Jonathan Dusting

Shear-Induced Unfolding of Lysozyme Monitored In Situ

Conformational changes due to externally applied physiochemical parameters, including pH, temperature, solvent composition, and mechanical forces, have been extensively reported for numerous proteins. However, investigations on the effect of fluid shear flow on protein conformation remain inconclusive despite its importance not only in the research of protein dynamics but also for biotechnology applications where processes such as pumping, filtration, and mixing may expose protein solutions to changes in protein structure. By combining particle image velocimetry and Raman spectroscopy, we have successfully monitored reversible, shear-induced structural changes of lysozyme in well-characterized flows. Shearing of lysozyme in water altered the proteins backbone structure, whereas similar shear rates in glycerol solution affected the solvent exposure of side-chain residues located toward the exterior of the lysozyme alpha-domain. The results demonstrate the importance of measuring conformational changes in situ and of quantifying fluid stresses by the three-dimensional shear tensor to establish reversible unfolding or misfolding transitions occurring due to flow exposure.

The effect of red blood cell aggregation on velocity and cell-depleted layer characteristics of blood in a bifurcating microchannel

Red blood cell (RBC) aggregation is a multifaceted phenomenon, and whether it is generally beneficial or deleterious remains unclear. In order to better understand its effect on microvascular blood flow, the phenomenon must be studied in complex geometries, as it is strongly dependent on time, flow, and geometry. The cell-depleted layer (CDL) which forms at the walls of microvessels has been observed to be enhanced by aggregation; however, details of the characteristics of the CDL in complex regions, such as bifurcations, require further investigation. In the present study, a microchannel with a T-junction was used to analyze the influence of aggregation on the flow field and the CDL. Micro-PIV using RBCs as tracers provided high resolution cell velocity data. CDL characteristics were measured from the same data using a newly developed technique based on motion detection. Skewed and sharpened velocity profiles in the daughter branches were observed, contrary to the behavior of a continuous Newtonian fluid. RBC aggregation was observed to increase the skewness, but decrease the sharpening, of the velocity profiles in the daughter branches. The CDL width was found to be significantly greater, with a wider distribution, in the presence of aggregation and the mean width increased proportionally with the reciprocal of the fraction of flow entering the daughter branch. Aggregation also significantly increased the roughness of the interface between the CDL and the RBC core. The present results provide further insight into how RBC aggregation may affect the flow in complex geometries, which is of importance in both understanding its functions invivo, and utilizing it as a tool in microfluidic devices.

Hematocrit, viscosity and velocity distributions of aggregating and non-aggregating blood in a bifurcating microchannel

Microscale blood flow is characterised by heterogeneous distributions of hematocrit, viscosity and velocity. In microvascular bifurcations, cells are unevenly distributed between the branches, and this effect can be amplified in subsequent branches depending on a number of parameters. We propose an approach to infer hematocrit profiles of human blood flowing through a bifurcating microchannel. The influence of aggregation, induced by the addition of Dextran 2000 to the samples, is also considered. Averaged values indicate plasma skimming, particularly in the presence of red blood cell (RBC) aggregation. Using an empirical model, the hematocrit profiles are used to estimate local relative viscosity distributions. Simulations are used to predict how the non-uniform viscosity influences the velocity profiles. Comparing these data to velocity profiles of RBCs measured using particle image velocimetry provides validation of the model. It is observed that aggregation blunts velocity profiles after a long straight section of channel. Downstream of the bifurcation, skewing of the velocity profiles is detected, which is enhanced by aggregation. The proposed methodology is capable of providing hitherto unreported information on important aspects of microscale blood rheology.

