Inertial picobalance reveals fast mass fluctuations in mammalian cells.

The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a 'picobalance'), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

Mechanical control of mitotic progression in single animal cells.

Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback-controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, intermediate forces where cells resist confinement (50-100 nN), and yield forces (>100 nN) where a significant decline in cell height impinges on microtubule spindle function, thereby inhibiting mitotic progression. Yield forces are coincident with a nonlinear drop in cell height potentiated by persistent blebbing and loss of cortical F-actin homogeneity. Our results suggest that a buildup of actomyosin-dependent cortical tension and intracellular pressure precedes mechanical failure, or herniation, of the cell cortex at the yield force. Thus, we reveal how the mechanical properties of mitotic cells and their response to external forces are linked to mitotic progression under conditions of mechanical confinement.

Fast Diagnostics of BRAF Mutations in Biopsies from Malignant Melanoma.

According to the American skin cancer foundation, there are more new cases of skin cancer than the combined incidence of cancers of the breast, prostate, lung, and colon each year, and malignant melanoma represents its deadliest form. About 50% of all cases are characterized by a particular mutation BRAF(V600E) in the BRAF (Rapid Acceleration of Fibrosarcoma gene B) gene. Recently developed highly specific drugs are able to fight BRAF(V600E) mutated tumors but require diagnostic tools for fast and reliable mutation detection to warrant treatment efficiency. We completed a preliminary clinical trial applying cantilever array sensors to demonstrate identification of a BRAF(V600E) single-point mutation using total RNA obtained from biopsies of metastatic melanoma of diverse sources (surgical material either frozen or fixated with formalin and embedded in paraffin). The method is faster than the standard Sanger or pyrosequencing methods and comparably sensitive as next-generation sequencing. Processing time from biopsy to diagnosis is below 1 day and does not require PCR amplification, sequencing, and labels.

Piezoresistive Membrane Surface Stress Sensors for Characterization of Breath Samples of Head and Neck Cancer Patients.

For many diseases, where a particular organ is affected, chemical by-products can be found in the patient's exhaled breath. Breath analysis is often done using gas chromatography and mass spectrometry, but interpretation of results is difficult and time-consuming. We performed characterization of patients' exhaled breath samples by an electronic nose technique based on an array of nanomechanical membrane sensors. Each membrane is coated with a different thin polymer layer. By pumping the exhaled breath into a measurement chamber, volatile organic compounds present in patients' breath diffuse into the polymer layers and deform the membranes by changes in surface stress. The bending of the membranes is measured piezoresistively and the signals are converted into voltages. The sensor deflection pattern allows one to characterize the condition of the patient. In a clinical pilot study, we investigated breath samples from head and neck cancer patients and healthy control persons. Evaluation using principal component analysis (PCA) allowed a clear distinction between the two groups. As head and neck cancer can be completely removed by surgery, the breath of cured patients was investigated after surgery again and the results were similar to those of the healthy control group, indicating that surgery was successful.

Real-time viscosity and mass density sensors requiring microliter sample volume based on nanomechanical resonators.

A microcantilever based method for fluid viscosity and mass density measurements with high temporal resolution and microliter sample consumption is presented. Nanomechanical cantilever vibration is driven by photothermal excitation and detected by an optical beam deflection system using two laser beams of different wavelengths. The theoretical framework relating cantilever response to the viscosity and mass density of the surrounding fluid was extended to consider higher flexural modes vibrating at high Reynolds numbers. The performance of the developed sensor and extended theory was validated over a viscosity range of 1-20 mPa·s and a corresponding mass density range of 998-1176 kg/m(3) using reference fluids. Separating sample plugs from the carrier fluid by a two-phase configuration in combination with a microfluidic flow cell, allowed samples of 5 µL to be sequentially measured under continuous flow, opening the method to fast and reliable screening applications. To demonstrate the study of dynamic processes, the viscosity and mass density changes occurring during the free radical polymerization of acrylamide were monitored and compared to published data. Shear-thinning was observed in the viscosity data at higher flexural modes, which vibrate at elevated frequencies. Rheokinetic models allowed the monomer-to-polymer conversion to be tracked in spite of the shear-thinning behavior, and could be applied to study the kinetics of unknown processes.

Development of robust and standardized cantilever sensors based on biotin/NeutrAvidin coupling for antibody detection.

