DNA translocation through short nanofluidic channels under asymmetric pulsed electric field

Investigation of single molecule DNA dynamics in confined environments has led to important applications in DNA analysis, separation, and sequencing. Here, we studied the electrophoretic transport of DNA molecules through nanochannels shorter than the DNA contour length and calculated the associated translocation time curves. We found that the longer T4 DNA molecules required a longer time to traverse a fixed length nanochannel than shorter λ DNA molecules and that the translocation time decreased with increasing electric field which agreed with theoretical predictions. We applied this knowledge to design an asymmetric electric pulse and demonstrate the different responses of λ and T4 DNA to the pulses. We used Brownian dynamics simulations to corroborate our experimental results on DNA translocation behaviour. This work contributes to the fundamental understanding of polymer transport through nanochannels and may help in designing better separation techniques in the future.     

Stretching DNA by electric field and flow field in microfluidic devices: An experimental validation to the devices designed with computer simulations

We examined the performance of three microfluidic devices for stretching DNA. The first device is a microchannel with a contraction, and the remaining two are the modifications to the first. The modified designs were made with the help of computer simulations [C. C. Hsieh and T. H. Lin, Biomicrofluidics 5(4), 044106 (2011) and C. C. Hsieh, T. H. Lin, and C. D. Huang, Biomicrofluidics 6, 044105 (2012)] and they were optimized for operating with electric field. In our experiments, we first used DC electric field to stretch DNA. However, the experimental results were not even in qualitative agreement with our simulations. More detailed investigation revealed that DNA molecules adopt a globular conformation in high DC field and therefore become more difficult to stretch. Owing to the similarity between flow field and electric field, we turned to use flow field to stretch DNA with the same devices. The evolution patterns of DNA conformation in flow field were found qualitatively the same as our prediction based on electric field. We analyzed the maximum values, the evolution and the distributions of DNA extension at different Deborah number in each device. We found that the shear and the hydrodynamic interaction have significant influence on the performance of the devices.     

Simulation of single DNA molecule stretching and immobilization in a de-wetting two-phase flow over micropillar-patterned surface

We investigate single DNA stretching dynamics in a de-wetting flow over micropillars using Brownian dynamics simulation. The Brownian dynamics simulation is coupled with transient flow field computation through a numerical particle tracking algorithm. The droplet formation on the top of the micropillar during the de-wetting process creates a flow pattern that allows DNA to stretch across the micropillars. It is found that DNA nanowire forms if DNA molecules could extend across the stagnation point inside the connecting water filament before its breakup. It also shows that DNA locates closer to the top wall of the micropillar has higher chance to enter the flow pattern of droplet formation and thus has higher chance to be stretched across the micropillars. Our simulation tool has the potential to become a design tool for DNA manipulation in complex biomicrofluidic devices.     

Fluctuations of DNA mobility in nanofluidic entropic traps

We studied the mobility of DNA molecules driven by an electric field through a nanofluidic device containing a periodic array of deep and shallow regions termed entropic traps. The mobility of a group of DNA molecules was measured by fluorescent video microscopy. Since the depth of a shallow region is smaller than the DNA equilibrium size, DNA molecules are trapped for a characteristic time and must compress themselves to traverse the boundary between deep and shallow regions. Consistent with previous experimental results, we observed a nonlinear relationship between the mobility and electric field strength, and that longer DNA molecules have larger mobility. In repeated measurements under seemingly identical conditions, we measured fluctuations in the mobility significantly larger than expected from statistical variation. The variation was more pronounced for lower electric field strengths where the trapping time is considerable relative to the drift time. To determine the origin of these fluctuations, we investigated the dependence of the mobility on several variables: DNA concentration, ionic strength of the solvent, fluorescent dye staining ratio, electroosmotic flow, and electric field strength. The mobility fluctuations were moderately enhanced in conditions of reduced ionic strength and electroosmotic flow.     

