Stream Butterfly Effect Free

[Map Rousch]

Asshown in the highly idealized schematic drawing below, wedesigned a system that would be capable of plugging directly into anair sampler, incorporating pumps P and flow control modulesinterconnecting three sequential microfluidic components: asedimentation device A to remove the large and rapidly sedimentinginterferent particles such as sand and dust, an electrokineticelement B to purify the target analyte, and an electrophoreticconcentrator C to increase the concentration of the analyte.

In the original design device, B was to be a zone electrophoresis(ZE) device. Such a device would have required precise positioning ofentrances and outlets to allow accurate operation. As it developed,it was possible to use isoelectric focusing (IEF) as an alternativeto ZE, thereby allowing devices B and C to be consolidated into asingle device.

Initial modeling of transport across channels in our laboratory,particularly before we gained access to Microcosm's FlumeCAD packageof software, was exclusively with 1D models of transport in channels.In these simulations all effects of dispersion of flow velocity wereignored, and the variation in flow rates along the optical path wereignored. Because the Poiseuille flow profile in ducts under positivedisplacement pumping is complex, the motion of particles from oneregion in the microfluidic device to another is also complex. Topredict separation and monitoring of chemicals and larger particlesin microchannels, the exact shape of the interdiffusion zone must beknown. For this reason more detailed modeling was undertaken. Thismodeling initially included just diffusion of species perpendicularto the flow direction.

It immediately became obvious that distribution in solute residence time in the channels leads to differential lateral diffusion near the walls and along the device midline. For example, if two fluids enter the device shown at left from the two input channels, their contents will interdiffuse, but that interdiffusion will appear to progress further in laminae immediately adjacent to the device upper and lower surfaces, than along the device midline. The green zone is a crude representation of the region in which interdiffusion between the two streams has occurred. We call this phenomenon the "butterfly effect".

A 2D computational model for simulation of analyte diffusion in the parallel flows of analytes as in H-filters and T-sensors was developed in our laboratory. This model was coded into MATLAB and run on a PC. It allowed for consideration of the true flow velocities in microchannels.

Note that there are other sources of complication, including thefact that at the contact point of the two fluids their velocity is 0.Fluid accelerates along the device midline until fully developed flowoccurs at a predictable distance downstream. These factors areparticularly important considering that we quantify theinterdiffusion zone by imaging. This line of modeling expanded toinclude not only diffusion but also chemical reaction and physicalcomplexation of multiple species in the channels; this allowed thedevelopment of quantitative models of such processes as immunoassaysin microchannels that agree very well with experimental data.

Additional modeling efforts in the group were directed towardprediction of electrophoresis and isoelectric focusing inmicrochannels. The first step in the modeling was to accuratelydepict the development of pH gradients in channels under theinfluence of electrochemical activity caused by passing a currentperpendicular to the flow (vide infra). This modeling involvedsimilar features to the diffusion-model above, except that it addedcreation of species at the channel wall, and electrophoresis of allcharged species in the presence of an imposed electric field. Thisled to some startlingly good agreement with experimental data.

Example of early 1D modeling of the positional dependence of pH and the Na+ ion concentration in a microchannel under the influence of current across that channel. No buffer was used in this simulation

A later time in the evolution of the pH and Na+ ion profiles in the same microchannel. Note that very sharp pH jump that persists at long times in the channel. This was confirmed experimentally as well.

The first separation device that studied was that originallyproposed--a simple microfluidic device that would remove the rapidlysedimenting and large particles from the feed stream bysedimentation. Many experiments were carried out in small systems andmonitored by optical microscopy. We then designed polymeric laminatedevices that were capable of separating rapidly sedimenting particlesfrom a stream of slowly sedimenting particles at flow rates (100L/sec) that would be compatible with existing commerciallyavailable air samples.

The initial device had an internal volume of about 100 L,and had walls of PMMA. It was demonstrated at the PI meeting inPittsburgh, where it worked for 4 hours as driven by one of the NMPVpumps (see picture later in the report).

Concept for sedimentation separation devices. The one at left was the concept for the original design. The one at center incorporated at least one gas permeable PDMS membrane for extraction of gases from the input solution.