Susceptibility of Different Proteins to Flow-Induced Conformational Changes Monitored with Raman Spectroscopy

By directly monitoring stirred protein solutions with Raman spectroscopy, the reversible unfolding of proteins caused by fluid shear is examined for several natural proteins with varying structural properties and molecular weight. While complete denaturation is not observed, a wide range of spectral variances occur for the different proteins, indicating subtle conformational changes that appear to be protein-specific. A number of significant overall trends are apparent from the study. For globular proteins, the overall extent of spectral variance increases with protein size and the proportion of beta-structure. For two less structured proteins, fetuin and alpha-casein, the observed changes are of relatively low magnitude, despite the greater molecular structural mobility of these proteins. This implies that other protein-specific factors, such as posttranslational modifications, may also be significant. Individual band changes occurring in the spectral profiles of each individual protein are also discussed in detail.

Spatial variation of blood viscosity: Modelling using shear fields measured by a μPIV based technique

The spatial characteristics of blood viscosity were investigated by combining a newly developed constitutive equation with shear deformation fields calculated from velocity measurements obtained by a μPIV based technique. Blood at physiological hematocrit levels and in the presence of aggregation was sheared in a narrow gap plate-plate geometry and the velocity and aggregation characteristics were determined from images captured using a high resolution camera. Changes in the microstructure of blood caused by aggregation were observed to affect the flow characteristics. At low shear rates, high aggregation and network formation caused the RBC motion to become essentially two-dimensional. The measured velocity fields were used to estimate the magnitude of shear which was subsequently used in conjunction with the new model to assess the spatial variation of viscosity across the flow domain. It was found that the non-uniform microstructural characteristics of blood influence its viscosity distribution accordingly. The viscosity of blood estimated in the core of the examined flow, using a zero-gradient core velocity profile assumption, was found to be significantly higher than the overall effective viscosity determined using other velocity profile assumptions.

Coupled human erythrocyte velocity field and aggregation measurements at physiological haematocrit levels

Simultaneous measurement of erythrocyte (RBC) velocity fields and aggregation properties has been successfully performed using an optical shearing microscope and Particle Image Velocimetry (PIV). Blood at 45% haematocrit was sheared at rates of 5.4< or =gamma < or = 252 s(-1) and imaged using a high speed camera. The images were then processed to yield aggregation indices and flow velocities. Negligible levels of aggregation were observed for gamma > or = 54.0 s(-1), while high levels of aggregation and network formation occurred for gamma < or = 11.7 s(-1). The results illustrate that the velocity measurements are dependent on the extent of RBC aggregation. High levels of network formation cause the velocities at gamma > or = 5.4 s(-1) to deviate markedly from the expected solid body rotation profile. The effect of aggregation level on the PIV accuracy was assessed by monitoring the two-dimensional (2D) correlation coefficients. Lower levels of aggregation result in poorer image correlation, from which it can be inferred that PIV accuracy is reduced. Moreover, aggregation is time-dependent, and consequently PIV accuracy may decrease during recording as the cells break up. It is therefore recommended that aggregation and its effects are taken into account in future when undertaking blood flow studies using PIV. The simplicity of the technique, which requires no lasers, filters, or special pretreatments, demonstrates the potential wide-spread applicability of the data acquisition system for accurate blood flow PIV and aggregation measurement.

Detecting the Early Onset of Shear-Induced Fibril Formation of Insulin in situ

A new approach is presented for detecting the early onset of amyloid fibril formation of insulin in a fluidic environment. The fibrillogenesis of insulin in a well-characterized Taylor-Couette flow cell was analyzed in situ using Raman spectroscopy in combination with principal components analysis (PCA). Raman spectra recorded using a 532.5 nm excitation laser revealed a more rapid fibrillogenesis process during the first 90 min of shearing than previously reported for samples exposed to flow. Bands corresponding to intermolecular H-bonded β-sheet structure of insulin at 1678, 1630, and 1625 cm(-1) observed in the Raman difference spectra between unsheared insulin and sheared insulin show an increase in intensity as a function of shear exposure time, which is characteristic of fibril formation, with the first changes detected after 10 min. Additional analysis of samples removed from the flow cell after specific time periods provided conformation of the flow-enhanced fibrillogenesis process, including the detection of early fibril formation after only 1 min of shearing. FT-IR spectra of the insulin solutions showed evolution of bands at 1673 and 1633 cm(-1) from an increase in H-bonded β-turn and β-sheet structures, respectively, while fluorescence emission spectra detected the presence of a new emission band at 482 nm. TEM images confirmed the early onset of fibril formation at 1 min shear exposure, before a maturation and concentration increase of fibrils with further shearing. This study highlights the ability of fluid flows to accelerate insulin fibril formation, which has important implications for biotechnology applications such as the purification process of insulin therapeutic drugs in the pharmaceutical industry, as well as the use of optical-based methods for detecting fibrillogenesis.

