Emerging Roles of Microfluidics in Brain Research: From Cerebral Fluids Manipulation to Brain-on-a-Chip and Neuroelectronic Devices Engineering.

Remarkable progress made in the past few decades in brain research enables the manipulation of neuronal activity in single neurons and neural circuits and thus allows the decipherment of relations between nervous systems and behavior. The discovery of glymphatic and lymphatic systems in the brain and the recently unveiled tight relations between the gastrointestinal (GI) tract and the central nervous system (CNS) further revolutionize our understanding of brain structures and functions. Fundamental questions about how neurons conduct two-way communications with the gut to establish the gut-brain axis (GBA) and interact with essential brain components such as glial cells and blood vessels to regulate cerebral blood flow (CBF) and cerebrospinal fluid (CSF) in health and disease, however, remain. Microfluidics with unparalleled advantages in the control of fluids at microscale has emerged recently as an effective approach to address these critical questions in brain research. The dynamics of cerebral fluids (i.e., blood and CSF) and novel in vitro brain-on-a-chip models and microfluidic-integrated multifunctional neuroelectronic devices, for example, have been investigated. This review starts with a critical discussion of the current understanding of several key topics in brain research such as neurovascular coupling (NVC), glymphatic pathway, and GBA and then interrogates a wide range of microfluidic-based approaches that have been developed or can be improved to advance our fundamental understanding of brain functions. Last, emerging technologies for structuring microfluidic devices and their implications and future directions in brain research are discussed.

On-demand modulation of 3D-printed elastomers using programmable droplet inclusions.

One of the key thrusts in three-dimensional (3D) printing and direct writing is to seamlessly vary composition and functional properties in printed constructs. Most inks used for extrusion-based printing, however, are compositionally static and available approaches for dynamic tuning of ink composition remain few. Here, we present an approach to modulate extruded inks at the point of print, using droplet inclusions. Using a glass capillary microfluidic device as the printhead, we dispersed droplets in a polydimethylsiloxane (PDMS) continuous phase and subsequently 3D printed the resulting emulsion into a variety of structures. The mechanical characteristics of the 3D-printed constructs can be tuned in situ by varying the spatial distribution of droplets, including aqueous and liquid metal droplets. In particular, we report the use of poly(ethylene glycol) diacrylate (PEGDA) aqueous droplets for local PDMS chemistry alteration resulting in significant softening (85% reduced elastic modulus) of the 3D-printed constructs. Furthermore, we imparted magnetic functionality in PDMS by dispersing ferrofluid droplets and rationally designed and printed a rudimentary magnetically responsive soft robotic actuator as a functional demonstration of our droplet-based strategy. Our approach represents a continuing trend of adapting microfluidic technology and principles for developing the next generation of additive manufacturing technology.

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Inhibition of Band 3 tyrosine phosphorylation: a new mechanism for treatment of sickle cell disease.

Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.

Piezo1 regulates mechanotransductive release of ATP from human RBCs.

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Flow-Regulated Growth of Titanium Dioxide (TiO2 ) Nanotubes in Microfluidics.

Electrochemical anodization of titanium (Ti) in a static, bulk condition is used widely to fabricate self-organized TiO2 nanotube arrays. Such bulk approaches, however, require extended anodization times to obtain long TiO2 nanotubes and produce only vertically aligned nanotubes. To date, it remains challenging to develop effective strategies to grow long TiO2 nanotubes in a short period of time, and to control the nanotube orientation. Here, it is shown that the anodic growth of TiO2 nanotubes is significantly enhanced (≈16-20 times faster) under flow conditions in microfluidics. Flow not only controls the diameter, length, and crystal orientations of TiO2 nanotubes, but also regulates the spatial distribution of nanotubes inside microfluidic devices. Strikingly, when a Ti thin film is deposited on silicon substrates and anodized in microfluidics, both vertically and horizontally aligned (relative to the bottom substrate) TiO2 nanotubes can be produced. The results demonstrate previously unidentified roles of flow in the regulation of growth of TiO2 nanotubes, and provide powerful approaches to effectively grow long, oriented TiO2 nanotubes, and construct hierarchical TiO2 nanotube arrays on silicon-based materials.

