Continuous Flow Microfluidic Separations

The goal of this research is to investigate novel methods for separating microscopic particles. Sorting complex mixtures of microscopic particles into subsets is a common process in many fields of research such as pharmaceuticals, environmental science, food science, military, cosmetics, agriculture and bio-medicine. This sorting step is often the most time consuming and laborious part of sample preparation. Microfluidics are especially appropriate for separations at this scale because they offer significant advantages over conventional cell separation techniques such as reduced labor, less reagent usage, improved repeatability, smaller lab footprint, lower capital equipment cost and small sample sizes.;We first investigated an aqueous two phase system (ATPS) as a means to separate erythrocytes from leukocytes. The goal was to reduce the erythrocyte population that can interfere with other assays which typically target the much rarer leukocytes. Each cell type has particular surface properties and net charge; within an ATPS each phase has a unique surface energy and charge. When a heterogeneous mixture of cells is placed in an ATPS, the distinctive characteristics of each cell determine its interaction with the phases, resulting in cell separation by type. The laminar flow characteristic of microfluidic devices was used to create zero, one, and two stable interfaces between the polymer streams. Three different patterns of polymer streams were evaluated for their effectiveness in concentrating leukocytes: immiscible PEG-PEG-Dex, immiscible Dex-PEG-Dex, and miscible PEG-PBS-Dex. The most effective configuration was the Dex-PEG-Dex stream pattern which on average increased the ratio of leukocytes to erythrocytes by a factor of 9.13 over unconcentrated blood.;Particle flux is an important factor for many microfluidics applications (e.g., separations) and up to this point little attention has been paid to particle introduction. Delivery of a constant flux of particles into a microfluidic device is a challenge. Injecting particles from syringe pumps, through small bore tubing, into microfluidic devices produces a varying and complex particle flux over time. A few methods have been reported in the literature for introducing particles into microfluidic devices. We characterized some of these methods and found that particle flux varied widely over time, by as much as seven fold. We then presented some useful methods for producing a constant rate of particles to a microfluidic chip. We found the most effective method for providing a constant flux was to use a density-matching solution and to eliminate all tubing.;The most common method for separating leukocytes from whole blood is through chemical lysis. However, lysis is a harsh method of separation and can alter the results of downstream analyses. In order to minimize the effect of lysis on the leukocytes we fabricated a microfluidic cytometer to characterize erythrocyte lysis kinetics. Diffusive transport coupled with laminar flow was used to control the concentration and exposure time of the lysis reagent to erythrocytes. Standard clinical practice is to expose erythrocytes to lysis reagent for 10 min. Under optimum conditions we achieved complete erythrocyte lysis of a blood sample in 0.7 seconds. A maximum lysis reaction rate of 1.55 sec-1 was extrapolated from the data. Lysis began after 0.2 seconds and could be initiated with a lysis reagent concentration of 1.0% (68.5 mM).;A second particle we focused on was microbubbles. Microbubbles are useful in contrast enhancement of ultrasound imaging and there is potential for other uses such as drug delivery. These applications would benefit if the microbubbles were of uniform size. However, techniques for making microbubbles produce a wide range of sizes or bubbles of the same size but of insufficient quantity. Microfluidic particle separations using secondary flows were investigated for sorting microbubbles. These methods have been shown to be effective at separating solid particles but were found to damage the bubbles. An alternative means of generating secondary flows to avoid damage to bubbles was proposed.;Finally, we fabricated a microfluidic device that sorted microbubbles based on their buoyancy to capture a subset of bubbles of 6 -- 7.5 mum in diameter. Microbubbles with a wide range of diameters were introduced to the device and rose through a fixed width buffer stream at a rate proportional to their diameter. Traps at the top of the channel were located at distances from the bubble introduction point so as to collect bubbles of specific sizes. The bubbles collected over six experiments showed an average size of 6.36 mum with a poly-dispersity index of 11.0%.