Simulation of electrophoretic stretching of DNA in a microcontraction using an obstacle array for conformational preconditioning

Recently our group has reported experiments using an obstacle array to precondition the conformations of DNA molecules to facilitate their stretch in a microcontraction. Based upon previous successes simulating electrophoretic stretching in microcontractions without obstacles, we use our simulation model to study the deformation of DNA chains in a microcontraction preceded by an array of cylindrical obstacles. We compare our data to the experimental results and find good qualitative, and even quantitative, agreement concerning the behavior of the chains in the array; however, the simulations overpredict the mean stretch of the chains as they leave the contraction. We examine the amount of stretch gained between leaving the array and reaching the end of the contraction and speculate that the differences seen are caused by nonlinear electrokinetic effects that become important in the contraction due to a combination of field gradients and high field strengths.     

Size-dependent trajectories of DNA macromolecules due to insulative dielectrophoresis in submicrometer-deep fluidic channels

In this paper, we demonstrate for the first time that insulative dielectrophoresis can induce size-dependent trajectories of DNA macromolecules. We experimentally use λ (48.5 kbp) and T4GT7 (165.6 kbp) DNA molecules flowing continuously around a sharp corner inside fluidic channels with a depth of 0.4 μm. Numerical simulation of the electrokinetic force distribution inside the channels is in qualitative agreement with our experimentally observed trajectories. We discuss a possible physical mechanism for the DNA polarization and dielectrophoresis inside confining channels, based on the observed dielectrophoresis responses due to different DNA sizes and various electric fields applied between the inlet and the outlet. The proposed physical mechanism indicates that further extensive investigations, both theoretically and experimentally, would be very useful to better elucidate the forces involved at DNA dielectrophoresis. When applied for size-based sorting of DNA molecules, our sorting method offers two major advantages compared to earlier attempts with insulative dielectrophoresis: Its continuous operation allows for high-throughput analysis, and it only requires electric field strengths as low as ∼10 V∕cm.     

Electrotaxis of oral squamous cell carcinoma cells in a multiple-electric-field chip with uniform flow field

We report a new design of microfluidic chip (Multiple electric Field with Uniform Flow chip, MFUF chip) to create multiple electric field strengths (EFSs) while providing a uniform flow field simultaneously. MFUF chip was fabricated from poly-methyl methacrylates (PMMA) substrates by using CO₂ laser micromachining. A microfluidic network with interconnecting segments was utilized to de-couple the flow field and the electric field (EF). Using our special design, different EFSs were obtained in channel segments that had an identical cross-section and therefore a uniform flow field. Four electric fields with EFS ratio of 7.9:2.8:1:0 were obtained with flow velocity variation of only 7.8% CV (coefficient of variation). Possible biological effect of shear force can therefore be avoided. Cell behavior under three EFSs and the control condition, where there is no EF, was observed in a single experiment. We validated MFUF chip performance using lung adenocarcinoma cell lines and then used the chip to study the electrotaxis of HSC-3, an oral squamous cell carcinoma cell line. The MFUF chip has high throughput capability for studying the EF-induced cell behavior under various EFSs, including the control condition (EFS = 0).     

Study of flow behaviors on single-cell manipulation and shear stress reduction in microfluidic chips using computational fluid dynamics simulations

Various single-cell retention structures (SCRSs) were reported for analysis of single cells within microfluidic devices. Undesirable flow behaviors within micro-environments not only influence single-cell manipulation and retention significantly but also lead to cell damage, biochemical heterogeneity among different individual cells (e.g., different cell signaling pathways induced by shear stress). However, the fundamentals in flow behaviors for single-cell manipulation and shear stress reduction, especially comparison of these behaviors in different microstructures, were not fully investigated in previous reports. Herein, flow distribution and induced shear stress in two different single-cell retention structures (SCRS I and SCRS II) were investigated in detail to study their effects on single-cell trapping using computational fluid dynamics (CFD) methods. The results were successfully verified by experimental results. Comparison between these two SCRS shows that the wasp-waisted configuration of SCRS II has a better performance in trapping and manipulating long cylinder-shaped cardiac myocytes and provides a safer “harbor” for fragile cells to prevent cell damage due to the shear stress induced from strong flows. The simulation results have not only explained flow phenomena observed in experiments but also predict new flow phenomena, providing guidelines for new chip design and optimization, and a better understanding of the cell micro-environment and fundamentals of microfluidic flows in single-cell manipulation and analysis.     

