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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]

Improved methods are needed for routine, inexpensive monitoring of biomarkers that could facilitate earlier detection and characterization of cancer. Suspended microchannel resonators (SMRs) are highly sensitive, batch-fabricated microcantilevers with embedded microchannels that can directly quantify adsorbed mass via changes in resonant frequency. As in other label-free detection methods, biomolecular measurements in complex media such as serum are challenging due to high background signals from nonspecific binding. In this report, we demonstrate that carboxybetaine-derived polymers developed to adsorb directly onto SMR Si[O.sub.2] surfaces act as uitralow fouling and functionalizable surface coatings. Coupled with a reference microcantilever, this approach enables detection of activated leukocyte cell adhesion molecule (ALCAM), a model cancer biomarker, in undiluted serum with a limit of detection of 10 ng/mL. 10.1021/ac9027356

We investigate the buoyant mass of bacterial cells in real time with the suspended microchannel resonator (SMR) as the population recovers from an osmotic shock. The density of the culture medium is chosen such that the bacteria initially have a positive buoyant mass which becomes negative as they recover from the hyperosmotic stress. This behavior can be used to differentiate between an antibiotic-resistant and an antibiotic-susceptible strain of the pathogenic bacteria Citrobacter rodentium, and we propose a general approach for exploiting the high precision of the SMR for rapid detection of antibiotic resistance.

Measurements of the mass and surface charge of microparticles are employed in the characterization of many types of colloidal dispersions. The suspended microchannel resonator (SMR) is capable of measuring individual particle masses with femtogram resolution. Here, we employ the high sensitivity of the SMR resonance frequency to changes in particle position, relative to the cantilever tip, to determine the electrophoretic mobility of discrete particles in an applied electric field. When a sinusoidal electric field is applied to the suspended microchannel, the transient resonance frequency shift corresponding to a particle transit can be analyzed by digital signal processing to extract both the buoyant mass and electrophoretic mobility of each particle. These parameters, together with the mean particle density, can be used to compute the size, absolute mass, and surface charge of discrete microspheres, leading to a true representation of the mean and polydispersity of these quantifies for a population. We have applied this technique to an aqueous suspension of two types of polystyrene microspheres, to differentiate them based on their absolute mass and their surface charge. The integrated measurement of electrophoretic mobility using the SMR is determined to be quantitative, based on comparison with commercial instruments, and exhibits favorable scaling properties that will ultimately enable measurements from mammalian cells.

Universal detectors that maintain high sensitivity as the detection volume is reduced to the subnanoliter scale can enhance the utility of miniaturized total analysis systems ([mu]-TAS). Here the unique scaling properties of the suspended microchannel resonator (SMR) are exploited to show universal detection in a 10 pL analysis volume with a density detection limit of ~1 [micro]g/[cm.sup.3] (10 Hz bandwidth) and a dynamic range of 6 decades. Analytes with low IN extinction coefficients such as polyethylene glycol (PEG) 8 kDa, glucose, and glycine are measured with molar detection limits of 0.66, 13.5, and 31.6 [micro]M, respectively. To demonstrate the potential for real-time monitoring, gel titration chromatography was used to separate different molecular weights of PEG as the SMR acquired a chromatogram by measuring the eluate density. This work suggests that the SMR could offer a simple and sensitive universal detector for various separation systems from liquid chromatography to capillary electrophoresis. Moreover, since the SMR is itself a microfluidic channel, it can be directly integrated into [mu]-TAS without compromising overall performance.

Author(s): Thomas P. Burg [1, 5]; Michel Godin [1, 5]; Scott M. Knudsen [1]; Wenjiang Shen [3]; Greg Carlson [3]; John S. Foster [3]; Ken Babcock [3, 4]; Scott R. [...]

There is a great need in experimental biology for tools to study interactions between biological molecules and to profile expressure 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 * [cm.sup.2]) 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. Index Terms--Biomedical transducers, chemical transducers, density measurement, Q factor, mass sensor, microbalance, vacuum packaging.

We present a robust and simple method for direct, label-free PCR product quantification using an integrated microelectronic sensor. The field-effect sensor can sequentially detect the intrinsic charge of multiple unprocessed PCR products and does not require sample processing or additional reagents in the PCR mixture. The sensor measures nucleic acid concentration in the PCR relevant range and specifically detects the PCR products over reagents such as Taq polymerase and nucleotide monomers. The sensor can monitor the product concentration at various stages of PCR and can generate a readout that resembles that of a real-time fluorescent measurement using an intercalating dye but without its potential inhibition artifacts. The device is mass-produced using standard semiconductor processes, can be reused for months, and integrates all sensing components directly on-chip. As such, our approach establishes a foundation for the direct integration of PCR-based in vitro biotechnologies with microelectronics.

We report label-free protein detection using a microfabricated cantilever-based sensor that is functionalized with DNA aptamers to act as receptor molecules. The sensor utilizes two adjacent cantilevers that constitute a sensor/ reference pair and allows direct detection of the differential bending between the two cantilevers. One cantilever is functionalized with aptamers selected for Taq DNA polymerase while the other is blocked with single-stranded DNA. We have found that the polymerase-aptamer binding induces a change in surface stress, which causes a differential cantilever bending that ranges from 3 to 32 nm depending on the ligand concentration. Protein recognition on the sensor surface is specific and has a concentration dependence that is similar to that in solution.

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 thermomechanical noise level of 0.008 [Angstrom]/[Hz.sup.1/2] over the frequency range of 40-1000 Hz. [781] Index Terms--AFM technology, BioMEMS, biosensor, chemical-biological functionalization, diffraction, interdigitated, silicon nitride.

High-resolution atomic force microscopy (AFM) of soft or fragile samples requires a cantilever with a low spring constant and a sharp tip. We have developed a novel process for making such cantilevers from silicon nitride with oxidation-sharpened silicon tips. First, we made and sharpened silicon tips on a silicon wafer. Next, we deposited a thin film of silicon nitride over the tips and etched it to define nitride cantilevers and to remove it from the tips so that they protruded through the cantilevers. Finally, we etched from the back side to release the cantilevers by removing the silicon substrate. We characterized the resulting cantilevers by imaging them with a scanning electron microscope, by measuring their thermal noise spectra, and by using them to image a test sample in contact mode. A representative cantilever had a spring constant of ~0.06 N/m, and the tip had a radius of 9.2 nm and a cone angle of 36[degrees] over 3 [micro]m of tip length. These cantilevers are capable of higher resolution imaging than commercially available nitride cantilevers with oxidation-sharpened nitride tips, and they are especially useful for imaging large vertical features. [735] Index Terms--Atomic force microscopy (AFM), microfabricated cantilevers, microfabricated tips, micromachining, oxidation sharpening.

A high-resolution accelerometer with a bulk-micromachined silicon proof mass and an interferometric position sensor was developed for measuring vibratory accelerations. The interferometer consists of interdigitated fingers that are alternately attached to the proof mass and support substrate. Illuminating the fingers with coherent light generates a series of diffracted beams. The intensity of a given beam depends on the out-of-plane separation between the proof mass fingers and support fingers. Proof masses with mechanical resonances ranging from 80 Hz to 1 kHz were fabricated with a two mask process involving two deep reactive ion etches, an oxide etch stop, and a polyimide protective layer. The structures were packaged with a laser diode and photodiode into 8.6-[cm.sup.3] acrylic housings. The 80-Hz resonant proof mass has a noise equivalent acceleration of 40 ng/rt Hz and a dynamic range of 85 dB at 40 Hz. [730]

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]

We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function—including Granzyme B—are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology.

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]

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]