Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum.

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

Engineering Analysis of Non-Braided Polycaprolactone Bioresorbable Flow Diverters for Aneurysms.

This paper reports a nonbraided, bioresorbable polycaprolactone (PCL) flow diverter (FD) for the endovascular treatment of aneurysms. Bioresorbable FDs can reduce the risk associated with the permanent metallic FDs as they are resorbed by the body after curing of aneurysms. PCL FDs were designed and fabricated using an in-house hybrid electromelt spinning-fused deposition fabrication unit. Flow diverter's properties, surface qualities, and mechanical characteristics of PCL FDs of 50%, 60%, and 70% porosities were studied using scanning electron microscope (SEM), atomic force microscopy (AFM), and high precision universal testing machine (UTM). The deployability through a clinically relevant catheter was demonstrated in a PDMS aneurysm model. The angiographic visibility of the developed PCL FDs was evaluated using BaSO4 and Bi2O3 coatings of various concentration. The average strut thicknesses were 74.12 ± 6.63 µm, 63.07 ± 1.26 µm, and 56.82 ± 2.09 µm for PCL FDs with 50%, 60%, and 70% porosities, respectively. They average pore areas for the 50%, 60% and 70% porosities FDs were 0.055 ± 0.0056 mm2, 0. 0605 ± 0.0065 mm2, and 0.0712 ± 0.012 mm2, respectively. The surface quality was great with an RMS roughness value of 14.45 nm. The tensile, radial strength, and flexibility were found to be satisfactory and comparable to the nonbraided coronary stents. The developed PCL FDs were highly flexible and demonstrated to be deployable through conventional delivery system as low as 4 Fr catheters in a PDMS aneurysm model. The visibility under X-ray increases with the increasing concentration of coating materials BaSO4 and Bi2O3. The visibility intensity was slightly higher with Bi2O3 coating of PCL FDs. The overall results of the engineering analysis of the developed nonbraided PCL FDs are promising.

Experimental Analysis of Laser Micromachining of Microchannels in Common Microfluidic Substrates.

Laser micromachining technique offers a promising alternative method for rapid production of microfluidic devices. However, the effect of process parameters on the channel geometry and quality of channels on common microfluidic substrates has not been fully understood yet. In this research, we studied the effect of laser system parameters on the microchannel characteristics of Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and microscope glass substrate-three most widely used materials for microchannels. We also conducted a cell adhesion experiment using normal human dermal fibroblasts on laser-machined microchannels on these substrates. A commercial CO2 laser system consisting of a 45W laser tube, circulating water loop within the laser tube and air cooling of the substrate was used for machining microchannels in PDMS, PMMA and glass. Four laser system parameters - speed, power, focal distance, and number of passes were varied to fabricate straight microchannels. The channel characteristics such as depth, width, and shape were measured using a scanning electron microscope (SEM) and a 3D profilometer. The results show that higher speed produces lower depth while higher laser power produces deeper channels regardless of the substrate materials. Unfocused laser machining produces wider but shallower channels. For the same speed and power, PDMS channels were the widest while PMMA channels were the deepest. Results also showed that the profiles of microchannels can be controlled by increasing the number of passes. With an increased number of passes, both glass and PDMS produced uniform, wider, and more circular channels; in contrast, PMMA channels were sharper at the bottom and skewed. In rapid cell adhesion experiments, PDMS and glass microchannels performed better than PMMA microchannels. This study can serve as a quick reference in material-specific laser-based microchannel fabrications.

Effect of Process Parameters and Material Properties on Laser Micromachining of Microchannels.

Laser micromachining has emerged as a promising technique for mass production of microfluidic devices. However, control and optimization of process parameters, and design of substrate materials are still ongoing challenges for the widespread application of laser micromachining. This article reports a systematic study on the effect of laser system parameters and thermo-physical properties of substrate materials on laser micromachining. Three dimensional transient heat conduction equation with a Gaussian laser heat source was solved using finite element based Multiphysics software COMSOL 5.2a. Large heat convection coefficients were used to consider the rapid phase transition of the material during the laser treatment. The depth of the laser cut was measured by removing material at a pre-set temperature. The grid independent analysis was performed for ensuring the accuracy of the model. The results show that laser power and scanning speed have a strong effect on the channel depth, while the level of focus of the laser beam contributes in determining both the depth and width of the channel. Higher thermal conductivity results deeper in cuts, in contrast the higher specific heat produces shallower channels for a given condition. These findings can help in designing and optimizing process parameters for laser micromachining of microfluidic devices.

Electrodeformation of Vesicles Suspended in a Liquid Medium.

Deformation of flexible vesicles suspended in a fluid medium due to an applied electric field can provide valuable insight into deformation dynamics at a very small scale. In an electric field, the response of the vesicle membrane is strongly influenced by the conductivity of surrounding fluid, vesicle size and shape, and the magnitude of applied field. We studied the electrodeformation of vesicles immersed in a fluid media under a DC electric field. An immersed interface method is used to solve the electric field over the domain with conductive or non-conductive vesicles while an immersed boundary method is employed to solve fluid flow, fluid-solid interaction, membrane mechanics and vesicle deformation. Initial force analysis on the membrane surface reveals almost linear influence of vesicle size, but the vesicle size does not affect the long-term deformation which is consistent with experimental evidence. Highly nonlinear effect of the applied field as well as the conductivity ratios inside and outside of the vesicle are observed. Results also point towards an early linear deformation regime followed by an equilibrium stage for the membranes. Modeling results suggest that electrodeforming vesicles can create unique external flows for different conductivity ratios. Moreover, significant influence of the initial aspect ratio of the vesicle on the force distribution is observed across a range of conductivity ratios.

Modeling and simulation of dielectrophoretic particle-particle interactions and assembly.

Electric field induced particle-particle interactions and assembly are of great interest due to their useful applications in micro devices. The behavior of particles becomes more complex if multiple particles interact with each other at the same time. In this paper, we present a numerical study of two dimensional DC dielectrophoresis based particle-particle interactions and assembly for multiple particles using a hybrid immersed interface-immersed boundary method. The immersed interface method is employed to capture the physics of electrostatics in a fluid media with suspended particles. Particle interaction based dielectrophoretic forces are obtained using Maxwell's stress tensor without any boundary or volume integration. This electrostatic force distribution mimics the actual physics of the immersed particles in a fluid media. The corresponding particle response and hydrodynamic interactions are captured through the immersed boundary method by solving the transient Navier-Stokes equations. The interaction and assembly of multiple electrically similar and dissimilar particles are studied for various initial positions and orientations. Numerical results show that in a fluid media, similar particles form a chain parallel to the applied electric field, whereas dissimilar particles form a chain perpendicular to the applied electric field. Irrespective of initial position and orientation, particles first align themselves parallel or perpendicular to the electric field depending on the similarity or dissimilarity of particles. The acceleration and deceleration of particles are also observed and analyzed at different phases of the assembly process. This comprehensive study can be used to explain the multiple particle interaction and assembly phenomena observed in experiments.

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.