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As the use of nanoparticles is expanding in many industrial sectors, pharmaceuticals, cosmetics among others, flow-through characterization techniques are often required for in-line metrology. Among the parameters of interest, the concentration and mass of nanoparticles can be informative for yield, aggregates formation or even compliance with regulation. The Suspended Nanochannel Resonator (SNR) can offer mass resolution down to the attogram scale precision in a flow-through format. However, since the readout has been based on the optical lever, operating more than a single resonator at a time has been challenging. Here we present a new architecture of SNR devices with piezoresistive sensors that allows simultaneous readout from multiple resonators. To enable this architecture, we push the limits of nanofabrication to create implanted piezoresistors of nanoscale thickness (∼100 nm) and implement an algorithm for designing SNRs with dimensions optimized for maintaining attogram scale precision. Using 8-in. processing technology, we fabricate parallel array SNR devices which contain ten resonators. While maintaining a precision similar to that of the optical lever, we demonstrate a throughput of 40 000 particles per hour-an order of magnitude improvement over a single device with an analogous flow rate. Finally, we show the capability of the SNR array device for measuring polydisperse solutions of gold particles ranging from 20 to 80 nm in diameter. We envision that SNR array devices will open up new possibilities for nanoscale metrology by measuring not only synthetic but also biological nanoparticles such as exosomes and viruses.

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.

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.

Microbes are an essential component of marine food webs and biogeochemical cycles, and therefore precise estimates of their biomass are of significant value. Here, we measured single-cell biomass distributions of isolates from several numerically abundant marine bacterial groups, including Pelagibacter (SAR11), Prochlorococcus and Vibrio using a microfluidic mass sensor known as a suspended microchannel resonator (SMR). We show that the SMR can provide biomass (dry mass) measurements for cells spanning more than two orders of magnitude and that these estimates are consistent with other independent measures. We find that Pelagibacterales strain HTCC1062 has a median biomass of 11.9±0.7 fg per cell, which is five- to twelve-fold smaller than the median Prochlorococcus cell's biomass (depending upon strain) and nearly 100-fold lower than that of rapidly growing V. splendidus strain 13B01. Knowing the biomass contributions from various taxonomic groups will provide more precise estimates of total marine biomass, aiding models of nutrient flux in the ocean.

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.

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.

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.

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.

Common techniques used to synchronize eukaryotic cells in the cell cycle often impose metabolic stress on the cells or physically select for size rather than age. To address these deficiencies, a minimally perturbing method known as the "baby machine" was developed previously. In the technique, suspension cells are attached to a membrane, and as the cells divide, the newborn cells are eluted to produce a synchronous population of cells in the G1 phase of the cell cycle. However, the existing "baby machine" is only suitable for cells which can be chemically attached to a surface. Here, we present a microfluidic "baby machine" in which cells are held onto a surface by pressure differences rather than chemical attachment. As a result, our method can in principle be used to synchronize a variety of cell types, including cells which may have weak or unknown surface attachment chemistries. We validate our microfluidic "baby machine" by using it to produce a synchronous population of newborn L1210 mouse lymphocytic leukemia cells in G1 phase.

We present two methods by which single cells can be mechanically trapped and continuously monitored within the suspended microchannel resonator (SMR) mass sensor. Since the fluid surrounding the trapped cell can be quickly and completely replaced on demand, our methods are well suited for measuring changes in cell size and growth in response to drugs or other chemical stimuli. We validate our methods by measuring the density of single polystyrene beads and Saccharomyces cerevisiae yeast cells with a precision of approximately 10(-3) g cm(-3), and by monitoring the growth of single mouse lymphoblast cells before and after drug treatment.

Dissipation of mechanical energy underlies the sensitivity of many nanomechanical devices, with environmental effects often having a significant effect. One case of practical relevance is the interaction of elastic beam resonators with fluid, which is known to dramatically increase energy dissipation. Recently, we investigated energy dissipation in a different class of elastic beam resonator that embeds a microfluidic channel in its interior. In this paper, we examine the effect of the beam material Poisson ratio on these devices and discover that it can strongly affect energy dissipation--this is in direct contrast to conventional cantilever beams immersed in fluid. Increasing the Poisson ratio in these microfluidic devices is found to decrease energy dissipation, with the incompressible material limit providing minimum energy dissipation. Our paper establishes that, in this limit, placement of the fluid channel away from the beam neutral axis has negligible effect on energy dissipation in many cases of practical interest. The physical implications of these findings are discussed, and a detailed comparison with available experimental results is provided.

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.

Nanomechanical resonators enable a range of precision measurements in air or vacuum, but strong viscous damping makes applications in liquid challenging. Recent experiments have shown that fluid damping is greatly reduced in fluidic embedded-channel microcantilevers. Here we report the discovery of nonmonotonic energy dissipation due to the fluid in such devices, which leads to the intriguing prospect of enhancing the quality factor upon miniaturization. These observations elucidate the physical mechanisms of energy dissipation in embedded-channel resonators and thus provide the basis for numerous applications in nanoscience and biology.

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 quantities 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.