Electrode distance regulates the anodic growth of titanium dioxide (TiO2) nanotubes.

Electrochemical anodization of titanium has been used widely to produce self-organized TiO2 nanotube arrays. Many experimental parameters, such as anodizing voltage and electrolyte composition, have been investigated extensively in the anodic growth of TiO2 nanotubes. The effect of electrode distance on the anodic growth of TiO2 nanotube arrays, however, remains elusive. This could be an important problem when in situ growth of TiO2 nanotubes is required in microdevices. Here, we show that decreasing the electrode distance at a constant anodizing voltage enhances the anodic growth of TiO2 nanotubes and the change of nanotube structures becomes more sensitive to the electrode distance at high voltages. We further demonstrate the correlation between electrode distance and current density during the anodic growth of TiO2 nanotubes and suggest that the change of current density regulated by electrode distance controls the growth of TiO2 nanotubes. The present study offers an effective approach to enhance the production of TiO2 nanotube arrays without changing the anodizing voltage and electrolyte composition and thus provides useful insights to the anodic growth of TiO2 nanotubes at reduced electrode distances.

Vitamin D receptor pathway is required for probiotic protection in colitis.

Low expression of vitamin D receptor (VDR) and dysfunction of vitamin D/VDR signaling are reported in patients with inflammatory bowel disease (IBD); therefore, restoration of VDR function to control inflammation in IBD is desirable. Probiotics have been used in the treatment of IBD. However, the role of probiotics in the modulation of VDR signaling to effectively reduce inflammation is unknown. We identified a novel role of probiotics in activating VDR activity, thus inhibiting inflammation, using cell models and VDR knockout mice. We found that the probiotics Lactobacillus rhamnosus strain GG (LGG) and Lactobacillus plantarum (LP) increased VDR protein expression in both mouse and human intestinal epithelial cells. Using the VDR luciferase reporter vector, we detected increased transcriptional activity of VDR after probiotic treatment. Probiotics increased the expression of the VDR target genes, such as antimicrobial peptide cathelicidin, at the transcriptional level. Furthermore, the role of probiotics in regulating VDR signaling was tested in vivo using a Salmonella-colitis model in VDR knockout mice. Probiotic treatment conferred physiological and histologic protection from Salmonella-induced colitis in VDR(+/+) mice, whereas probiotics had no effects in the VDR(-/-) mice. Probiotic treatment also enhanced numbers of Paneth cells, which secrete AMPs for host defense. These data indicate that the VDR pathway is required for probiotic protection in colitis. Understanding how probiotics enhance VDR signaling and inhibit inflammation will allow probiotics to be used effectively, resulting in innovative approaches to the prevention and treatment of chronic inflammation.

Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release.

RBCs are known to release ATP, which acts as a signaling molecule to cause dilation of blood vessels. A reduction in the release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. Furthermore, reduced deformation of RBCs has been correlated with myocardial infarction and coronary heart disease. Because ATP release has been linked to cell deformation, we undertook a multiscale approach to understand the links between single RBC dynamics, ATP release, and macroscopic viscosity all at physiological shear rates. Our experimental approach included microfluidics, ATP measurements using a bioluminescent reaction, and rheology. Using microfluidics technology with high-speed imaging, we visualize the deformation and dynamics of single cells, which are known to undergo motions such as tumbling, swinging, tanktreading, and deformation. We report that shear thinning is not due to cellular deformation as previously believed, but rather it is due to the tumbling-to-tanktreading transition. In addition, our results indicate that ATP release is constant at shear stresses below a threshold (3 Pa), whereas above the threshold ATP release is increased and accompanied by large cellular deformations. Finally, performing experiments with well-known inhibitors, we show that the Pannexin 1 hemichannel is the main avenue for ATP release both above and below the threshold, whereas, the cystic fibrosis transmembrane conductance regulator only contributes to deformation-dependent ATP release above the stress threshold.