As the operation of the first sedimentation separation deviceprogressed at the Pittsburgh PI meeting, it became filled withbubbles because of saturation of the fluid in the channel with air.These bubbles became pinned within the PMMA device and wereimpossible to dislodge. This resulted in "channeling" of the flowthrough the maze of bubbles, as well as a reduction in the effectivefluid internal volume of the device, accelerating the flow throughthe device. This set of problems was solved in the design of the nextgeneration of sedimentation separators, at the cost of a greatincrease in the complexity of the device. This 13-layer laminateseparated particles by sedimentation rate at 100 l/min andremoved any trapped gas to prevent disturbance of the flow byadherent bubbles.

These electrokinetic flow cells were mounted in custom holders onour 2 Zeiss inverted optical microscopes. These holders allow opticalmonitoring of the cells while connected to Kloehn computer-controlledsyringe pumps. Ultimately as many as 6 pumps could be connected to asingle device using conventional PEEK HPLC tubing.

Mounted on the inverted microscope is the test device, which can be viewed in epifluorescence or transilluminated. The replaceable "sandwich" of glass slide, cover slips and gold electrodes is clamped within the aluminum parts shown.

Viewed from above, note the red wires connected to the electrodes. PEEK tubing (Upchurch) is connected via 0-dead-volume connectors of our own design. Four of six possible inlet ports are connected to this device.

By adding electrodes to the walls of a T-sensor, we found that we could use electrical forces to move biological particles (from proteins to parasites) from side to side in channels. The simplest form of electrokinetic separation, zone electrophoresis (ZE) is based on differences in electrophoretic mobility of the particles to move analytes from one side of a channel to the other a different rates. This generally requires injection of the mixture in the channel along a line.

An intermediate version of the electrokinetic laminate device. Gold sputtered on Mylar was sandwiched between layers of Mylar and held together with pre-applied adhesive layers. This device is approximately the size of a standard microscope slide.

A later version of the electrokinetic laminate device in which the gold electrodes were replaced by palladium electrodes sandwiched between layers of Mylar, held together with pre-applied adhesive layers. Inter-electrode gap was either 2.5 or 1.27 mm.

Biological particles have surfaces with positive and negative titratable groups. Above a critical voltage, electrolysis of water at the surfaces of the electrodes creates a pH gradient across the channel perpendicular to the flow direction--alkaline at the cathode, acid at the anode. In isoelectric focusing (IEF), a particle migrates electrophoretically until it reaches fluid at its isoelectric pH (where it has no net charge). Isoelectric focusing is superior to "simple" electrophoresis because particles come to rest at a particular position in the channel, simplifying separation.

In a microfluidic channel between two electrodes, into which a buffer solution is pumped, we predicted that the initial uniform pH of the solution will be converted to a stable pH gradient as the rate of creation of acid and base would be just matched by their rates of recombination along the channel midline. The proteins, bacteria, or other (relatively large) charged particles would respond to both the field and the pH and equilibrate after the establishment of the pH gradient. They could then be sorted into separate outlet streams for analysis or storage. It was also possible that they could be identified optically by their position between the two electrodes.

We rapidly found that high concentrations of conventional pHindicator dyes could be used to monitor the pH in channels no thickerthan a few hundred m. Initial attempts to use sophisticatedratiometric fluorescent indicators like SNARF met with limitedsuccess. We ultimately developed a fully quantitative method formapping the channel pH using optical absorptive dyes such as thosedescribed below. We found that when the concentration of the bufferwas kept at or below about 1 mM, it was possible to generate pHgradients with wide ranges.

Formation of bubbles in the microchannels would have been a severeproblem. We found that as long as the potential between theelectrodes was kept below about 2.3V, bubbles did not form.Conversion from Au to Pd electrodes increased the current almost10-fold, with no apparent negative effects. This does not mean thatO2 and H2 were not present. In fact, we foundevidence of high partial pressures of O2 in the channels,particularly in static fluid.

Note that the pH gradient stabilized in the second window, andremained rock steady until the flow was distorted by the fact thatall the fluid was exiting through a single port. This was extremelyencouraging.

3a8082e126