10,000-fold concentration increase in proteins in a cascade microchip using anionic ITP by a 3-D numerical simulation with experimental results

This paper describes both the experimental application and 3-D numerical simulation of isotachophoresis (ITP) in a 3.2 cm long "cascade" poly(methyl methacrylate) (PMMA) microfluidic chip. The microchip includes 10 × reductions in both the width and depth of the microchannel, which decreases the overall cross-sectional area by a factor of 100 between the inlet (cathode) and outlet (anode). A 3-D numerical simulation of ITP is outlined and is a first example of an ITP simulation in three dimensions. The 3-D numerical simulation uses COMSOL Multiphysics v4.0a to concentrate two generic proteins and monitor protein migration through the microchannel. In performing an ITP simulation on this microchip platform, we observe an increase in concentration by over a factor of more than 10,000 due to the combination of ITP stacking and the reduction in cross-sectional area. Two fluorescent proteins, green fluorescent protein and R-phycoerythrin, were used to experimentally visualize ITP through the fabricated microfluidic chip. The initial concentration of each protein in the sample was 1.995 μg/mL and, after preconcentration by ITP, the final concentrations of the two fluorescent proteins were 32.57 ± 3.63 and 22.81 ± 4.61 mg/mL, respectively. Thus, experimentally the two fluorescent proteins were concentrated by over a factor of 10,000 and show good qualitative agreement with our simulation results.     

10,000-fold concentration increase of the biomarker cardiac troponin I in a reducing union microfluidic chip using cationic isotachophoresis

This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10,000. The starting concentration of cTnI was 2.3 μg mL?1 and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL?1. To the author's knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.     

The spatial patterns through diffusion-driven instability in a predator-prey model

Studies on stability mechanism and bifurcation analysis of a system of interacting populations by the combined effect of self and cross-diffusion become an important issue in ecology. In the current investigation, we derive the conditions for existence and stability properties of a predator-prey model under the influence of self and cross-diffusion. Numerical simulations have been carried out in order to show the significant role of self and cross-diffusion coefficients and other important parameters of the system. Various contour pictures of spatial patterns through Turing instability are portrayed and analysed in order to substantiate the applicability of the present model. Finally, the paper ends with an extended discussion of biological implications of our findings.     

Effect of surface irregularities on unsteady pulsatile flow in a compliant artery

Of concern in the paper is an analytical study of pulsatile blood flow in an irregular stenosed arterial segment through a mathematical model. The model is two-dimensional and axisymmetric with an outline of the stenosis obtained from a three-dimensional casting of a mildly stenosed artery [L. Back, Y. Cho, D. Crawford, R. Cuffel, Effect of mild atherosclerosis on flow resistance in a coronary artery casting of man, J. Biomech. Eng. 106 (1984) 48–53]. The combined influence of an asymmetric shape and surface irregularities of the constriction has been explored in a computational study of blood flow through arterial stenosis with 48% areal occlusion. The moving wall of the artery is included to be anisotropic, linear, viscoelastic, incompressible circular cylindrical membrane shell. The effect of the surrounding connective tissues on the motion of the arterial wall is also paid due attention. Results are also obtained for a smooth stenosis model and also for a stenosis model representative by the cosine curve. An extensive quantitative analysis has been performed in non-uniform non-staggered grids through numerical computations for the effect of surface irregularities on the flow velocity, the flux, the resistive impedance and on the wall shear stress through their graphical representations so as to validate the applicability of such an improved mathematical model.     

Dynamics of a predator–prey model with disease in the predator

The present investigation deals with a predator–prey model with disease that spreads among the predator species only. The predator species is split out into two groups—the susceptible predator and the infected predator both of which feeds on prey species. The stability and bifurcation analyses are carried out and discussed at length. On the basis of the normal form theory and center manifold reduction, the explicit formulae are derived to determine stability and direction of Hopf bifurcating periodic solution. An extensive quantitative analysis has been performed in order to validate the applicability of our model under consideration. Copyright © 2013 John Wiley & Sons, Ltd.     