A cantilever-based protein biosensor has been developed providing a customizable multilayer platform for the detection of antibodies. It consists of a biotin-terminated PEG layer pre-functionalized on the gold-coated cantilever surface, onto which NeutrAvidin is adsorbed through biotin/NeutrAvidin specific binding. NeutrAvidin is used as a bridge layer between the biotin-coated surface and the biotinylated biomolecules, such as biotinylated bovine serum albumin (biotinylated BSA), forming a multilayer sensor for direct antibody capture. The cantilever biosensor has been successfully applied to the detection of mouse anti-BSA (m-IgG) and sheep anti-BSA(s-IgG) antibodies. As expected, the average differential surface stress signals of about 5.7 ± 0.8 × 10(-3) N/m are very similar for BSA/m-IgG and BSA/s-IgG binding, i.e., they are independent of the origin of the antibody. A statistic evaluation of 112 response curves confirms that the multilayer protein cantilever biosensor shows high reproducibility. As a control test, a biotinylated maltose binding protein was used for detecting specificity of IgG, the result shows a signal of bBSA layer in response to antibody is 5.8 × 10(-3) N/m compared to bMBP. The pre-functionalized biotin/PEG cantilever surface is found to show a long shelf-life of at least 40 days and retains its responsivity of above 70% of the signal when stored in PBS buffer at 4 °C. The protein cantilever biosensor represents a rapid, label-free, sensitive and reliable detection technique for a real-time protein assay.

Optimization of DNA hybridization efficiency by pH-driven nanomechanical bending.

The accessibility and binding affinity of DNA are two key parameters affecting the hybridization efficiency in surface-based biosensor technologies. Better accessibility will result in a higher hybridization efficiency. Often, mixed ssDNA and mercaptohexanol monolayers are used to increase the hybridization efficiency and accessibility of surface-bound oligonucleotides to complementary target DNA. Here, no mercaptohexanol monolayer was used. We demonstrate by differential microcantilever deflection measurements at different pH that the hybridization efficiency peaks between pH 7.5 and 8.5. At low pH 4.5, hydration and electrostatic forces led to tensile surface stress, implying the reduced accessibility of the bound ssDNA probe for hybridization. In contrast, at high pH 8.5, the steric interaction between neighboring ssDNA strands was decreased by higher electrostatic repulsive forces, bending the microcantilever away from the gold surface to provide more space for the target DNA. Cantilever deflection scales with pH-dependent surface hybridization efficiency because of high target DNA accessibility. Hence, by changing the pH, the hybridization efficiency is adjusted.

Rapid Bacteria Detection from Patients' Blood Bypassing Classical Bacterial Culturing.

Sepsis is a life-threatening condition mostly caused by a bacterial infection resulting in inflammatory reaction and organ dysfunction if not treated effectively. Rapid identification of the causing bacterial pathogen already in the early stage of bacteremia is therefore vital. Current technologies still rely on time-consuming procedures including bacterial culturing up to 72 h. Our approach is based on ultra-rapid and highly sensitive nanomechanical sensor arrays. In measurements we observe two clearly distinguishable distributions consisting of samples with bacteria and without bacteria respectively. Compressive surface stress indicates the presence of bacteria. For this proof-of-concept, we extracted total RNA from EDTA whole blood samples from patients with blood-culture-confirmed bacteremia, which is the reference standard in diagnostics. We determined the presence or absence of bacterial RNA in the sample through 16S-rRNA hybridization and species-specific probes using nanomechanical sensor arrays. Via both probes, we identified two clinically highly-relevant bacterial species i.e., Escherichia coli and Staphylococcus aureus down to an equivalent of 20 CFU per milliliter EDTA whole blood. The dynamic range of three orders of magnitude covers most clinical cases. We correctly identified all patient samples regarding the presence or absence of bacteria. We envision our technology as an important contribution to early and sensitive sepsis diagnosis directly from blood without requirement for cultivation. This would be a game changer in diagnostics, as no commercial PCR or POCT device currently exists who can do this.

High-resolution mass measurements of single budding yeast reveal linear growth segments.

The regulation of cell growth has fundamental physiological, biotechnological and medical implications. However, methods that can continuously monitor individual cells at sufficient mass and time resolution hardly exist. Particularly, detecting the mass of individual microbial cells, which are much smaller than mammalian cells, remains challenging. Here, we modify a previously described cell balance ('picobalance') to monitor the proliferation of single cells of the budding yeast, Saccharomyces cerevisiae, under culture conditions in real time. Combined with optical microscopy to monitor the yeast morphology and cell cycle phase, the picobalance approaches a total mass resolution of 0.45 pg. Our results show that single budding yeast cells (S/G2/M phase) increase total mass in multiple linear segments sequentially, switching their growth rates. The growth rates weakly correlate with the cell mass of the growth segments, and the duration of each growth segment correlates negatively with cell mass. We envision that our technology will be useful for direct, accurate monitoring of the growth of single cells throughout their cycle.

Groundwater sources for the Mataranka Springs (Northern Territory, Australia).

