Session Q41: Biofluids

Fool's Gold Footprinting: microfluidic probing of nucleic acids

We describe a microfluidic device containing a mineral matrix capable of rapidly generating hydroxyl radicals that enables high-resolution structural studies of nucleic acids. Hydroxyl radicals cleave the solvent accessible backbone of DNA and RNA; the cleavage products can be detected with as fine as single nucleotide resolution. Protection from hydroxyl radical cleavage (footprinting) can identify sites of protein binding or the presence of tertiary structure. Here we report preparation of micron sized particles of iron sulfide (pyrite) and fabrication of a microfluidic prototype that together generate enough hydroxyl radicals within 20 ms to cleave DNA sufficiently for a footprinting analysis to be conducted. This prototype enables the development of high-throughput and/or rapid reaction devices with which to probe nucleic acid folding dynamics and ligand binding.   

Dislocation dynamics and bacterial growth

Recent experiments have revealed remarkable phenomena in the growth mechanisms of rod-shaped bacteria: proteins associated with the cell wall growth move at constant velocity in circles oriented approximately along the cell circumference (Garner et al., Science 2011, Dom\'inguez-Escobar et al., Science 2011, Deng et al., PNAS 2011). We view these dislocations in the partially ordered peptidoglycan structure, and study theoretically the dynamics of these interacting dislocations on the surface of a cylinder. The physics of the nucleation of these dislocations and the resulting dynamics within the model show surprising effects arising from the cylindrical geometry, which are predicted to have important implications on the growth mechanism. We also discuss how long range elastic interactions affect the dynamics of the fraction of active dislocations in the environment.   

Signal Relay During Cell Migration

We developed a signal relay model to quantify the effect of intercellular communication in presence of an external signal, during the motion of groups of Dictyostelium discoideum cells. A key parameter is the ratio of amplitude of the cAMP (cyclic adenosine monophosphate) a signaling chemical secreted from individual cells versus the external cAMP field, which defines a time scale. Another time scale is set by the degradation rate of the cAMP. In our simulations, the competition between these two time scales results rich dynamics including uniform motion, as well as streaming and clustering instabilities. The simulations are compared to experiments for a wide range of different external signal strengths for both cells that secrete cAMP and a mutant which cannot relay cAMP. Under different strength of external linear cAMP gradient, the wild type cells form streams and exhibit clustering due to the intercellular signaling through individual cAMP secretion. In contrast, cells lacking signal relay move relatively straight. We find that the model captures both independent motion and the formation of aggregates when cells relay the signal.   

Intracellular Transport in Beta Cells - from Anti-Corellated to Active

The intracellular transport along micro-tubules is the main focus of this research. We study the transport of insulin granules inside Beta cells. By developing new technique for the analysis of single 2D trajectories we observe a transition in the transport behavior from anti-correlated to active as a function of time. We further use the observed effect in order to discriminate between possible scenarios of active transport through disordered media as models of efficient intracellular transport.   

Acoustic streaming in the cochlea under compressive bone conduction excitation

This work examines the acoustic streaming in the cochlea. A model will be developed to examine the steady flow over a flexible boundary that is induced by compressive excitation of the cochlear capsule. A stokeslet based analysis of oscillatory flows was used to model fluid motion. The influence of evanescent modes on the pressure field is considered as the limit of the aspect ratio epsilon approaches zero. We will show a uniformly valid solution in space.   

Subtle exchange model of flow depended on the blood cell shape to enhance the micro-circulation in capillary

In general the exchange of gases or other material in capillary system is conceptualized by the diffusion effect. But in this model, we investigate a micro-flow pattern by simulation and computation on a micro-exchange model in which the blood cell is a considered factor, especially on its shape. It shows that the cell benefits the circulation while it is moving in the capillary. In the study, the flow detail near the cell surface is mathematically analyzed, such that the Navier-Stokes equations are applied and the viscous factor is also briefly considered. For having a driven force to the motion of micro-circulation, a breathing mode is suggested to approximately compute on the flow rate in the blood capillary during the transfer of cell. The rate is also used to estimate the enhancement to the circulation in additional to the outcome of diffusion. Moreover in the research, the shape change of capillary wall under pressure influence is another element in the beginning calculation for the effect in the assistance to cell motion.   

Modeling fluid diffusion in cerebral white matter with random walks in complex environments

Recent studies with diffusion MRI have shown new aspects of geometric order in the brain, including complex path coherence within the cerebral cortex, and organization of cerebral white matter and connectivity across multiple scales. The main assumption of these studies is that water molecules diffuse along myelin~sheaths of neuron axons in the white matter and thus the anisotropy of their diffusion tensor observed by MRI can provide information about the direction of the axons connecting different parts of the brain. We model the diffusion of particles confined in the space of between the bundles of cylindrical obstacles representing fibrous structures of various orientations. We have investigated the directional properties of the diffusion, by studying the angular distribution of the end point of the random walks as a function of their length, to understand the scale over which the distribution randomizes.~We will show evidence of qualitative change in the behavior of the diffusion for different volume fractions of obstacles. Comparisons with three-dimensional MRI images will be illustrated.   

Adhesion of a Cylindrical Bacterium in the Presence of DLVO Potential

A single cigar shape bacterium attaches to a rigid substrate (e.g. sand surface). In the presence of electrostatic double layers and van der Waals attraction according to the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the bacterium glycoprotein shell deforms and may settle in either the primary or secondary energy minimum depending on whether it has sufficient energy to overcome the repulsive energy barrier. The adhesion-detachment mechanics is derived using a computational approach, and the followings are obtained: (i) relation between the applied load and contact area with the substrate, (ii) deformed profile at equilibrium, (iii) mechanical stress distribution in the shell, (iv) critical compressive load to force the shell going from secondary energy minimum to primary, and (v) ``pull-off'' forces to detach the shell from substrate. The model leads to better understanding of bacteria adhesion-aggregation-transportation, and has significant relevance to environmental and medical sciences.   