The effects of membrane cholesterol and simvastatin on red blood cell deformability and ATP release.

It is known that deformation of red blood cells (RBCs) is linked to ATP release from the cells. Further, membrane cholesterol has been shown to alter properties of the cell membrane such as fluidity and bending stiffness. Membrane cholesterol content is increased in some cardiovascular diseases, for example, in individuals with acute coronary syndromes and chronic stable angina, and therefore, because of the potential clinical relevance, we investigated the influence of altered RBC membrane cholesterol levels on ATP release. Because of the correlation between statins and reduced membrane cholesterol in vivo, we also investigated the effects of simvastatin on RBC deformation and ATP release. We found that reducing membrane cholesterol increases cell deformability and ATP release. We also found that simvastatin increases deformability by acting directly on the membrane in the absence of the liver, and that ATP release was increased for cells with enriched cholesterol after treatment with simvastatin.

Coated gas bubbles for the continuous synthesis of hollow inorganic particles.

We present a microfluidic approach for the controlled encapsulation of individual gas bubbles in micrometer-diameter aqueous droplets with high gas volume fractions and demonstrate this approach to making a liquid shell, which serves as a template for the synthesis of hollow inorganic particles. In particular, we find that an increase in the viscosity of the aqueous phase facilitates the encapsulation of individual gas bubbles in an aqueous droplet and allows control of the thickness of a thin aqueous shell. Furthermore, because such droplets contain a finite amount of water, uncontrolled hydrolysis reactions between reactive inorganic precursors and bulk water can be avoided. We demonstrate this approach by introducing reactive inorganic precursors, such as silane and titanium butoxide, for sol-gel reactions downstream from the formation of the bubble in a droplet and consequently fabricate hollow particles of silica or titania in one continuous flow process. These approaches provide a route to controlling double-emulsion-type gas-liquid microstructures and offer a new fabrication method for thin-shell-covered microbubbles and hollow microparticles.

Microfluidic generation of droplets with a high loading of nanoparticles.

Microfluidic approaches for controlled generation of colloidal clusters, for example, via encapsulation of colloidal particles in droplets, have been used for the synthesis of functional materials including drug delivery carriers. Most of the studies, however, use a low concentration of an original colloidal suspension (<10 wt %). Here we demonstrate microfluidic approaches for directly making droplets with moderate (10-25 wt %) and high (>60 wt %) particle concentrations. Three types of microfluidic devices, PDMS flow-focusing, PDMS T-junction, and microcapillary devices, are investigated for direct encapsulation of a high concentration of polystyrene (PS) nanoparticles in droplets. In particular, it is shown that PDMS devices fabricated by soft lithography can generate droplets from a 25 wt % PS suspension, whereas microcapillary devices made from glass capillary tubes are able to produce droplets from a 67 wt % PS nanoparticle suspension. When the PS concentration is between 0.6 and 25 wt %, the size of the droplets is found to change with the oil-to-water flow rate ratio and is independent of the concentration of particles in the initial suspensions. Drop sizes from ~12 to 40 µm are made using flow rate ratios Q(oil)/Q(water) from 20 to 1, respectively, with either of the PDMS devices. However, clogging occurs in PDMS devices at high PS concentrations (>25 wt %) arising from interactions between the PS colloids and the surface of PDMS devices. Glass microcapillary devices, on the other hand, are resistant to clogging and can produce droplets continuously even when the concentration of PS nanoparticles reaches 67 wt %. We believe that our findings indicate useful approaches and guidelines for the controlled generation of emulsions filled with a high loading of nanoparticles, which are useful for drug delivery applications.