Elasto-inertial particle focusing under the viscoelastic flow of DNA solution in a square channel

Particle focusing is an essential step in a wide range of applications such as cell counting and sorting. Recently, viscoelastic particle focusing, which exploits the spatially non-uniform viscoelastic properties of a polymer solution under Poiseuille flow, has attracted much attention because the particles are focused along the channel centerline without any external force. Lateral particle migration in polymer solutions in square channels has been studied due to its practical importance in lab-on-a-chip applications. However, there are still many questions about how the rheological properties of the medium alter the equilibrium particle positions and about the flow rate ranges for particle focusing. In this study, we investigated lateral particle migration in a viscoelastic flow of DNA solution in a square microchannel. The elastic property is relevant due to the long relaxation time of a DNA molecule, even when the DNA concentration is extremely low. Further, the shear viscosity of the solution is essentially constant irrespective of shear rate. Our current results demonstrate that the particles migrate toward the channel centerline and the four corners of a square channel in the dilute DNA solution when the inertia is negligible (elasticity-dominant flow). As the flow rate increases, the multiple equilibrium particle positions are reduced to a single file along the channel centerline, due to the elasto-inertial particle focusing mechanism. The current results support that elasto-inertial particle focusing mechanism is a universal phenomenon in a viscoelastic fluid with constant shear viscosity (Boger fluid). Also, the effective flow rate ranges for three-dimensional particle focusing in the DNA solution were significantly higher and wider than those for the previous synthetic polymer solution case, which facilitates high throughput analysis of particulate systems. In addition, we demonstrated that the DNA solution can be applied to focus a wide range of particle sizes in a single channel and also align red blood cells without any significant deformation.     

两端锚定的单链聚电解质在交流电场中的滞后效应

在均匀的交流电场作用下,应用布朗动力学模拟方法,研究了两端锚定的柔性和半柔性的单链聚电解质的动力学行为。研究结果表明,链段的位置随交流电场的变化表现出类似于磁滞回线的滞后效应,随着交流电的频率减小,这种滞后效应运渐消失,成为一条单一的曲线。本项目针对两端锚定的聚电解质随交流电场变化的动力学行为的研究,文献中未见报道。     

A microfluidic model to study fluid dynamics of mucus plug rupture in small lung airways

Fluid dynamics of mucus plug rupture is important to understand mucus clearance in lung airways and potential effects of mucus plug rupture on epithelial cells at lung airway walls. We established a microfluidic model to study mucus plug rupture in a collapsed airway of the 12th generation. Mucus plugs were simulated using Carbopol 940 (C940) gels at concentrations of 0.15%, 0.2%, 0.25%, and 0.3%, which have non-Newtonian properties close to healthy and diseased lung mucus. The airway was modeled with a polydimethylsiloxane microfluidic channel. Plug motion was driven by pressurized air. Global strain rates and shear stress were defined to quantitatively describe plug deformation and rupture. Results show that a plug needs to overcome yield stress before deformation and rupture. The plug takes relatively long time to yield at the high Bingham number. Plug length shortening is the more significant deformation than shearing at gel concentration higher than 0.15%. Although strain rates increase dramatically at rupture, the transient shear stress drops due to the shear-thinning effect of the C940 gels. Dimensionless time-averaged shear stress, T_xy, linearly increases from 3.7 to 5.6 times the Bingham number as the Bingham number varies from 0.018 to 0.1. The dimensionless time-averaged shear rate simply equals to T_xy/2. In dimension, shear stress magnitude is about one order lower than the pressure drop, and one order higher than yield stress. Mucus with high yield stress leads to high shear stress, and therefore would be more likely to cause epithelial cell damage. Crackling sounds produced with plug rupture might be more detectable for gels with higher concentration.     

