Search

Egg (oocyte) vitrification is the dominant method for preserving fertility for women of reproductive age. However, the method is typically performed by hand, requiring precise (∼0.1 to 10 μL) and time-sensitive (∼1 s) liquid exchange of cryoprotectants (CPA) around eggs as well as fine handling of eggs (∼100 μm) for immersion into liquid nitrogen (LN2). Here, we developed a microfluidic platform for programmable vitrification. Our platform is based on a millimeter-sized hanging droplet inside which a given egg is suspended and subjected to liquid exchanges within seconds. After programmable exposures to CPA, the egg is extracted from the liquid–air interface of the droplet using a motorized fine-tip instrument and immersed into LN2 for vitrification. To benchmark our platform with the manual method, we vitrified over a hundred mouse eggs and found comparable percentages (∼95%) for post-vitrification survivability. In addition, our platform performs real-time microscopy of the egg thereby enabling future studies where its morphology may be linked to functional outcomes. Our study contributes to the ongoing efforts to enhance the automation of embryology techniques towards broader applications in reproductive medicine both for clinical and research purposes. [ABSTRACT FROM AUTHOR]

Leukemias and their bone marrow microenvironments undergo dynamic changes over the course of disease. However, little is known about the circulation kinetics of leukemia cells, nor the impact of specific factors on the clearance of circulating leukemia cells (CLCs) from the blood. To gain a basic understanding of CLC dynamics over the course of disease progression and therapeutic response, we apply a blood exchange method to mouse models of acute leukemia. We find that CLCs circulate in the blood for 1–2 orders of magnitude longer than solid tumor circulating tumor cells. We further observe that: (i) leukemia presence in the marrow can limit the clearance of CLCs in a model of acute lymphocytic leukemia (ALL), and (ii) CLCs in a model of relapsed acute myeloid leukemia (AML) can clear faster than their untreated counterparts. Our approach can also directly quantify the impact of microenvironmental factors on CLC clearance properties. For example, data from two leukemia models suggest that E-selectin, a vascular adhesion molecule, alters CLC clearance. Our research highlights that clearance rates of CLCs can vary in response to tumor and treatment status and provides a strategy for identifying basic processes and factors that govern the kinetics of circulating cells. A blood exchange system reveals that leukemia cells clear faster in the blood than circulating tumor cells from solid tumors and that clearance rates can vary in response to tumor and treatment status. [ABSTRACT FROM AUTHOR]

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1–3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.An in vitro system that recapitulates temporal characteristics of embryonic development demonstrates that the different rates of mouse and human embryonic development stem from differences in metabolic rates and—further downstream—the global rate of protein synthesis. [ABSTRACT FROM AUTHOR]

Rotational dynamics often challenge physical intuition while enabling unique realizations, from the rotor of a gyroscope that maintains its orientation regardless of the outer gimbals, to a tennis racket that rotates around its handle when tossed face-up in the air. In the context of inertial sensing, which can measure mass with atomic precision, rotational dynamics are normally considered a complication hindering measurement interpretation. Here, we exploit the rotational dynamics of a microfluidic device to develop a modality in inertial sensing. Combining theory with experiments, we show that this modality measures the volume of a rigid particle while normally being insensitive to its density. Paradoxically, particle density only emerges when fluid viscosity becomes dominant over inertia. We explain this paradox via a viscosity-driven, hydrodynamic coupling between the fluid and the particle that activates the rotational inertia of the particle, converting it into a 'viscous flywheel'. This modality now enables the simultaneous measurement of particle volume and mass in fluid, using a single, high-throughput measurement. Balances for nanoparticles such as resonating fluid-filled cantilevers usually probe only mass through changes in oscillation frequency. Katsikis and Collis et al. tap information from previously ignored rotational motion to simultaneously measure particle mass and volume. [ABSTRACT FROM AUTHOR]

Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Sizedependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2. Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency. [ABSTRACT FROM AUTHOR]

Energy dissipation experienced by vibrating microcantilever beams immersed in fluid is strongly dependent on the mode of vibration, with quality factors typically increasing with mode number. Recently, we examined energy dissipation in a new class of cantilever device that embeds a microfluidic channel in its interior-the fundamental mode of vibration only was considered. Due to its importance in practice, we examine the effect of mode number on energy dissipation in these microfluidic beam resonators. Interestingly, and in contrast to other cantilever devices, we find that the quality factor typically decreases with increasing mode number. We explore the underlying physical mechanisms leading to this counterintuitive behavior, and provide a detailed comparison to experimental measurements for which good agreement is found. [ABSTRACT FROM AUTHOR]

