Scott R. Manalis

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.

Making it stick: convection, reaction and diffusion in surface-based biosensors

The past decade has seen researchers develop and apply novel technologies for biomolecular detection, at times approaching hard limits imposed by physics and chemistry. In nearly all sensors, the transport of target molecules to the sensor can play as critical a role as the chemical reaction itself in governing binding kinetics, and ultimately performance. Yet rarely does an analysis of the interplay between diffusion, convection and reaction motivate experimental design or interpretation. Here we develop a physically intuitive and practical understanding of analyte transport for researchers who develop and employ biosensors based on surface capture. We explore the qualitatively distinct behaviors that result, develop rules of thumb to quickly determine how a given system will behave, and derive order-of-magnitude estimates for fundamental quantities of interest, such as fluxes, collection rates and equilibration times. We pay particular attention to collection limits for micro- and nanoscale sensors, and highlight unexplained discrepancies between reported values and theoretical limits.

Electronic detection of DNA by its intrinsic molecular charge.

We report the selective and real-time detection of label-free DNA using an electronic readout. Microfabricated silicon field-effect sensors were used to directly monitor the increase in surface charge when DNA hybridizes on the sensor surface. The electrostatic immobilization of probe DNA on a positively charged poly-l-lysine layer allows hybridization at low ionic strength where field-effect sensing is most sensitive. Nanomolar DNA concentrations can be detected within minutes, and a single base mismatch within 12-mer oligonucleotides can be distinguished by using a differential detection technique with two sensors in parallel. The sensors were fabricated by standard silicon microtechnology and show promise for future electronic DNA arrays and rapid characterization of nucleic acid samples. This approach demonstrates the most direct and simple translation of genetic information to microelectronics.

Suspended microchannel resonators for biomolecular detection

We present a resonant mass sensor for specific biomolecular detection in a subnanoliter fluid volume. The sensing principle is based on measuring shifts in resonance frequency of a suspended microfluidic channel upon accumulation of molecules on the inside walls of the device. Confining the fluid to the inside of a hollow cantilever enables direct integration with conventional microfluidic systems, significantly increases sensitivity by eliminating high damping and viscous drag, and allows the resonator to be actuated by electrostatic forces. Fluid density measurements reveal a mass resolution of 10−17 g/μm2 in a 4 mHz–4 Hz bandwidth. To demonstrate biomolecular detection, we present real-time measurements of the specific binding between avidin and biotinylated bovine serum albumin. Based on these measurements, we expect that changes in surface mass loading on the order of 10−19 g/μm2 can be detected in an optimized system.

Terabit-per-square-inch data storage with the atomic force microscope

An areal density of 1.6 Tbits/in.2 has been achieved by anodically oxidizing titanium with the atomic force microscope (AFM). This density was made possible by (1) single-wall carbon nanotubes selectively grown on an AFM cantilever, (2) atomically flat titanium surfaces on α-Al2O3 (1012), and (3) atomic scale force and position control with the tapping-mode AFM. By combining these elements, 8 nm bits on 20 nm pitch are written at a rate of 5 kbit/s at room temperature in air.

Measurement of mass, density, and volume during the cell cycle of yeast

Cell growth comprises changes in both mass and volume—two processes that are distinct, yet coordinated through the cell cycle. Understanding this relationship requires a means for measuring each of the cell’s three basic physical parameters: mass, volume, and the ratio of the two, density. The suspended microchannel resonator weighs single cells with a precision in mass of 0.1% for yeast. Here we use the suspended microchannel resonator with a Coulter counter to measure the mass, volume, and density of budding yeast cells through the cell cycle. We observe that cell density increases prior to bud formation at the G1/S transition, which is consistent with previous measurements using density gradient centrifugation. To investigate the origin of this density increase, we monitor relative density changes of growing yeast cells. We find that the density increase requires energy, function of the protein synthesis regulator target of rapamycin, passage through START (commitment to cell division), and an intact actin cytoskeleton. Although we focus on basic cell cycle questions in yeast, our techniques are suitable for most nonadherent cells and subcellular particles to characterize cell growth in a variety of applications.

Measuring single-cell density

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.

Characterizing deformability and surface friction of cancer cells

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 cells 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 constrictions 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.

Measuring the mass, density, and size of particles and cells using a suspended microchannel resonator

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 10−4g∕cm3.

Vacuum-Packaged Suspended Microchannel Resonant Mass Sensor for Biomolecular Detection

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/(ngmiddotcm2) 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