An electrohydrodynamic flow in ac electrowetting

In ac electrowetting, hydrodynamic flows occur within a droplet. Two distinct flow patterns were observed, depending on the frequency of the applied electrical signal. The flow at low-frequency range was explained in terms of shape oscillation and a steady streaming process in conjunction with contact line oscillation. The origin of the flow at high-frequency range has not yet been explained. We suggest that the high-frequency flow originated mainly from the electrothermal effect, in which electrical charge is generated due to the gradient of electrical conductivity and permittivity, which is induced by the Joule heating of fluid medium. To support our argument, we analyzed the flow field numerically while considering the electrical body force generated by the electrothermal effect. We visualized the flow pattern and measured the flow velocity inside the droplet. The numerical results show qualitative agreement with experimental results with respect to electric field and frequency dependence of flow velocity. The effects of induced-charge electro-osmosis, natural convection, and the Marangoni flow are discussed.     

复杂流体流动的耗散粒子动力学研究进展

综述了耗散粒子动力学在复杂流体流动方面的研究进展,包括利用耗散粒子动力学研究液滴变形及破碎行为、微通道中DNA分子悬浮液的流动以及毛细血管中红细胞凝聚机理等方面的研究成果。同时,也简要介绍了并行计算技术在耗散粒子动力学方面的应用。     

Polymer brushes with nanoinclusions under shear: A molecular dynamics investigation

We use molecular dynamics simulations with a dissipative particle dynamics thermostat to study the behavior of nanosized inclusions (colloids) in a polymer brush under shear whereby the solvent is explicitly included in the simulation. The brush is described by a bead-spring model for flexible polymer chains, grafted on a solid substrate, while the polymer-soluble nanoparticles in the solution are taken as soft spheres whose diameter is about three times larger than that of the chain segments and the solvent. We find that the brush number density profile, as well as the density profiles of the nanoinclusions and the solvent, remains insensitive to strong shear although the grafted chains tilt in direction of the flow. The thickness of the penetration layer of nanoinclusions, as well as their average concentration in the brush, stays largely unaffected even at the strongest shear. Our result manifests the remarkable robustness of polymer brushes with embedded nanoparticles under high shear which could be of importance for technological applications.     

Examination of the role of transient receptor potential vanilloid type 4 in endothelial responses to shear forces

Shear stress is the major mechanical force applied on vascular endothelial cells by blood flow, and is a crucial factor in normal vascular physiology and in the development of some vascular pathologies. The exact mechanisms of cellular mechano-transduction in mammalian cells and tissues have not yet been elucidated, but it is known that mechanically sensitive receptors and ion channels play a crucial role. This paper describes the use of a novel and efficient microfluidic device to study mechanically-sensitive receptors and ion channels in vitro, which has three independent channels from which recordings can be made and has a small surface area such that fewer cells are required than for conventional flow chambers. The contoured channels of the device enabled examination of a range of shear stresses in one field of view, which is not possible with parallel plate flow chambers and other previously used devices, where one level of flow-induced shear stress is produced per fixed flow-rate. We exposed bovine aortic endothelial cells to different levels of shear stress, and measured the resulting change in intracellular calcium levels ([Ca²⁺]_i) using the fluorescent calcium sensitive dye Fluo-4AM. Shear stress caused an elevation of [Ca²⁺]_i that was proportional to the level of shear experienced. The response was temperature dependant such that at lower temperatures more shear stress was required to elicit a given level of calcium signal and the magnitude of influx was reduced. We demonstrated that shear stress-induced elevations in [Ca²⁺]_i are largely due to calcium influx through the transient receptor potential vanilloid type 4 ion channel.     