Measuring the size of micron-scale particles plays a central role in the biological sciences and in a wide range of industrial processes. A variety of size parameters, such as particle diameter, volume, and mass, can be measured using electrical and optical techniques. Suspended microchannel resonators (SMRs) are microfluidic devices that directly measure particle mass by detecting a shift in resonance frequency as particles flow through a resonating microcantilever beam. While these devices offer high precision for sizing particles by mass, throughput is fundamentally limited by the small dimensions of the resonator and the limited bandwidth with which changes in resonance frequency can be tracked. Here, we introduce two complementary technical advancements that vastly increase the throughput of SMRs. First, we describe a deconvolution-based approach for extracting mass measurements from resonance frequency data, which allows an SMR to accurately measure a particle's mass approximately 16-fold faster than previously possible, increasing throughput from 120 particles/min to 2000 particles/min for our devices. Second, we describe the design and operation of new devices containing up to 16 SMRs connected fluidically in parallel and operated simultaneously on the same chip, increasing throughput to approximately 6800 particles/min without significantly degrading precision. Finally, we estimate that future systems designed to combine both of these techniques could increase throughput by nearly 200-fold compared to previously described SMR devices, with throughput potentially as high as 24 000 particles/min. We envision that increasing the throughput of SMRs will broaden the range of applications for which mass-based particle sizing can be employed. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) has benefited from significant advancements in treatment that have improved outcomes and reduced morbidity. However, the disease remains incurable and is characterized by high rates of drug resistance and relapse. Consequently, methods to select the most efficacious therapy are of great interest. Here we utilize a functional assay to assess the ex vivo drug sensitivity of single multiple myeloma cells based on measuring their mass accumulation rate (MAR). We show that MAR accurately and rapidly defines therapeutic susceptibility across human multiple myeloma cell lines to a gamut of standard-of-care therapies. Finally, we demonstrate that our MAR assay, without the need for extended culture ex vivo, correctly defines the response of nine patients to standard-of-care drugs according to their clinical diagnoses. This data highlights the MAR assay in both research and clinical applications as a promising tool for predicting therapeutic response using clinical samples. [ABSTRACT FROM AUTHOR]

The physical characteristics of the T cell receptor (TCR)-peptide-major histocompatibility complex (pMHC) interaction are known to play a central role in determining T cell function in the initial stages of the adaptive immune response. State-of-the-art assays can probe the kinetics of this interaction with single-molecularbond resolution, but this precision typically comes at the cost of low throughput, since the complexity of these measurements largely precludes "scaling up."Here, we explore the feasibility of detecting specific TCR-pMHC interactions by flowing T cells past immobilized pMHC and measuring the reduction in cell speed due to the mechanical force of the receptor-ligand interaction. To test this new fluidic measurement modality, we fabricated a microfluidic device in which pMHC-coated beads are immobilized in hydrodynamic traps along the length of a serpentine channel. As T cells flow past the immobilized beads, their change in speed is tracked via microscopy. We validated this approach using two model systems: primary CD8+ T cells from an OT-1 TCR transgenic mouse with beads conjugated with H-2Kb:SIINFEKL, and Jurkat T cells with beads conjugated with anti-CD3 and anti-CD28 antibodies. [ABSTRACT FROM AUTHOR]

Assays that can determine the response of tumor cells to cancer therapeutics could greatly aid the selection of drug regimens for individual patients. However, the utility of current functional assays is limited, and predictive genetic biomarkers are available for only a small fraction of cancer therapies. We found that the single-cell mass accumulation rate (MAR), profiled over many hours with a suspended microchannel resonator, accurately defined the drug sensitivity or resistance of glioblastoma and B-cell acute lymphocytic leukemia cells. MAR revealed heterogeneity in drug sensitivity not only between different tumors, but also within individual tumors and tumor-derived cell lines. MAR measurement predicted drug response using samples as small as 25 μl of peripheral blood while maintaining cell viability and compatibility with downstream characterization. MAR measurement is a promising approach for directly assaying single-cell therapeutic responses and for identifying cellular subpopulations with phenotypic resistance in heterogeneous tumors. [ABSTRACT FROM AUTHOR]

Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10-12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4-20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes. [ABSTRACT FROM AUTHOR]

Human red blood cells (RBCs) lose ∼30% of their volume and ∼20% of their hemoglobin (Hb) content during their ∼100-day lifespan in the bloodstream. These observations are well-documented, but the mechanisms for these volume and hemoglobin loss events are not clear. RBCs shed hemoglobin-containing vesicles during their life in the circulation, and this process is thought to dominate the changes in the RBC physical characteristics occurring during maturation. We combine theory with single-cell measurements to investigate the impact of vesiculation on the reduction in volume, Hb mass, and membrane. We show that vesicle shedding alone is sufficient to explain membrane losses but not volume or Hb losses. We use dry mass measurements of human RBCs to validate the models and to propose that additional unknown mechanisms control volume and Hb reduction and are responsible for ∼90% of the observed reduction. RBC population characteristics are used in the clinic to monitor and diagnose a wide range of conditions including malnutrition, inflammation, and cancer. Quantitative characterization of cellular maturation processes may help in the early detection of clinical conditions where maturation patterns are altered. [ABSTRACT FROM AUTHOR]

Cell size, measured as either volume or mass, is a fundamental indicator of cell state. Far more tightly regulated than size is density, the ratio between mass and volume, which can be used to distinguish between cell populations even when volume and mass appear to remain constant. Here we expand upon a previous method for measuring cell density involving a suspended microchannel resonator (SMR). We introduce a new device, the dual SMR, as a high-precision instrument for measuring single-cell mass, volume, and density using two resonators connected by a serpentine fluidic channel. The dual SMR designs considered herein demonstrate the critical role of channel geometry in ensuring proper mixing and damping of pressure fluctuations in microfluidic systems designed for precision measurement. We use the dual SMR to compare the physical properties of two well-known cancer cell lines: human lung cancer cell H1650 and mouse lymphoblastic leukemia cell line L1210. [ABSTRACT FROM AUTHOR]

Physical characterization of nanoparticles is required for a wide range of applications. Nanomechanical resonators can quantify the mass of individual particles with detection limits down to a single atom in vacuum. However, applications are limited because performance is severely degraded in solution. Suspended micro- and nanochannel resonators have opened up the possibility of achieving vacuum-level precision for samples in the aqueous environment and a noise equivalent mass resolution of 27 attograms in 1-kHz bandwidth was previously achieved by Lee et al. [(2010) Nano Lett 10(7):2537-2542]. Here, we report on a series of advancements that have improved the resolution by more than 30-fold, to 0.85 attograms in the same bandwidth, approaching the thermomechanical noise limit and enabling precise quantification of particles down to 10 nm with a throughput of more than 18,000 particles per hour. We demonstrate the potential of this capability by comparing the mass distributions of exosomes produced by different cell types and by characterizing the yield of self-assembled DNA nanoparticle structures. [ABSTRACT FROM AUTHOR]

We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell’s buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell’s water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell’s dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density – the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein), we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells, changes in growth conditions cause substantial shifts in dry density, suggesting concurrent changes in the protein, nucleic acid and lipid content of the cell. [ABSTRACT FROM AUTHOR]

Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell's passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces. [ABSTRACT FROM AUTHOR]

We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stability over several hours. We also wrote custom software, detailed here, to analyze instrument data files collected by either this continuous method or standard, periodic sampling. We use the continuous method to measure the growth rate of yeast in G1 during a prolonged arrest and, in different samples, the dependency of growth rate on cell size and cell cycle position in arrested and proliferating cells. We also quantify with high time resolution the response of mouse lymphoblast cell culture to drug treatment. This method provides a technique for continuous measurement of cell size that is applicable to a large variety of cell types and greatly expands the set of analysis tools available for the Coulter counter. [ABSTRACT FROM AUTHOR]

We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL-1. We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes. [ABSTRACT FROM AUTHOR]

An intrinsic uncertainty in particle mass sensing with the suspended microchannel resonator results from variation in a particle's position near the free end of the resonator. To circumvent this error we employ the second flexural bending mode. This mode exhibits additional frequency peaks while particles pass over the antinode, a point where the frequency shift is insensitive to the lateral position of the particle. We measure polystyrene beads with the first and second modes and confirm that the second mode sensing provides a narrower mass histogram. For 3 μm diameter beads, second mode sensing at the antinode improves the coefficient of variation in buoyant mass from 1.76% to 1.05% for population measurements and from 1.40% to 0.53% for a single trapped particle. [ABSTRACT FROM AUTHOR]

