Generation of controllable gaseous H2S concentrations using microfluidics.

Hydrogen sulfide (H2S) plays an important role as an intercellular and intracellular signaling molecule, yet its targets are not well understood. As a molecule it easily evaporates and it is hard to acquire stable concentration for in vitro studies, constituting a major problem for the field to identify its downstream targets and function. Here we develop a microfluidic system that can provide consistent and controllable H2S levels in contrast to the current method of delivering large bolus doses to cells. The system relies on the permeability of H2S gas through a polydimethylsiloxane thin membrane. A hydrogen sulfide donor, sodium hydrosulfide, is perfused in the microchannels below the gas permeable membrane and gaseous H2S diffuses across the membrane, providing a stable concentration for up to 5 hours. Using electrochemical sensors within 3 ppm range, we found that H2S concentration was dependent on two parameters, the concentration of H2S donor, sodium hydrosulfide and the flow rate of the solution in the microchannels. Additionally, different H2S concentration profiles can be obtained by alternating the flow rate, providing an easy means to control the H2S concentration. Our approach constitutes a unique method for H2S delivery for in vitro and ex vivo studies and is ideally suited to identify novel biological processes and cellular mechanisms regulated by H2S.

Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density Lipoproteins.

OBJECTIVE: Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process. APPROACH AND RESULTS: Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells. CONCLUSIONS: DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.

Bubble removal with the use of a vacuum pressure generated by a converging-diverging nozzle.

Bubbles are an intrinsic problem in microfluidic devices and they can appear during the initial filling of the device or during operation. This report presents a generalizable technique to extract bubbles from microfluidic networks using an adjacent microfluidic negative pressure network over the entire microfluidic channel network design. We implement this technique by superimposing a network of parallel microchannels with a vacuum microfluidic channel and characterize the bubble extraction rates as a function of negative pressure applied. In addition, we generate negative pressure via a converging-diverging (CD) nozzle, which only requires inlet gas pressure to operate. Air bubbles generated during the initial liquid filling of the microfluidic network are removed within seconds and their volume extraction rate is calculated. This miniaturized vacuum source can achieve a vacuum pressure of 7.23 psi which corresponds to a bubble extraction rate of 9.84 pL/s, in the microfluidic channels we characterized. Finally, as proof of concept it is shown that the bubble removal system enables bubble removal on difficult to fill microfluidic channels such as circular or triangular shaped channels. This method can be easily integrated into many microfluidic experimental protocols.

Vacuum pressure generation via microfabricated converging-diverging nozzles for operation of automated pneumatic logic.

Microfluidic devices with integrated pneumatic logic enable automated fluid handling without requiring external control instruments. These chips offer the additional advantage that they may be powered by vacuum and do not require an electricity source. This work describes a microfluidic converging-diverging (CD) nozzle optimized to generate vacuum at low input pressures, making it suitable for microfluidic applications including powering integrated pneumatic logic. It was found that efficient vacuum pressure was generated for high aspect ratios of the CD nozzle constriction (or throat) width to height and diverging angle of 3.6(o). In specific, for an inlet pressure of 42.2 psia (290.8 kPa) and a volumetric flow rate of approximately 1700 sccm, a vacuum pressure of 8.03 psia (55.3 kPa) was generated. To demonstrate the capabilities of our converging - diverging nozzle device, we connected it to a vacuum powered peristaltic pump driven by integrated pneumatic logic and obtained tunable flow rates from 0 to 130 µL/min. Finally, we demonstrate a proof of concept system for use where electricity and vacuum pressure are not readily available by powering a CD nozzle with a bicycle tire pump and pressure regulator. This system is able to produce a stable vacuum sufficient to drive pneumatic logic, and could be applied to power automated microfluidics in limited resource settings.