Dynamics of shear-induced ATP release from red blood cells.

Adenosine triphosphate (ATP) is a regulatory molecule for many cell functions, both for intracellular and, perhaps less well known, extracellular functions. An important example of the latter involves red blood cells (RBCs), which help regulate blood pressure by releasing ATP as a vasodilatory signaling molecule in response to the increased shear stress inside arterial constrictions. Although shear-induced ATP release has been observed widely and is believed to be triggered by deformation of the cell membrane, the underlying mechanosensing mechanism inside RBCs is still controversial. Here, we use an in vitro microfluidic approach to investigate the dynamics of shear-induced ATP release from human RBCs with millisecond resolution. We demonstrate that there is a sizable delay time between the onset of increased shear stress and the release of ATP. This response time decreases with shear stress, but surprisingly does not depend significantly on membrane rigidity. Furthermore, we show that even though the RBCs deform significantly in short constrictions (duration of increased stress <3 ms), no measurable ATP is released. This critical timescale is commensurate with a characteristic membrane relaxation time determined from observations of the cell deformation by using high-speed video. Taken together our results suggest a model wherein the retraction of the spectrin-actin cytoskeleton network triggers the mechanosensitive ATP release and a shear-dependent membrane viscosity controls the rate of release.

Suppression of dendrite growth by cross-flow in microfluidics.

Formation of rough, dendritic deposits is a critical problem in metal electrodeposition processes and could occur in next-generation, rechargeable batteries that use metallic electrodes. Electroconvection, which originates from the coupling of the imposed electric field and a charged fluid near an electrode surface, is believed to be responsible for dendrite growth. However, few studies are performed at the scale of fidelity where root causes and effective strategies for controlling electroconvection and dendrite growth can be investigated in tandem. Using microfluidics, we showed that forced convection across the electrode surface (cross-flow) during electrodeposition reduced metal dendrite growth (97.7 to 99.4%) and delayed the onset of electroconvective instabilities. Our results highlighted the roles of forced convection in reducing dendrite growth and electroconvective instabilities and provided a route toward effective strategies for managing the consequences of instability in electrokinetics-based processes where electromigration dominates ion diffusion near electrodes.

The dynamic behavior of chemically "stiffened" red blood cells in microchannel flows.

The rigidity of red blood cells (RBCs) plays an important role in whole blood viscosity and is correlated with several cardiovascular diseases. Two chemical agents that are commonly used to study cell deformation are diamide and glutaraldehyde. Despite diamide's common usage, there are discrepancies in the literature surrounding diamide's effect on the deformation of RBCs in shear and pressure-driven flows; in particular, shear flow experiments have shown that diamide stiffens cells, while pressure-driven flow in capillaries did not give this result. We performed pressure-driven flow experiments with RBCs in a microfluidic constriction and quantified the cell dynamics using high-speed imaging. Diamide, which affects RBCs by cross-linking spectrin skeletal membrane proteins, did not reduce deformation and showed an unchanged effective strain rate when compared to healthy cells. In contrast, glutaraldehyde, which is a non-specific fixative that acts on all components of the cell, did reduce deformation and showed increased instances of tumbling, both of which are characteristic features of stiffened, or rigidified, cells. Because glutaraldehyde increases the effective viscosity of the cytoplasm and lipid membrane while diamide does not, one possible explanation for our results is that viscous effects in the cytoplasm and/or lipid membrane are a dominant factor in dictating dynamic responses of RBCs in pressure-driven flows. Finally, literature on the use of diamide as a stiffening agent is summarized, and provides supporting evidence for our conclusions.

Long-lived photogenerated states of alpha-oligothiophene-acridinium dyads have triplet character.

