surface stress generated during the antibody attachment and blocking steps. The statistical correlation is harnessed to identify immobilization failure or success, and thus enhancing the specificity and sensitivity of the assay. This procedure enables achieving rates of true positives and true negatives of 90% and 91% respectively. The detection limit is of 10 ng mL(-1) (250 pM) that is similar to the detection limit obtained in our enzyme-linked immunosorbent assay (ELISA) and at least two orders of magnitude smaller than that achieved with well-established label-free biosensors such as a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) sensor.

Nanomechanical Sensing for Mass Flow Control in Nanowire-Based Open Nanofluidic Systems.

Open nanofluidic systems, where liquids flow along the outer surface of nanoscale structures, provide otherwise unfeasible capabilities for extremely miniaturized liquid handling applications. A critical step toward fully functional applications is to obtain quantitative mass flow control. We demonstrate the application of nanomechanical sensing for this purpose by integrating voltage-driven liquid flow along nanowire open channels with mass detection based on flexural resonators. This approach is validated by assembling the nanowires with microcantilever resonators, enabling high-precision control of larger flows, and by using the nanowires as resonators themselves, allowing extremely small liquid volume handling. Both implementations are demonstrated by characterizing voltage-driven flow of ionic liquids along the surface of the nanowires. We find a voltage range where mass flow rate follows a nonlinear monotonic increase, establishing a steady flow regime for which we show mass flow control at rates from below 1 ag/s to above 100 fg/s and precise liquid handling down to the zeptoliter scale. The observed behavior of mass flow rate is consistent with a voltage-induced transition from static wetting to dynamic spreading as the mechanism underlying liquid transport along the nanowires.

Square Membrane Resonators Supporting Degenerate Modes of Vibration for High-Throughput Mass Spectrometry of Single Bacterial Cells.

In nanomechanical mass spectrometry, sensing devices are commonly placed in the vacuum environment and a stream of analytes is directed toward the sensor surface for measurement. Beam structures, such as double-clamped nanobeams and nanocantilevers, are commonly used due to their low inertial mass and the simplicity of the analytical models for mass extraction. The drawback of such structures is their low capture areas, compromising the capture efficiency and throughput of this technique. Bi-axisymmetric resonators, such as ultrathin square or circular membranes, arise as an optimal geometry to maximize capture efficiency while minimizing the device inertial mass. However, these structures present degenerate mechanical modes, whose frequency perturbations upon analyte adsorption are not well described by commonly used models. Furthermore, prior knowledge of the vibration mode shapes of the sensor is crucial for the correct calculation of the analyte's mass, and the mode shape of degenerate modes may change significantly after every adsorption event. In this work, we present an accurate analytical theory to describe the effect of mass adsorption on the degenerate modes of square membrane resonators and propose two different methods based on the new theory to update the vibration mode shapes after every adsorption event. Finally, we illustrate the problem experimentally obtaining the mass and adsorption position of individual Escherichia coli K-12 bacterial cells on commercial square silicon nitride membranes fabricated with very small tolerances.

Quantification of the surface stress in microcantilever biosensors: revisiting Stoney's equation.

Microcantilever biosensors in the static operation mode translate molecular recognition into a surface stress signal. Surface stress is derived from the nanomechanical cantilever bending by applying Stoney's equation, derived more than 100 years ago. This equation ignores the clamping effect on the cantilever deformation, which induces significant errors in the quantification of the biosensing response. This leads to discrepancies in the surface stress induced by biomolecular interactions in measurements with cantilevers with different sizes and geometries. So far, more accurate solutions have been precluded by the formidable complexity of the theoretical problem that involves solving the two-dimensional biharmonic equation. In this paper, we present an accurate and simple analytical expression to quantify the response of microcantilever biosensors. The equation exhibits an excellent agreement with finite element simulations and DNA immobilization experiments on gold-coated microcantilevers.

Stepwise motion of a microcantilever driven by the hydrolysis of viral ATPases.

The biomolecular machines involved in DNA packaging by viruses generate one of the highest mechanical powers observed in nature. One component of the DNA packaging machinery, called the terminase, has been proposed as the molecular motor that converts chemical energy from ATP hydrolysis into mechanical movement of DNA during bacteriophage morphogenesis. However, the conformational changes involved in this energy conversion have never been observed. Here we report a real-time measurement of ATP-induced conformational changes in the terminase of bacteriophage T7 (gp19). The recording of the cantilever bending during its functionalization shows the existence of a gp19 monolayer arrangement confirmed by atomic force microscopy of the immobilized proteins. The ATP hydrolysis of the gp19 terminase generates a stepped motion of the cantilever and points to a mechanical cooperative effect among gp19 oligomers. Furthermore, the effect of ATP can be counteracted by non-hydrolyzable nucleotide analogs.

Imaging the surface stress and vibration modes of a microcantilever by laser beam deflection microscopy.

There is a need for noninvasive techniques for simultaneous imaging of the stress and vibration mode shapes of nanomechanical systems in the fields of scanning probe microscopy, nanomechanical biological and chemical sensors and the semiconductor industry. Here we show a novel technique that combines a scanning laser, the beam deflection method and digital multifrequency excitation and analysis for simultaneous imaging of the static out-of-plane displacement and the shape of five vibration modes of nanomechanical systems. The out-of-plane resolution is at least 100 pm Hz⁻¹/² and the lateral resolution, which is determined by the laser spot size, is 1-1.5 µm. The capability of the technique is demonstrated by imaging the residual surface stress of a microcantilever together with the shape of the first 22 vibration modes. The vibration behavior is compared with rigorous finite element simulations. The technique is suitable for major improvements in the imaging of liquids, such as higher bandwidth and enhanced spatial resolution.

High-throughput determination of dry mass of single bacterial cells by ultrathin membrane resonators.

How bacteria are able to maintain their size remains an open question. Techniques that can measure the biomass (dry mass) of single cells with high precision and high-throughput are demanded to elucidate this question. Here, we present a technological approach that combines the transport, guiding and focusing of individual bacteria from solution to the surface of an ultrathin silicon nitride membrane resonator in vacuum. The resonance frequencies of the membrane undergo abrupt variations at the instants where single cells land on the membrane surface. The resonator design displays a quasi-symmetric rectangular shape with an extraordinary capture area of 0.14 mm2, while maintaining a high mass resolution of 0.7 fg (1 fg = 10-15 g) to precisely resolve the dry mass of single cells. The small rectangularity of the membrane provides unprecedented frequency density of vibration modes that enables to retrieve the mass of individual cells with high accuracy by specially developed inverse problem theory. We apply this approach for profiling the dry mass distribution in Staphylococcus epidermidis and Escherichia coli cells. The technique allows the determination of the dry mass of single bacterial cells with an accuracy of about 1% at an unparalleled throughput of 20 cells/min. Finally, we revisit Koch & Schaechter model developed during 60 s to assess the intrinsic sources of stochasticity that originate cell size heterogeneity in steady-state populations. The results reveal the importance of mass resolution to correctly describe these mechanisms.