The Mataranka Springs Complex is the headwater of the iconic Roper River of northern Australia. Using environmental tracers measured in springs and nearby boreholes, the origin of groundwater contributing to the springs was evaluated to help assess the impact of proposed groundwater extraction in the Cambrian Limestone Aquifer (CLA) for irrigation agriculture and for hydraulic fracturing in the Beetaloo Sub-basin (an anticipated world-class unconventional gas reserve). Major ions, Sr, 87Sr/86Sr, δ18O-H2O, δ2H-H2O, 3H, 14C-DIC were consistent with regional groundwater from the Daly and Georgina basins of the CLA as the sources of water sustaining the major springs (Rainbow and Bitter) and one of the minor springs (Warloch Pond). However, 3H = 0.34 TU in another minor spring (Fig Tree) indicated an additional contribution from a young (probably local) source. High concentrations of radiogenic 4He (> 10-7 cm3 STP g-1) at Rainbow Spring, Bitter Spring and in nearby groundwater also indicated an input of deeper, older groundwater. The presence of older groundwater within the CLA demonstrates the need for an appropriate baseline characterisation of the vertical exchange of groundwater in Beetaloo Sub-basin ahead of unconventional gas resource development.

Rapid and Ultrasensitive Detection of Mutations and Genes Relevant to Antimicrobial Resistance in Bacteria.

The worldwide emergence of multidrug-resistant (MDR) bacteria is associated with significant morbidity, mortality, and healthcare costs. Rapid and accurate diagnostic methods to detect antibiotic resistance are critical for antibiotic stewardship and infection control measurements. Here a cantilever nanosensor-based diagnostic assay is shown to detect single nucleotide polymorphisms (SNPs) and genes associated with antibiotic resistance in Gram negative (Pseudomonas aeruginosa) and positive (Enterococcus faecium) bacteria, representing frequent causes for MDR infections. Highly specific RNA capture probes for SNPs (ampRD135G or ampRG154R ) or resistance genes (vanA, vanB, and vanD) allow to detect the binding of bacterial RNA within less than 5 min. Serial dilutions of bacterial RNA indicate an unprecedented sensitivity of 10 fg µL-1 total RNA corresponding to less than ten bacterial cells for SNPs and 1 fg µL-1 total RNA for vanD detection equivalent to single bacterial cell sensitivity.

Sub-ppm detection of vapors using piezoresistive microcantilever array sensors.

The performance of microfabricated piezoresistive cantilever array sensors has been evaluated using various vapors of volatile organic compounds including alkanes with different chain length from 5 (n-pentane) to 14 (n-tetradecane). We demonstrate that piezoresistive microcantilever array sensors have the selectivity of discriminating individual alkanes in a homologous series as well as common volatile organic compounds according to principal component analysis. We developed a new method to evaluate the sensitivity, taking advantage of the low vapor pressures of alkanes with longer chains, such as n-dodecane, n-tridecane and n-tetradecane, under saturated vapor conditions. This method reveals sub-ppm sensitivity and the cantilever response is found to follow the mass of evaporated analytes as calculated using a quantitative model based on the Langmuir evaporation model.

Modeling sediment oxygen demand in a highly productive lake under various trophic scenarios.

Hypolimnetic oxygen depletion in lakes is a widespread problem and is mainly controlled by the sediment oxygen uptake (SOU) and flux of reduced substances out of the sediments (Fred). Especially in eutrophic lakes, Fred may constitute a major fraction of the areal hypolimnetic mineralization rate, but its size and source is often poorly understood. Using a diagenetic reaction-transport model supported by a large data set of sediment porewater concentrations, bulk sediment core data and lake monitoring data, the behavior of Fred was simulated in eutrophic Lake Baldegg. Transient boundary conditions for the gross sedimentation of total organic carbon and for hypolimnetic O2 concentrations were applied to simulate the eutrophication and re-oligotrophication history of the lake. According to the model, Fred is dominated by methanogenesis, where up to70% to the total CH4 is produced from sediments older than 20 years deposited during the time of permanent anoxia between 1890 and 1982. An implementation of simplified seasonal variations of the upper boundary conditions showed that their consideration is not necessary for the assessment of annual average fluxes in long-term simulations. Four lake management scenarios were then implemented to investigate the future development of Fred and SOU until 2050 under different boundary conditions. A comparison of three trophic scenarios showed that further reduction of the lake productivity to at least a mesotrophic state is required to significantly decrease Fred and SOU from the present state. Conversely, a termination of artificial aeration at the present trophic state would result in high rates of organic matter deposition and a long-term increase of Fred from the sediments of Lake Baldegg.

Microcantilever sensors.

