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Two-phase porous media flow is important in many applications from drug delivery to groundwater diffusion and oil recovery and is of particular interest to biomedical diagnostic test developers using cellulose and nitrocellulose membranes with limited fluid sample volumes. This work presents a new two-phase porous media flow model based on the incompressible Navier−Stokes equation. The model aims to address the limitations of existing methods by incorporating a partial saturation distribution in porous media to account for limited fluid volumes. The basic parameters of the model are the pore size distribution and the contact angle. To validate the model, we solved five analytical solutions and compared them to corresponding experimental data. The experimentally measured penetration length data agreed with the model predictions, demonstrating model accuracy. Our findings suggest that this new two-phase porous media flow model can provide a valuable tool for researchers developing fluidic assays in paper and other porous media. 

Abstract Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins. Graphical Abstract 

Background: Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. We have previously demonstrated that labeling the in vivo murine proteome is feasible via injection of azidohomoalanine (Aha), an ncAA and methionine (Met) analog, without the need for Met depletion. Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues. Results: To address these gaps, we created a deterministic, compartmental model of the kinetic transport and incorporation of Aha in mice. Model results demonstrate the ability to predict Aha distribution and protein labeling in a variety of tissues and dosing paradigms. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing plasma and liver metabolomes following various Aha dosing regimens. We show that Aha administration induces minimal metabolic alterations in mice. Conclusions: Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We expect this model to be a useful tool to guide future experiments utilizing this technique to study proteomic responses to stimuli.