Photoinduced processes, leading to charge-transfer states with extended lifetimes, are of key importance for solar-energy-conversion applications. Utilizing external heavy-atom effect allowed us to photogenerate long-lived transients of electron donor-acceptor dyads. For an electron acceptor and a principal chromophore of the dyads, we selected N-methylacridinium, and for electron donors thiophene, bithiophene, and terthiophene were selected. While the photoinduced charge transfer, mediated by the investigated dyads, occurred in the picosecond time domain, the lifetime of the transients extended to the microsecond time domain. We ascribed the relatively long lifetimes to the triplet character of the observed transients. An increase in the size of the donor lowered the energy of the charge-transfer states of the dyads. When the energy level of the acridinium triplet lies below the energy level of the charge-transfer state, the locally excited triplet accounted for the long-lived transient. For the conjugates with charge-transfer states lying below all other excited states, the long-lived transients were, indeed, the charge-transfer species.

Oxygen tension-mediated erythrocyte membrane interactions regulate cerebral capillary hyperemia.

The tight coupling between cerebral blood flow and neural activity is a key feature of normal brain function and forms the basis of functional hyperemia. The mechanisms coupling neural activity to vascular responses, however, remain elusive despite decades of research. Recent studies have shown that cerebral functional hyperemia begins in capillaries, and red blood cells (RBCs) act as autonomous regulators of brain capillary perfusion. RBCs then respond to local changes of oxygen tension (PO2) and regulate their capillary velocity. Using ex vivo microfluidics and in vivo two-photon microscopy, we examined RBC capillary velocity as a function of PO2 and showed that deoxygenated hemoglobin and band 3 interactions on RBC membrane are the molecular switch that responds to local PO2 changes and controls RBC capillary velocity. Capillary hyperemia can be controlled by manipulating RBC properties independent of the neurovascular unit, providing an effective strategy to treat or prevent impaired functional hyperemia.

Enzymatic reactions in microfluidic devices: Michaelis-Menten kinetics.

Kinetic rate constants for enzymatic reactions are typically measured with a series of experiments at different substrate concentrations in a well-mixed container. Here we demonstrate a microfluidic technique for measuring Michaelis-Menten rate constants with only a single experiment. Enzyme and substrate are brought together in a coflow microfluidic device, and we establish analytically and numerically that the initial concentration of product scales with the distance x along the channel as x5/2. Measurements of the initial rate of product formation, combined with the quasi-steady rate of product formation further downstream, yield the rate constants. We corroborate the x5/2 scaling result experimentally using the bioluminescent reaction between ATP and luciferase/luciferin as a model system.

Enhanced separation of aged RBCs by designing channel cross section.

Prolonged storage will alter the biophysical properties of red blood cells (RBCs), and it decreases the quality of stored blood for blood transfusion. It has been known that less deformable aged RBCs can be separated by margination, but the recognition of the storage time from the separation efficiency of the stiff RBCs is still a challenge. In this study, we realized enhanced separation of aged RBCs from normal RBCs by controlling the channel cross section and demonstrated that the storage time can be deduced from the percentage of the separated RBCs in the stored RBCs. This separation technology helps to reveal the regulation of time on the RBC aging mechanism and offer a new method to separate stiffened cells with high efficiency.

Photoinduced electron transfer in arylacridinium conjugates in a solid glass matrix.

The photophysical properties of a series of 9-arylacridinium conjugates in solid glass matrices composed of sucrose octaacetate have been determined. The fluorescence of the charge-shift states is significantly enhanced because of the retardation of nonradiative pathways for back-electron transfer. Changes of more than 3 orders of magnitude in back-electron-transfer rates (sucrose octaacetate glass vs conventional solvents at room temperature) were observed. Transient spectra displayed long-lived charge-shift species in the microsecond time regime for thianthrene acridinium conjugates. The rate retardation is associated with slow solvation times for surrounding solvent layers in the solid matrix. The red-edge effect (excitation wavelength-dependent fluorescence) for the arylacridinium ions in solid glass confirms the microheterogeneity of the sucrose octaacetate medium.