ENGINEERING IMAGING : USING PARTICLE IMAGE VELOCIMETRY TO SEE PHYSIOLOGY IN A NEW LIGHT

1 Despite the array of sophisticated imaging techniques available for biological applications, none of the standard biomedical techniques adequately provides the capability to measure motion and flow. Those techniques currently in use are particularly lacking in spatial and temporal resolution. 2 Herein, we introduce the technique of particle image velocimetry. This technique is a well‐established tool in engineering research and industry. Particle image velocimetry is continuing to develop and has an increasing number of variants. 3 Three case studies are presented: (i) the use of microparticle image velocimetry to study flow generated by high‐frequency oscillatory ventilation in a human airway model; (ii) the use of stereoparticle image velocimetry to study stirred cell and tissue culture devices; and (iii) a three‐dimensional X‐ray particle image velocimetry technique used to measure flow in an in vitro vascular flow model. 4 The case studies highlight the vast potential of applying the engineering technique of particle image velocimetry and its many variants to current research problems in physiology.

Quantifying local characteristics of velocity, aggregation and hematocrit of human erythrocytes in a microchannel flow.

The effect of erythrocyte aggregation on blood viscosity and microcirculatory flow is a poorly understood area of haemodynamics, especially with relevance to serious pathological conditions. Advances in microfluidics have made it possible to study the details of blood flow in the microscale, however, important issues such as the relationship between the local microstructure and local flow characteristics have not been investigated extensively. In the present study an experimental system involving simple brightfield microscopy has been successfully developed for simultaneous, time-resolved quantification of velocity fields and local aggregation of human red blood cells (RBC) in microchannels. RBCs were suspended in Dextran and phosphate buffer saline solutions for the control of aggregation. Local aggregation characteristics were investigated at bulk and local levels using statistical and edge-detection image processing techniques. A special case of aggregating flow in a microchannel, in which hematocrit gradients were present, was studied as a function of flowrate and time. The level of aggregation was found to strongly correlate with local variations in velocity in both the bulk flow and wall regions. The edge detection based analysis showed that near the side wall large aggregates are associated with regions corresponding to high local velocities and low local shear. On the contrary, in the bulk flow region large aggregates occurred in regions of low velocity and high erythrocyte concentration suggesting a combined effect of hematocrit and velocity distributions on local aggregation characteristics. The results of this study showed that using multiple methods for aggregation quantification, albeit empirical, could help towards a robust characterisation of the structural properties of the fluid.

On the quasiperiodic state in a moderate aspect ratio Taylor–Couette flow

The transition pathway leading to chaotic flow in a Taylor–Couette vessel of aspect ratio γ=11.2 and radius ratio η=0.81 with only the inner cylinder rotating has been studied using time-resolved particle image velocimetry. A hitherto unreported sequence of transitions, whereby the flow changes from wavy vortex flow to a quasiperiodic state of high modulation frequency, is identified using spatially resolved spectral analysis. The nature of the modulated wavy vortex flow is detailed using proper orthogonal decomposition, through which it is revealed that the modulation is similar to a fast moving azimuthal wave (FMAW). In contrast with previous observations of the FMAW at a much higher aspect ratio, the mode appears directly after wavy vortex flow and as a precursor to the CVF regime. The FMAW is also associated with codirectional vorticity fluctuations on each Taylor vortex core that diminish in strength close to the endwalls.