Onset of channeling during DNA electrophoresis in a sparse ordered post array

The “channeling hypothesis” of DNA electrophoresis in sparse, ordered arrays of posts predicts that the DNA will move through the array relatively unhindered if (i) the spacing between the posts is larger than the DNA coil and (ii) the electric field lines are straight. We tested this hypothesis by studying the electrophoretic separation of a small plasmid DNA (pUC19, 2686 base pairs) and a large, linear DNA (λ-DNA, 48 500 base pairs) in a hexagonal array of 1 μm diameter posts with a pitch of 7 μm. At low electric field strengths, these DNAs are separated due to the long-lived, rope-over-pulley collisions of λ-DNA with the posts. The resolution is lost as the electric field increases due to the onset of channeling by the λ-DNA. Using a diffusive model, we show that channeling arises at low electric fields due to the finite size of the array. This channeling is not intrinsic to the system and is attenuated by increasing the size of the array. Higher electric fields lead to intrinsic channeling, which is attributed to the disparate time scales for a rope-over-pulley collision and transverse diffusion between collisions. The onset of channeling is a gradual process, in agreement with extant Brownian dynamics simulation data. Even at weak electric fields, the electrophoretic mobility of λ-DNA in the array is considerably higher than would be expected if the DNA frequently collided with the posts.     

A microfluidic shear device that accommodates parallel high and low stress zones within the same culturing chamber

Fluid shear stress (FSS) plays a critical role in regulating endothelium function and maintaining vascular homeostasis. Current microfluidic devices for studying FSS effects on cells either separate high shear stress zone and low shear stress zone into different culturing chambers, or arranging the zones serially along the flow direction, which complicates subsequent data interpretation. In this paper, we report a diamond shaped microfluidic shear device where the high shear stress zone and the low shear stress zone are arranged in parallel within one culturing chamber. Since the zones with different shear stress magnitudes are aligned normal to the flow direction, the cells in one stress group are not substantially affected by the flow-induced cytokine/chemokine releases by cells in the other group. Cell loading experiments using human umbilical vein endothelial cells show that the device is able to reveal stress magnitude-dependent and loading duration-dependent cell responses. The co-existence of shear stress zones with varied magnitudes within the same culturing chamber not only ensures that all the cells are subject to the identical culturing conditions, but also allows the resemblance of the differential shear stress pattern in natural arterial conditions. The device is expected to provide a new solution for studying the effects of heterogeneous hemodynamic patterns in the onset and progression of various vascular diseases.     

Study for optical manipulation of a surfactant-covered droplet using lattice Boltzmann method

In this study, we simulated deformation and surfactant distribution on the interface of a surfactant-covered droplet using optical tweezers as an external source. Two optical forces attracted a single droplet from the center to both sides. This resulted in an elliptical shape deformation. The droplet deformation was characterized as the change of the magnitudes of surface tension and optical force. In this process, a non-linear relationship among deformation, surface tension, and optical forces was observed. The change in the local surfactant concentration resulting from the application of optical forces was also analyzed and compared with the concentration of surfactants subjected to an extensional flow. Under the optical force influence, the surfactant molecules were concentrated at the droplet equator, which is totally opposite to the surfactants behavior under extensional flow, where the molecules were concentrated at the poles. Lastly, the quasi-equilibrium surfactant distribution was obtained by combining the effects of the optical forces with the extensional flow. All simulations were executed by the lattice Boltzmann method which is a powerful tool for solving micro-scale problems.     

Multi-scale approach for the rheological characteristics of emulsions using molecular dynamics and lattice Boltzmann method

An emulsion system was simulated under simple shear rates to analyze its rheological characteristics using a hierarchical multi-scale approach. The molecular dynamics (MD) simulation was used to describe the interface of droplets in an emulsion. The equations derived from the MD simulation relative to interfacial tension, temperature, and surfactant concentration were applied as input parameters within lattice Boltzmann method (LBM) calculations. In the LBM simulation, we calculated the relative viscosity of an emulsion under a simple shear rate along with changes in temperature, shear rate, and surfactant concentration. The equations from the MD simulation showed that the interfacial tension of the droplets tended to decrease with an increase in temperature and surfactant concentration. The relative viscosity from the LBM simulation decreased with an increase in temperature. The shear thinning phenomena explaining the inverse proportion between shear rate and viscosity were observed. An increase in the surfactant concentration caused an increase in the relative viscosity for a decane-in-water emulsion, because the increased deformation caused by the decreased interfacial tension significantly influenced the wall shear stress.