Mechanical characterization of HIV‐1 with a solid‐state nanopore sensor

Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo‐type human immunodeficiency virus type 1 at sub‐micron scale. Non‐infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.     

Numerical simulation of generalized newtonian blood flow past a couple of irregular arterial stenoses

This paper looked at the numerical investigations of the generalized Newtonian blood flow through a couple of irregular arterial stenoses. The flow is treated to be axisymmetric, with an outline of the stenoses obtained from a three dimensional casting of a mild stenosed artery, so that the flow effectively becomes two‐dimensional. The Marker and Cell (MAC) method is developed for the governing unsteady generalized Newtonian equations in staggered grid for viscous incompressible flow in the cylindrical polar co‐ordinates system. The derived pressure‐Poisson equation was solved using Successive‐Over‐Relaxation (S.O.R.) method and the pressure‐velocity correction formulae have been derived. Computations are performed for the pressure drop, the wall shear stress distribution and the separation region. The presented computations show that in comparison to the corresponding Newtonian model the generalized Newtonian fluid experiences higher pressure drop, lower peak wall shear stress and smaller separation region. © 2010 Wiley Periodicals, Inc. Numer Methods Partial Differential Eq 27: 960–981, 2011     

Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis

This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both "peak mode" and "plateau mode" and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL(-1) in the presence of human serum proteins and obtain cTnI concentrations factors as high as ~ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms.     

Enhanced Fluorescence Anisotropy Assay for Human Cardiac Troponin I and T Detection

Human cardiac troponin I (hcTnI) and troponin T (hcTnT) are the biomarkers of choice for the diagnosis of cardiac diseases. In an effort to improve assay sensitivity, in this study we developed a novel approach to simultaneously detect hcTnI and hcTnT in homogenous solutions by monitoring enhanced-fluorescence-anisotropy changes. Specifically, our design was based on a competition assay by measuring anisotropy change of fluorophore-labeled peptides bound to primary monoclonal antibodies in the presence of nano-gold-modified secondary antibody in response to the presence of target proteins. Enhanced-fluorescence-anisotropy resulted from interaction between the primary antibody and the nano-gold-labeled secondary antibody, which significantly increased the size and decreased tumbling motion of the complex of peptide-antibodies. The measurements were performed to detect hcTnI and hcTnT either individually or simultaneously in a homogenous buffer solution and in the solutions containing human plasma. Our results showed that when fluorescence emission was monitored at a single wavelength selected by a monochromator the assay at all experimental conditions had excellent linear response to the target proteins within the concentration range of 0.5–40 nM. The detection limit is 0.5 nM for both hcTnI and hcTnT in the presence of human plasma. However, when fluorescence emission was monitored using a cutoff filter, the linear response of the assay to the target proteins is within 15–500 pM. The detection limit is 15 pM which is close to the recommended 99th percentile cutoff point for concentrations of hcTnI and hcTnT tests to discriminate healthy and diseased conditions. Homogenous nature, rapid response time, and easy implementation of our assay design make it a useful tool for disease biomarker and protein sensing.     

Modeling and simulation of nanoparticle separation through a solid-state nanopore

Recent experimental studies show that electrokinetic phenomena such as electroosmosis and electrophoresis can be used to separate nanoparticles on the basis of their size and charge using nanopore‐based devices. However, the efficient separation through a nanopore depends on a number of factors such as externally applied voltage, size and charge density of particle, size and charge density of membrane pore, and the concentration of bulk electrolyte. To design an efficient nanopore‐based separation platform, a continuum‐based mathematical model is used for fluid. The model is based on Poisson–Nernst–Planck equations along with Navier–Stokes equations for fluid flow and on the Langevin equation for particle translocation. Our numerical study reveals that membrane pore surface charge density is a vital parameter in the separation through a nanopore. In this study, we have simulated high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) as the sample nanoparticles to demonstrate the capability of such a platform. Numerical results suggest that efficient separation of HDL from LDL in a 0.2 M KCL solution (resembling blood buffer) through a 150 nm pore is possible if the pore surface charge density is ～ ?4.0 mC/m². Moreover, we observe that pore length and diameter are relatively less important in the nanoparticle separation process considered here.