The fluid-structure interaction of resonating microcantilevers immersed in fluid has been widely studied and is a cornerstone in nanomechanical sensor development. In many applications, fluid damping imposes severe limitations by strongly degrading the signal-to-noise ratio of measurements. Recently, Burg et al. (Nature, vol. 446, 2007, pp. 1066-1069) proposed an alternative type of microcantilever device whereby a microfluidic channel was embedded inside the cantilever with vacuum outside. Remarkably, it was observed that energy dissipation in these systems was almost identical when air or liquid was passed through the channel and was 4 orders of magnitude lower than that in conventional microcantilever systems. Here, we study the fluid dynamics of these devices and present a rigorous theoretical model corroborated by experimental measurements to explain these observations. In so doing, we elucidate the dominant physical mechanisms giving rise to the unique features of these devices. Significantly, it is found that energy dissipation is not a monotonic function of fluid viscosity, but exhibits oscillatory behaviour, as fluid viscosity is increased/decreased. In the regime of low viscosity, inertia dominates the fluid motion inside the cantilever, resulting in thin viscous boundary layers - this leads to an increase in energy dissipation with increasing viscosity. In the highviscosity regime, the boundary layers on all surfaces merge, leading to a decrease in dissipation with increasing viscosity. Effects of fluid compressibility also become significant in this latter regime and lead to rich flow behaviour. A direct consequence of these findings is that miniaturization does not necessarily result in degradation in the quality factor, which may indeed be enhanced. This highly desirable feature is unprecedented in current nanomechanical devices and permits direct miniaturization to enhance sensitivity to environmental changes, such as mass variations, in liquid. [ABSTRACT FROM AUTHOR]

We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the 'instantaneous' growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells. [ABSTRACT FROM AUTHOR]

Nanomechanical resonators enable the measurement of mass with extraordinary sensitivity. Previously, samples as light as 7 zeptograms (1 zg = 10-21 g) have been weighed in vacuum, and proton-level resolution seems to be within reach. Resolving small mass changes requires the resonator to be light and to ring at a very pure tone—that is, with a high quality factor. In solution, viscosity severely degrades both of these characteristics, thus preventing many applications in nanotechnology and the life sciences where fluid is required. Although the resonant structure can be designed to minimize viscous loss, resolution is still substantially degraded when compared to measurements made in air or vacuum. An entirely different approach eliminates viscous damping by placing the solution inside a hollow resonator that is surrounded by vacuum. Here we demonstrate that suspended microchannel resonators can weigh single nanoparticles, single bacterial cells and sub-monolayers of adsorbed proteins in water with sub-femtogram resolution (1 Hz bandwidth). Central to these results is our observation that viscous loss due to the fluid is negligible compared to the intrinsic damping of our silicon crystal resonator. The combination of the low resonator mass (100 ng) and high quality factor (15,000) enables an improvement in mass resolution of six orders of magnitude over a high-end commercial quartz crystal microbalance. This gives access to intriguing applications, such as mass-based flow cytometry, the direct detection of pathogens, or the non-optical sizing and mass density measurement of colloidal particles. [ABSTRACT FROM AUTHOR]

There is a great need in experimental biology for tools to study interactions between biological molecules and to profile expression levels of large numbers of proteins. This paper describes the fabrication, packaging and testing of a resonant mass sensor for the detection of biomolecules in a microfluidic format. The transducer employs a suspended microchannel as the resonating element, thereby avoiding the problems of damping and viscous drag that normally degrade the sensitivity of resonant sensors in liquid. Our device differs from a vibrating tube densitometer in that the channel is very thin, which enables the detection of molecules that bind to the channel walls; this provides a path to specificity via molecular recognition by immobilized receptors. The fabrication is based on a sacrificial polysilicon process with low-stress low-pressure chemical-vapor deposited (LPCVD) silicon nitride as the structural material, and the resonator is vacuum packaged on the wafer scale using glass frit bonding. Packaged resonators exhibit a sensitivity of 0.8 ppm/(ng·cm²) and a mechanical quality factor of up to 700. To the best of our knowledge, this quality factor is among the highest so far reported for resonant sensors with comparable surface mass sensitivity in liquid. [ABSTRACT FROM AUTHOR]

