Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer.

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.

Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores.

UNLABELLED: 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. IMPORTANCE: Spores of Bacillus species cause food spoilage and disease and are extremely resistant to standard decontamination methods. This hardiness is partly due to spores' extremely low permeability to chemicals, including water. We present a method to directly monitor the uptake of molecules into B. subtilis spores by weighing spores in fluid. The results demonstrate the exchange of core water with subsecond resolution and show a correlation between water permeability and the rate at which small molecules can initiate or inhibit germination in coat-damaged spores. The ability to directly measure the uptake of molecules in the context of spores with known structural or genetic deficiencies is expected to provide insight into the determinants of spores' extreme resistance.

Weighing of biomolecules, single cells and single nanoparticles in fluid.

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.

Quantifying particle coatings using high-precision mass measurements.

We present a general method to quantify coatings on microparticle surfaces based on the additional mass. Particle buoyant mass is determined in a solution with a density that is nearly equivalent to that of the core particle, reducing the magnitude and uncertainty of the measurement. Under these conditions, added material with a different density than that of the core is a larger fraction of the total buoyant mass of the coated particle. This method can resolve a buoyant mass difference between uncoated and coated particles of ~1 fg. For the protein layer on the 3 µm polystyrene spheres measured herein, this is equivalent to 1/10th of a full layer.

Inertial and viscous flywheel sensing of nanoparticles.

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.

Label-free biomarker sensing in undiluted serum with suspended microchannel resonators.

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 SiO(2) surfaces act as ultralow 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.

Determination of bacterial antibiotic resistance based on osmotic shock response.

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.

Rapid and high-precision sizing of single particles using parallel suspended microchannel resonator arrays and deconvolution.

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.

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.

High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays.

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.

Group I aptazymes as genetic regulatory switches.

BACKGROUND: Allosteric ribozymes (aptazymes) that have extraordinary activation parameters have been generated in vitro by design and selection. For example, hammerhead and ligase ribozymes that are activated by small organic effectors and protein effectors have been selected from random sequence pools appended to extant ribozymes. Many ribozymes, especially self-splicing introns, are known control gene regulation or viral replication in vivo. We attempted to generate Group I self-splicing introns that were activated by a small organic effector, theophylline, and to show that such Group I aptazymes could mediate theophylline-dependent splicing in vivo. RESULTS: By appending aptamers to the Group I self-splicing intron, we have generated a Group I aptazyme whose in vivo splicing is controlled by exogenously added small molecules. Substantial differences in gene regulation could be observed with compounds that differed by as little as a single methyl group. The effector-specificity of the Group I aptazyme could be rationally engineered for new effector molecules. CONCLUSION: Group I aptazymes may find applications as genetic regulatory switches for generating conditional knockouts at the level of mRNA or for developing economically viable gene therapies.

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 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.

Ribozyme-mediated signal augmentation on a mass-sensitive biosensor.

Mass-based detection methods such as the quartz crystal microbalance (QCM) offer an attractive option to label-based methods; however the sensitivity is generally lower by comparison. In particular, low-molecular-weight analytes can be difficult to detect based on mass addition alone. In this communication, we present the use of effector-dependent ribozymes (aptazymes) as reagents for augmenting small ligand detection on a mass-sensitive device. Two distinct aptazymes were chosen: an L1-ligase-based aptazyme (L1-Rev), which is activated by a small peptide (MW approximately 2.4 kDa) from the HIV-1 Rev protein, and a hammerhead cleavase-based aptazyme (HH-theo3) activated by theophylline (MW = 180 Da). Aptazyme activity was observed in real time, and low-molecular-weight analyte detection has been successfully demonstrated with both aptazymes.

Functional DNAzymes organized into two-dimensional arrays.

DNAzymes are catalytically active DNA molecules, which have previously been described in solution. Here, we organize these molecules into a series of two-dimensional (2D) arrays using a periodic arrangement of DNA structures based on the DNA double crossover motif. We demonstrate by means of atomic force microscopy that the DNAzymes are organized according to the design and that they retain their activity when attached in linear strings within the context of the 2D array.

Arginine-rich motifs present multiple interfaces for specific binding by RNA.

A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.

In vitro selection of ribozymes dependent on peptides for activity.

A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide- and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).

In vitro selection using modified or unnatural nucleotides.

The use of modified nucleotides in an RNA or DNA pool to be used for in vitro selection offers many potential advantages, such as the increased stability of the selected nucleic acid against nuclease degradation. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for preparing a modified RNA pool and verifying its suitability for in vitro selection; and protocols for selecting and amplifying a functionally enriched pool.

Simultaneous detection of diverse analytes with an aptazyme ligase array.

Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection.

Micromechanical detection of proteins using aptamer-based receptor molecules.

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.