Hydrodynamic behavior of tumor cells in a confined model microvessel

An important step in cancer metastasis is the hydrodynamic transport of circulating tumor cells (CTCs) through microvasculature. In vivo imaging studies in mice models show episodes of confined motion and trapping of tumor cells at microvessel bifurcations, suggesting that hydrodynamic phenomena are important processes regulating CTC dissemination. Our goal is to use microfluidics to understand the interplay between tumor cell rheology, confinement and fluid forces that may help to identify physical factors determining CTC transport. We use leukemia cells as model CTCs and mimic the in vivo setting by investigating their motion in a confined microchannel with an integrated microfluidic manometer to measure time variations in the excess pressure drop during cell motion. Using image analysis, variations in excess pressure drop, cell shape and cell velocity are simultaneously quantified. We find that the throughput of the technique is high enough (~ 100 cells/min) to assess tumor cell heterogeneity. Therefore, in addition to measuring the hydrodynamic response of tumor cells in confined channels, our results indicate that the microfluidic manometer device could be used for rapid mechanical phenotyping of tumor cells.   

Flow and rupture of vesicles in narrow channels

Small lipid bilayer vesicles, also known as liposomes, are used for drug delivery systems in vasculature. Consequently how they deform and when they become unstable and rupture (lose their inner contents) under capillary flow is of great interest. In addition vesicles with a filling fraction of 0.6 can be considered as a simple mechanical model of red blood cells. We use coarse-grained molecular dynamics (CGMD) simulations with explicit solvent to study lipid bilayer vesicles in 3D capillary flow with filling fractions of 1.0 and 0.6. The shapes of the vesicles obtained in these simulations compare well to other experimental and theoretical studies. Using CGMD allows the study of rupture. This is in contrast to the majority of other approaches which model the bilayer as a purely elastic surface and only allow the investigation of deformation. We look at the stress profiles of these vesicles as measured by the area expansion per lipid along the membrane, and determine the location and pressure of rupture for a given confinement ratio (diameter of the vesicle divided by diameter of the channel). We also discuss the subsequent loss of inner fluid content.   

Simulations of cardiovascular blood flow accounting for time dependent deformational forces

Cardiovascular disease is currently the leading cause of death in the United States, and early detection is critical. Despite advances in imaging technology, 50\% of these deaths occur suddenly and with no prior symptoms. The development and progression of coronary diseases such as atherosclerosis has been linked to prolonged areas of low endothelial shear stress (ESS); however, there is currently no way to measure ESS in vivo. We will present a patient specific fluid simulation that applies the Lattice Boltzmann equation to model the blood flow in the coronary arteries whose geometries are derived from computed tomography angiography data. Using large-scale supercomputers up to 294,912 processors, we can model a full heartbeat at the resolution of the red blood cells. We are investigating the time dependent deformational forces exerted on the arterial flows from the movement of the heart. The change in arterial curvature that occurs over a heartbeat has been shown to have significant impact on flow velocity and macroscopic quantities like shear stress. We will discuss a method for accounting for these resulting forces by casting them into a kinetic formalism via a Gauss-Hermite projection and their impact on ESS while maintaining the static geomtry obtained from CTA data.   

Using vortex corelines to analyze the hemodynamics of patient specific cerebral aneurysm models

We construct one-dimensional sets known as vortex corelines for computational fluid dynamic (CFD) simulations of blood flow in patient specific cerebral aneurysm models. These sets identify centers of swirling blood flow that may play an important role in the biological mechanisms causing aneurysm growth, rupture, and thrombosis. We highlight three specific applications in which vortex corelines are used to assess flow complexity and stability in cerebral aneurysms, validate numerical models against PIV-based experimental data, and analyze the effects of flow diverting devices used to treat intracranial aneurysms.   

Picoliter droplet-based digital peptide nucleic acid clamp PCR and dielectric sorting for low abundant K-ras mutations

Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality in the US, and the 5-year survival of metastatic CRC (mCRC) is less than 10{\%}. Although monoclonal antibodies against epidermal growth factor receptor (EGFR) provide incremental improvements in survival, approximately 40{\%} of mCRC patients with activating KRAS mutations won't benefit from this therapy. Peptide nucleic acid (PNA), a synthetic non-extendable oligonucleotides, can bind strongly to completely complementary wild-type KRAS by Watson-Crick base pairing and suppress its amplification during PCR, while any mutant allele will show unhindered amplification. The method is particularly suitable for the simultaneously detection of several adjoining mutant sites, just as mutations of codons 12 and 13 of KRAS gene where there are totally 12 possible mutation types. In this work, we describe the development and validation of this method, based on the droplet-based digital PCR. Using a microfluidic system, single target DNA molecule is compartmentalized in microdroplets together with PNA specific for wild-type KRAS, thermocycled and the fluorescence of each droplet was detected, followed by sorting and sequencing. It enables the precise determination of all possible mutant KRAS simultaneously, and the precise quantification of a single mutated KRAS in excess background unmutated KRAS.   

Liquid solution delivery through the pulled nanopipette combined with QTF-AFM system

Nanopipette is a versatile fluidic tool for biochemical analysis, controlled liquid delivery in bio-nanotechnology. However, most of the researches have been performed in solution based system, thus it is challenge to study nanofluidic properties of the liquid solution delivery through the nanopipette in ambient conditions. In this work, we demonstrated the liquid ejection, dispersion, and subsequent deposition of the nanoparticles via a 30 nm aperture pipette based on the quartz tuning fork -- atomic force microscope (QTF-AFM) combined nanopipette system.