Precision mass measurements in solution reveal properties of single cells and bioparticles

Precise characterization of biological materials ranging from single cells (~1-20 microns) to extracellular vesicles (20-200 nm) is of fundamental interest because of their biological and translational value. Here we discuss the value of precision mass measurements in solution for informing various physical and biological parameters, such as mass accumulation rate, longitudinal cell growth or cell density. We introduce how the limits of the single-particle mass measurements can be pushed down to nano-scale dimensions enabling the resolution of extracellular vesicles and viruses in solution. We believe with future advancements on the precision and throughput of this approach, the capability of analyzing biologically relevant particles in solution will have broad biological and translational impact

High-speed multiple-mode mass-sensing resolves dynamic nanoscale mass distributions

Simultaneously measuring multiple eigenmode frequencies of nanomechanical resonators can determine the position and mass of surface-adsorbed proteins, and could ultimately reveal the mass tomography of nanoscale analytes. However, existing measurement techniques are slow (<1 Hz bandwidth), limiting throughput and preventing use with resonators generating fast transient signals. Here we develop a general platform for independently and simultaneously oscillating multiple modes of mechanical resonators, enabling frequency measurements that can precisely track fast transient signals within a user-defined bandwidth that exceeds 500 Hz. We use this enhanced bandwidth to resolve signals from multiple nanoparticles flowing simultaneously through a suspended nanochannel resonator and show that four resonant modes are sufficient for determining their individual position and mass with an accuracy near 150 nm and 40 attograms throughout their 150-ms transit. We envision that our method can be readily extended to other systems to increase bandwidth, number of modes, or number of resonators.United States. Army Research Office (Grant W911NF-09-0001)Center for Integration of Medicine and Innovative Technology (Contract 09-440)National Science Foundation (U.S.) (Grant 1129359

Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores

We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ∼200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl₃ to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability.United States. Army Research Office (W911F-09-1-0286)United States. Army Research Office (W911NF-09-0001

Direct single-cell biomass estimates for marine bacteria via Archimedes’ principle

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.National Science Foundation (U.S.) (OCE-1129359)Simons Foundation (337262)United States. Army Research Office (W911NF-09-D-0001

Suspended nanochannel resonators at attogram precision

Nanomechanical resonators can quantify individual particles down to a single atom; however the applications are limited due to their degraded performance in solution. Suspended micro- and nanochannel resonators can achieve vacuum level performances for samples in solution since the target analyte flows through an integrated channel within the resonator. Here we report on a new generation suspended nanochannel resonator (SNR) that operates at approximately 2 MHz with quality factors between 10,000-20,000. The SNR is measured to have a mass sensitivity of 8.2 mHz/attogram. With an optimized oscillator system, we show that the resonator can be oscillated with a mass equivalent frequency stability of 0.85 attogram (4 parts-perbillion) at 1 kHz bandwidth, which is 1.8 times the calculated stability imposed by the thermal noise. We demonstrate the use of this mass resolution by quantifying the mass and concentration of nanoparticles down to 10 nm in solution

Synchronization of Distant Optical Clocks at the Femtosecond Level

The use of optical clocks/oscillators in future ultra-precise navigation, gravitational sensing, coherent arrays, and relativity experiments will require time comparison and synchronization over terrestrial or satellite free-space links. Here we demonstrate full unambiguous synchronization of two optical timescales across a free-space link. The time deviation between synchronized timescales is below 1 fs over durations from 0.1 s to 6500 s, despite atmospheric turbulence and kilometer-scale path length variations. Over several days, the time wander is 40 fs peak-to-peak. Our approach relies on the two-way reciprocity of a single-spatial-mode optical link, valid to below 225 attoseconds across a turbulent 4-km path. This femtosecond level of time-frequency transfer should enable optical networks using state-of-the-art optical clocks/oscillators.Comment: 19 pages, 9 figure

Drug sensitivity of single cancer cells is predicted by changes in mass accumulation rate

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.United States. National Institutes of Health (R01 CA170592)United States. National Institutes of Health (R33 CA191143)National Cancer Institute (U.S.) (U54 CA143874)United States. National Institutes of Health (NIH/NIGMS T32 GM008334

Intracellular Water Exchange for Measuring the Dry Mass, Water Mass and Changes in Chemical Composition of Living Cells

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 H[subscript 2]O-based fluid and a D[subscript 2]O-based fluid. Rapid exchange of intracellular H[subscript 2]O for D[subscript 2]O 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.National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874)National Institutes of Health (U.S.) (Center for Cell Division Process Grant P50GM6876)National Institutes of Health (U.S.) (Contract R01CA170592)United States. Army Research Office (Institute for Collaborate Biotechnologies Contract W911NF-09-D-0001