Microfabricated cantilevers have been used in atomic force microscopy for the topography imagingof non-conductive surfaces for more than 20 years. Cantilever beams without tips have proved theirapplicability in recent years as miniaturized, ultrasensitive, and fast-responding sensors for applicationsin chemistry, physics, biochemistry, and medicine. Microcantilever sensors respond by bending dueto the absorption of molecules. A shift in resonance frequency also occurs. They can be operatedin different environments such as gaseous environment, liquids, or vacuum. In gas, microcantileversensors can be operated as an artificial nose, whereby the bending pattern of a microfabricatedarray of eight polymer-coated silicon cantilevers is characteristic of the different vapors from solvents,flavors, and beverages. When operated in a liquid, microcantilever sensors are able to detectbiochemical reactions. Each cantilever is functionalized with a specific biochemical probe receptor,sensitive for detection of the corresponding target molecule. Applications lie in the fields of label-and amplification-free detection of DNA hybridization, the detection of proteins as well as antigen-antibodyreactions, and the detection of larger entities, such as bacteria and fungi.

Pedicle screw augmentation with bone cement enforced Vicryl mesh.

Achieving sufficient mechanical purchase of pedicle screws in osteoporotic or previously instrumented bone is technically and biologically challenging. Techniques using different kinds of pedicle screws or methods of cement augmentation have been used to address this challenge, but are associated with difficult revisions and complications. The purpose of this biomechanical trial was to investigate the use of biocompatible textile materials in combination with bone cement to augment pullout strength of pedicle screws while reducing the risk of cement extrusion. Pedicle screws (6/40 mm) were either augmented with standard bone-cement (Palacos LV + G) in one group (BC, n = 13) or with bone-cement enforced by Vicryl mesh in another group (BCVM, n = 13) in osteoporosis-like saw bone blocks. Pullout testing was subsequently performed. In a second experimental phase, similar experiments were performed using human cadaveric lumbar vertebrae (n = 10). In osteoporosis-like saw bone blocks, a mean screw pullout force of 350 N (±125) was significantly higher with the Bone cement (BC) compared to bone-cement enforced by Vicryl mesh (BCVM) technique with 240 N (±64) (p = 0.030). In human cadaveric lumbar vertebrae the mean screw pullout force was 784 ± 366 N with BC and not statistically different to BCVM with 757 ± 303 N (p = 0.836). Importantly, cement extrusion was only observed in the BC group (40%) and never with the BCVM technique. In vitro textile reinforcement of bone cement for pedicle screw augmentation successfully reduced cement extrusion compared to conventionally delivered bone cement. The mechanical strength of textile delivered cement constructs was more reproducible than standard cementing. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:212-216, 2018.

Resonator combined with a piezoelectric actuator for chemical analysis by force microscopy.

A high frequency silicon resonator for dynamic scanning force microscopy is combined with an integrated piezoelectric actuation element for large displacements. A high resonance frequency is required for imaging on the nanometer scale, and a large displacement is needed for the chemical analysis of the material at the end of the probe. The small piezoelectric resonator is formed at the end of a long piezoelectric actuator using a silicon micromachining technology. The resonator can be oscillated at 96.4 kHz, and the actuator generates a maximum displacement of 15 microm at the end of the probe. The dynamic-mode scanning force microscopy capability, using the integrated piezoelectric resonator, is demonstrated on a 2 microm pitch Au grating.

Micromechanical sensor for studying heats of surface reactions, adsorption, and cluster deposition processes.

We present a newly designed highly sensitive micromechanical sensor devoted to thermodynamic studies involving supported clusters. The thermally sensitive element of the sensor consists of a micromachined silicon cantilever array, onto which a thin metal film is evaporated. Due to the difference between the thermal expansion coefficients of silicon and the metal employed, thermal bending is observed when heat is exchanged with the cantilever. The sensitivity and the response time of the cantilever are studied as a function of the film material (gold or aluminum) and the thickness of the metal film. With our routinely prepared cantilevers, a minimum power of 120 nW is measurable with a submillisecond response time, corresponding to a limit of detection in the femtojoule range. The high sensitivity of the sensor is demonstrated by measuring the heat exchange which occurs during the deposition of clusters on the cantilever. Experimentally, we illustrate the 1,3-butadiene hydrogenation reaction using a cluster model catalysts created by soft-landing palladium clusters onto the cantilever surface.

Imaging modes of atomic force microscopy for application in molecular and cell biology.

Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

Label free analysis of transcription factors using microcantilever arrays.

We report the measurement of protein interaction with double-stranded DNA oligonucleotides using cantilever microarray technology. We investigated two different DNA-binding proteins, the transcription factors SP1 and NF-kappaB, using cantilever arrays as they allow label-free measurement of different biomolecular interactions in parallel. Double-stranded DNA oligonucleotides containing a specific binding site for a transcription factor were sensitized on gold-coated cantilevers. The binding of the transcription factor creates a surface stress, resulting in a bending of the cantilevers. Both transcription factors could be detected independently at concentrations of 80-100 nM. A concentration dependence of the bending signal was measured using concentrations from 100 to 400 nM of NF-kappaB. The experiments show that the recognition sequence of one transcription factor can serve as a reference for the other, highlighting the sequence specificity of transcription factor binding.