The overall goal of this model system, is to mimic the geometry and breathing motion of the Pulmonary Acinus for studying Acinar airflow patterns and airborne micro-particle trajectories. This method can help answer key questions in the field of Acinar Transport Phenomena. Such as, the effects of gravity, drug, and diffusion on particle deposition outcomes.
The main advantage of this technique is that experiments are done inside a one-to-one scale model rather than a scaled up model. This allows for accurate observation of particle Brownian motion. The implications of this technique extend towards inhalation therapy, because a better understanding of aerosolized drug deposition can be achieved.
To begin, use deep reactive ion etching of a silicon on insulator wafer to fabricate a master silicon wafer as described previously. Next, mix the PDMS and curing agent at a ten-to-one weight ratio inside a clean small container. Degas the mixture in a desiccator under vacuum until all of the air bubbles are removed.
Place the master wafer into a petri dish, and pour the degassed PDMS mixture to a height of approximately one millimeter above the master wafer. Degas the poured PDMS once again for at least 40 minutes, until all air bubbles are removed above the wafer, and bubbles below the wafer are minimized. Make sure that the wafer is as close as possible to the bottom of the plate.
If necessary, press the wafer gently to the bottom using two stirring sticks and degas once again. Then, bake the PDMS at 65 degrees Celsius for 20 minutes in a natural convection oven. While the PDMS is baking, file the barrel section of a plastic two milliliter syringe, using a fine grit sand paper to improve its adherence to PDMS.
In addition, use the sand paper to flatten the base of the syringe barrel by placing the sand paper on a flat surface and sliding the base of the syringe barrel on top of it. Clean off any debris from the syringe using pressurized air. Then place the barrel section of the syringe on top of the first PDMS layer with the large opening facing the surface of the PDMS.
Pour a second layer of PDMS on top of the first one, to a height of about five millimeters, while ensuring that the PDMS does not enter the barrel of the syringe. Then, degas the PDMS once again. Bake the entire set-up at 65 degrees Celsius for at least two hours in a natural convection oven to harden the PDMS.
Cut through the PDMS mold around the patterned region of the master wafer using a scalpel. While cutting, the scalpel should weakly touch the surface of the wafer. Then, gently insert a thin tool, such as wafer forceps, in the notch created by the scalpel, and peel off the PDMS cast from the master wafer.
Place the cast on a soft surface covered with aluminum foil, so that the patterned side faces up and punch a hole in the PDMS at the chamber inlet and channel inlet, using a one millimeter biopsy punch. Next, add some of the PDMS with curing agent to a clean glass slide and spin coat a thin layer at 3, 000 RPM's for 30 seconds. Then, bake the slide at 65 degrees Celsius for at least one hour.
Clean the slide and PDMS cast using scotch tape. Treat the surfaces of the coated glass slide and the molded PDMS with oxygen plasma using a corona treater. Note that each surface should be treated for at least one minute.
Then, gently press the surfaces together and bake them overnight at 65 degrees Celsius. Mix water suspended fluorescent polystyrene particles with water and glycerol in a glass vile to obtain a 64-to-36 volume to volume ratio of glycerol to water containing 0.25%particles by weight. Next, place a drop of the glycerol solution on top of the channel inlet and a drop of DI water on the chamber inlet.
Then, set the apparatus inside a desiccator and pull a vacuum for about five minutes. Before releasing the vacuum, wait for the bubbles that form in the drops of glycerol solution and DI water to pop. Upon vacuum release, the liquids are drawn into the voids inside the device.
If residual air remains inside the channels, eliminate it by applying external pressure on the fluids using a syringe and allowing the air to diffuse into the PDMS. Next, inject about two millimeters of deionized water into the top chamber until it is fully filled with water. Then, cover the chamber with a 19 gauge blunt syringe tip.
Cut the tip of another blunt 19 gauge syringe tip and insert this tip into the side chamber inlet. Connect both syringe tips to a one milliliter syringe using thin, Teflon tubing and a t-shaped connector. Make sure that the one milliliter syringe, Teflon tubing, t-shaped connector, and top chamber are all filled with water, without any bubbles.
Then, connect the one milliliter syringe to a syringe pump and program it to mimic a quiet title breathing cycle as described in the accompanying text protocol. In order to obtain micro-particle vilo symmetry images, place the device on to the stage of an inverted microscope. Then, turn on the double pulsed Nd:YAG laser and load the image analysis software on the computer.
While the device is being actuated, obtain a series of nine-to-twelve phase locked double frame images of the particle seeded flow, using a micro-particle image vilo symmetry system in accordance with the manufacturers'specifications. To achieve phase locked double frame images, acquire a double frame series at 10 hertz. Then, reorganize the data so that all framed pairs that are separated by a full cycle time form a new time series.
Use the sum of correlation algorithm to compute phase locked velocity vector map so the resulting flow field from the image series. Repeat this process several times with varying lag times between the first and second frames of each frame pair for resolving different flow regions inside the alveolar cavity. Next, use a data analysis program to stitch together the individual flow maps into a complete and high detailed map of flow patterns by averaging overlapping data points.
A critical feature of the micro-fluidic acinar platform presented here, is its ability to reproduce physiologically realistic breathing motions that give rise to physiological flow profiles and velocities within acinar ducts and within alveoli. Flow velocity profiles across the width of the channels show a steady drop of flow rates towards deeper acinar generations. Flow profiles near and within alveolar cavities show that flow magnitudes drop steeply along the opening of alveoli.
Resulting in flow velocity that are two to three orders of magnitudes slower inside alveoli compared to the ducts. In addition, flow patterns change considerably with increasing acinar generation. While generation one features a re-circulation zone, which roughly coincides with the center of the alveolas, generation three's characterized by a re-circulation zone which is shifted toward the proximal side of alveolas with a more open stream line pattern.
Finally, radial stream lines with no re-circulation zone are observed in device generation five. Once mastered, this technique can be done in a few hours if it is performed properly. Following this procedure, which enables flow visualization in acinar geometries, the platform can be used to track single airborne particles in order to explore the dynamics and the position of inhaled particles.