Heparin is a highly sulfated glycosaminoglycan that is used as an important clinical anticoagulant. Monitoring and control of the heparin level in a patient's blood during and after surgery is essential, but current clinical methods are limited to indirect and off-line assays. We have developed a silicon field-effect sensor for direct detection of heparin by its intrinsic negative charge. The sensor consists of a simple microfabricated electrolyte-insulator-silicon structure encapsulated within microfluidic channels. As heparin-specific surface probes the clinical heparin antagonist protamine or the physiological partner antithrombin III were used. The dose-response curves in 10% PBS revealed a detection limit of 0.001 units/ml, which is orders of magnitude lower than clinically relevant concentrations. We also detected heparin-based drugs such as the low-molecular-weight heparin enoxaparin (Lovenox) and the synthetic pentasaccharide heparin analog fondaparinux (Arixtra), which cannot be monitored by the existing near-patient clinical methods. We demonstrated the specificity of the antithrombin III functionalized sensor for the physiologically active pentasaccharide sequence. As a validation, we showed correlation of our measurements to those from a colorimetric assay for heparin-mediated anti-Xa activity. These results demonstrate that silicon field-effect sensors could be used in the clinic for routine monitoring and maintenance of therapeutic levels of heparin and heparin-based drugs and in the laboratory for quantitation of total amount and specific epitopes of heparin and other glycosaminoglycans. [ABSTRACT FROM AUTHOR]

We have micromachined a mechanical sensor that uses interferometry to detect the differential and absolute deflections of two adjacent cantilevers. The overall geometry of the device allows simple fluidic delivery to each cantilever to immobilize molecules for biological and chemical detection. We show that differential sensing is 50 times less affected by ambient temperature changes than the absolute, thus enabling a more reliable differentiation between specific cantilever bending and background effects. We describe the fabrication process and show results related to the dynamic characterization of the device as a differential sensor. The root-mean-squared (rms) sensor noise in water and air is ∼1 nm over the frequency range of 0.4-40 Hz. We also find that in air, the deflection resolution is limited only by the cantilever's thermome- chanical noise level of 0.008 Å/Hz[sup 1/2] over the frequency range of 40-1000 Hz. [ABSTRACT FROM AUTHOR]

Proposes a process for making silicon nitride cantilevers with oxidation-sharpened silicon tips for atomic force microscopy. Characterization of the cantilevers; Thermal noise spectra of the cantilevers; Tip quality.

Examines the development of a high-resolution accelerometer with a bulk-micromachined silicon proof mass and interferometric position sensor for measuring vibratory accelerations. Structure of the interferometer; Intensity of the beams; Fabrication of the proof masses with mechanical resonances.

Functional precision medicine aims to match individual cancer patients to optimal treatment through ex vivo drug susceptibility testing on patient-derived cells. However, few functional diagnostic assays have been validated against patient outcomes at scale because of limitations of such assays. Here, we describe a high-throughput assay that detects subtle changes in the mass of individual drug-treated cancer cells as a surrogate biomarker for patient treatment response. To validate this approach, we determined ex vivo response to temozolomide in a retrospective cohort of 69 glioblastoma patient-derived neurosphere models with matched patient survival and genomics. Temozolomide-induced changes in cell mass distributions predict patient overall survival similarly to O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and may aid in predictions in gliomas with mismatch-repair variants of unknown significance, where MGMT is not predictive. Our findings suggest cell mass is a promising functional biomarker for cancers and drugs that lack genomic biomarkers. [Display omitted] • In a retrospective study, ex vivo drug testing predicts GBM patient survival • Testing is based on detecting subtle changes in single-cell mass • Predictive power of functional testing is comparable to MGMT promoter methylation • Mass biomarker could be used in situations where genomic biomarkers are unavailable Stockslager et al. find in a retrospective study that functional drug susceptibility testing predicts the response of patients with glioblastoma to chemotherapy. By detecting subtle changes in tumor cell mass after ex vivo drug exposure, treatment response can be predicted with power comparable to the standard-of-care genomic biomarker. [ABSTRACT FROM AUTHOR]

We introduce a microfluidic system for simultaneously measuring single-cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 h of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1-S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis. [ABSTRACT FROM AUTHOR]

