A platform for stop flow gradient generation to investigate chemotaxis

The ability of artificial microswimmers to respond to external stimuli and the mechanistical details of their origins belong to the most disputed challenges in interdisciplinary science. Therein, the creation of chemical gradients is technically challenging, because they quickly level out due to diffusion. Inspired by pivotal stopped flow experiments in chemical kinetics, we show that microfluidics gradient generation combined with a pressure feedback loop for precisely controlling the stop of the flows, can enable us to study mechanistical details of chemotaxis of artificial Janus micromotors, based on a catalytic reaction. We find that these copper Janus particles display a chemotactic motion along the concentration gradient in both, positive and negative direction and we demonstrate the mechanical reaction of the particles to small forces deviations, explaining this behaviour.

Analysis of Static Molecular Gradients in a High-Throughput Drug Screening Microfluidic Assay

In this study, we thoroughly analyzed molecular gradient generation, its stability over time, and linearity in our high-throughput drug screening microfluidic assay (HTS). These parameters greatly affect the precision and accuracy of the device’s analytical protocol. As part of the research, we developed a mathematical model of dependence of the concentration profile on the initial concentrations of active substances in reservoirs and the number of tilts, as well as the dependence of the active substance concentration profiles in the culture chambers on the concentration profile of the reference dye in the indicator chamber. The mean concentration prediction error of the proposed equations ranged from 1.4% to 2.4% for the optimized parameters of the procedure and did not increase with the incubation time. The concentration profile linearity index, Pearson’s correlation coefficient reached −0.997 for 25 device tilts. The observed time stability of the profiles was very good. The mean difference between the concentration profile after 5 days of incubation and the baseline profile was only 7.0%. The newly created mathematical relationships became part of the new HTS biochip operating protocols, which are detailed in the article.

Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question “what is a virus?”.

Functional compartmentalization and metabolic separation in a prokaryotic cell

The prokaryotic cell is traditionally seen as a “bag of enzymes,” yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria. These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the “cytoplasm” or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO2 assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

Pixel-based open-space microfluidics for versatile surface processing

An increasing number of applications in biology, chemistry, and material sciences require fluid manipulation beyond what is possible with current automated pipette handlers, such as gradient generation, interface reactions, reagent streaming, and reconfigurability. In this article, we introduce the pixelated chemical display (PCD), a scalable strategy for highly parallel, reconfigurable liquid handling on open surfaces. Microfluidic “pixels” are created when a fluid stream injected above a surface is confined by neighboring identical fluid streams, forming a repeatable flow unit that can be used to tesselate a surface. PCDs generating up to 144 pixels are fabricated and used to project “chemical moving pictures” made of several reagents over both immersed and dry surfaces, without any physical barrier or wall. This work distinguishes itself from previous work in open-space microfluidics by presenting a device architecture where the number of confinement areas can be scaled to any size. Furthermore, it challenges the open-space tenet that the aspiration rate must be higher than the injection rate for reagents to be confined. Overall, this article sets the foundation for massively parallel surface processing using continuous flow streams and showcases possibilities in both wet and dry surface patterning and roll-to-roll processes.

Heparin MicroIslands to Promote Enhanced Diabetic Wound Healing Outcomes

A powerful tool to improve tissue integration with biomaterial scaffolds for the regeneration of damaged tissues is to promote cell migration using chemotactic gradients of growth factors. This approach has been realized by the exogenous delivery of growth factors, which unfortunately also limits the scaffold’s ability to meet each wound’s unique spatial and temporal regenerative needs. To address this limitation, we present a new approach to gradient generation by incorporating heparin microislands, which are spatially isolated heparin-containing microparticles that create chemotactic microgradients through reorganization of endogenous local growth factors. We incorporated heparin microislands within microporous annealed particle (MAP) scaffolds, which allows us to tune their incorporation ratiometrically to create a heterogenous microenvironment. In this manuscript, we demonstrate the ability of heparin microislands to organize uniform growth factors into spontaneous microgradients and control downstream cell migration in vitro. Further, we present their ability to significantly improve wound healing outcomes (epidermal regeneration and vascularization) in a diabetic wound model relative to two clinically relevant controls.

Systematic Investigation of Insulin Fibrillation on a Chip

A microfluidic protein aggregation device (microPAD) that allows the user to perform a series of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific conditions. Parallel processes of metering reagents, stepwise concentration gradient generation, and mixing are achieved simultaneously by pneumatic valves. Fibrillation of bovine insulin was selected to test the device. The effect of insulin and sodium chloride (NaCl) concentration on the formation of fibrillar structures was studied by observing the growth rate of partially folded protein, using the fluorescent marker Thioflavin-T. Moreover, dual gradients of different NaCl and hydrochloric acid (HCl) concentrations were formed, to investigate their interactive roles in the formation of insulin fibrils and spherulites. The chip-system provides a bird’s eye view on protein aggregation, including an overview of the factors that affect the process and their interactions. This microfluidic platform is potentially useful for rapid analysis of the fibrillation of proteins associated with many misfolding-based diseases, such as quantitative and qualitative studies on amyloid growth.