We present a mass-based readout for agglutination assays. The suspended microchannel resonator (SMR) is used to classify monomers and dimers that are formed during early stage aggregation, and to relate the total count to the analyte concentration. Using a model system of streptavidin functionalized microspheres and biotinylated antibody as the analyte, we obtain a dose-response curve over a concentration range of 0.63–630 nM and show that the results are comparable to what has been previously achieved by image analysis and conventional flow cytometry. [ABSTRACT FROM AUTHOR]

We demonstrate the measurement of mass, density, and size of cells and nanoparticles using suspended microchannel resonators. The masses of individual particles are quantified as transient frequency shifts, while the particles transit a microfluidic channel embedded in the resonating cantilever. Mass histograms resulting from these data reveal the distribution of a population of heterogeneously sized particles. Particle density is inferred from measurements made in different carrier fluids since the frequency shift for a particle is proportional to the mass difference relative to the displaced solution. We have characterized the density of polystyrene particles, Escherichia coli, and human red blood cells with a resolution down to 10-4 g/cm3. [ABSTRACT FROM AUTHOR]

The energetic demands of a cell are believed to increase during mitosis, but the rates of ATP synthesis and consumption during mitosis have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytic leukemia cells and demonstrate that mitochondria hyperpolarize from the G2/M transition until the metaphase-anaphase transition. This hyperpolarization was dependent on cyclin-dependent kinase 1 (CDK1) activity. By using an electrical circuit model of mitochondria, we quantify mitochondrial ATP synthesis rates in mitosis from the single-cell time-dynamics of mitochondrial membrane potential. We find that mitochondrial ATP synthesis decreases by approximately 50% during early mitosis and increases back to G2 levels during cytokinesis. Consistently, ATP levels and ATP synthesis are lower in mitosis than in G2 in synchronized cell populations. Overall, our results provide insights into mitotic bioenergetics and suggest that cell division is not a highly energy demanding process. ATP drives most cellular processes, although ATP production and consumption levels during mitosis remain unreported. Here, the authors combine metabolic measurements and modeling to quantify ATP levels and synthesis dynamics, revealing that ATP synthesis and consumption are lowered during mitosis. [ABSTRACT FROM AUTHOR]

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depolymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites. Cortical microtubules of Toxoplasma gondii are exceptionally stable, but it isn't known how they are anchored along membranes. Here, Harding et al. show that GAPM proteins localize to the inner membrane complex and are essential for maintaining the structural stability of parasites. [ABSTRACT FROM AUTHOR]

A fundamental trade-off between flow rate and measurement precision limits performance of many single-cell detection strategies, especially for applications that require biophysical measurements from living cells within complex and low-input samples. To address this, we introduce ‘active loading’, an automated, optically-triggered fluidic system that improves measurement throughput and robustness by controlling entry of individual cells into a measurement channel. We apply active loading to samples over a range of concentrations (1-1000 particles μL−1), demonstrate that measurement time can be decreased by up to 20-fold, and show theoretically that performance of some types of existing single-cell microfluidic devices can be improved by implementing active loading. Finally, we demonstrate how active loading improves clinical feasibility for acute, single-cell drug sensitivity measurements by deploying it to a preclinical setting where we assess patient samples from normal brain, primary and metastatic brain cancers containing a complex, difficult-to-measure mixture of confounding biological debris. Single-cell detection methods are limited by the trade-off between flow rate and measurement precision. Here the authors introduce active loading, an optically triggered microfluidic system to concentrate diluted cell samples, which reduces clogging and decreases processing time in single-cell assays. [ABSTRACT FROM AUTHOR]

Summary Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted. Graphical Abstract Highlights • Transcriptional amplification in trisomy 21 models is revealed by spike-in controls • Overexpression of HMGN1 (on 21q22) is necessary and sufficient for RNA effect • HMGN1 overexpression causes global H3K27 hyperacetylation, detected by ChIP-Rx • Transcriptome amplification enriches cell-type-specific programs of B cells/B-ALL How trisomy 21 contributes to Down syndrome phenotypes, including increased leukemia risk, is not well understood. Mowery et al. use per-cell normalization approaches to reveal global transcriptional amplification in Down syndrome models. HMGN1 overexpression is sufficient to induce these alterations and promotes lineage-associated transcriptional programs, signaling, and B cell progenitor phenotypes. [ABSTRACT FROM AUTHOR]