Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers

The overall goal of the following experiment is to analyze carryover volume between adjacent liquid segments within a lab and tube device using magnetic beads. This is achieved by creating the device using hydrophobic tubing, loaded with a syringe pump to consist of multiple liquid segments separated by an air valve and incorporating both magnetic beads and a fluorescent dye into one of the liquids. As a second step, the fluorescent intensity of the adjacent liquid segments is measured using a microscope after each transfer of the magnetic beads is performed using an external magnet.

Next numerical analysis is done on these fluorescent intensity measurements in order to calculate the carryover volume of the magnetic beads. The results show that analysis of the phase change in a multi-component system comprised of two liquids is feasible based on the ability to use magnetic beads as a method of transferring microliter sized liquid volumes between adjacent reservoirs. So as I hope you'll see today, we are working with this very exciting and and simple technique involving the use of magnetic beads in transferring fluids within a simple tube as opposed to more complex conventional microfluidic circuits.

And we, we've applied it initially to the investigation of face base between admissible fluids, and we are now expanding that to other, other applications, including diagnostic analysis of whole blood separating proteins from red blood cells, urine analysis, immunoassays, and so on. The main advantage of this technique over other existing methods like using microfluidic channels, is that it's simple and has cost-effective fabrication. It offers high user variability between different experiment types and it has consistent results.

Though this method can provide insight into applications requiring the movement of a minute, peak volumes, it can also be applied to other systems like blood separation, drug targeting, and contrast to agents. To prepare the tube first cut tubing into 15 centimeter segments, hang the tube segments vertically using tape, placing a paper towel underneath the tubes to collect the excess fluoro polymer solution. Inject 100 microliters of the fluoro polymer solution into the top opening of each tube segment using a pipette such that it will come into contact with the entire circumference of the inner wall.

Allow tube segments to hang in place for one hour to remove any excess amount of fluoro polymer solution. Then place the tube segments into an oven at 100 degrees Celsius for one hour to ane the fluoro polymer coating layer. Next, prepare the diluted magnetic bead solution by placing a 20 milliliter sample vial onto a micro balance and zeroing the balance.

Agitate the magnetic bead solution container and then withdraw 0.6 milliliters using a micro pipet. Dispense the pipetted solution into the sample vial on the balance and dispense 0.4 milliliters of distilled water into the sample vial. Also, prepare the fluorescent dye by dissolving 0.02 weight percent of dye into deionized water and vortexing the solution for one minute.

To prepare the tubing device, insert a female lure lock connector onto one end of the tubing. Place the tube into a lure lock syringe that has a three milliliter volume and 0.1 milliliter graduation. Place the syringe into the syringe pump and set the feed rate at two milliliters per hour.

Vortex the container with a magnetic bead solution before inserting 20 microliters into the tube using syringe pump withdrawal. After the test chamber, liquid insertion is concluded, withdraw six microliters of air into the tube. This volume of air will later form a valve in between the two liquid segments.

Once air gap insertion is completed, begin withdrawal of 180 microliters of liquid with fluorescent die. Place a second female lure lock connector onto the other end of the tube and remove the tube device from the syringe. Then place lure lock caps on both ends of the device.

Take the initial fluorescence intensity measurement of the test chamber and reservoir using the inverted microscope. Place the device over the top of the cube magnet such that the magnetic beads all segregate to one area in the test chamber. Transfer the beads to the reservoir by moving the device over the top of the magnet.

Once the magnetic bead cluster is transferred through the air gap and into the reservoir, agitate the magnetic beads by placing the device over the top of the magnet and rotating to release the liquid being trapped within the cluster until homogenization of the reservoir has been completed. Then place the device over the top of the magnet such that the magnetic beads in the reservoir all segregate to one area. Transfer the magnetic bead cluster back to the test chamber.

Once the cluster reaches the test chamber, agitate the magnetic beads by placing the device over the top of the magnet and rotating it. Release the trapped fluorescent liquid within until homogenization of the test chamber has been completed. Take fluorescent intensity measurements of both the test chamber and reservoir using the inverted microscope.

Repeat these steps until both liquid segments converge to similar fluorescence intensities. To prepare the tubing device, insert a female lure lock onto one end of the tubing and place the tube into a lure lock syringe. Then place the syringe into the syringe pump and set the feed rate at two milliliters per hour.

Insert 20 microliters of a one-to-one magnetic beads in water to C 12 E five mixed solution into the tube using syringe pump withdrawal. After test chamber, liquid insertion is concluded, withdraw six microliters of air into the tube following air gap insertion. Begin withdrawal of 180 microliters of pure C 12 E five surfactant to set up the optics.

First, move the syringe pump with the tubing device such that the test chamber with magnetic beads is in focus With the stereo microscope, cross polarizers are utilized. To enhance the contrast between the precipitated solid phase and liquid phase, place a sheet of polarizer film on top of an LED light source. Slide the LED light source underneath the tube attached to the syringe pump.

Attach another polarizer film to the lens of the stereo microscope using tape, be sure that the two polarizer films have a 90 degree offset from each other. Place the cube magnet next to the test chamber while the magnet is mounted onto a stand. Once the magnetic beads form a cluster, begin pumping liquids into the tube at a feed rate of two milliliters per hour, such that the magnetic bead cluster is moved from the test chamber across the air gap and into the surfactant reservoir chamber.

When the magnetic bead cluster reaches the midpoint of the reservoir chamber, stop the pumping on the syringe pump. Move the cube magnet away from the tube for magnetic beads to separate and reduce the diffusion time of the liquid trapped in the magnetic bead cluster. Once the diffusion and phase change of the liquid is completed, place the magnet back to its former destination by the reservoir, so the magnetic beads form into a cluster.

Using the syringe pump, withdraw the liquids such that the magnetic bead cluster is transferred from the surfactant reservoir across the air gap and back into the test chamber. After the magnetic bead cluster reaches the midpoint of the test chamber, stop the syringe pump, move the cube magnet away from the tube. Once the diffusion and phase change of the liquid is completed, place the magnet back to its former destination by the reservoir, so the magnetic beads form into a cluster.

Repeat these steps until the test chamber displays a phase change. Shown here is an image of the magnetic beads water, and the non ionic surfactant C 12 E five inside of the hydrophobic tubing. During the initial magnetic bead transfer process, the beads are held into place using an external cube magnet.

While the liquids are pumped to the left here, the beads are exiting the test chamber and entering into the C 12 E five. Once the magnetic beads enter, the surfactant water begins to diffuse away from the magnetic bead structure. After the magnetic beads have been in the surfactant chamber for five seconds, only a small amount of phase change is seen at the ten second mark.

The magnetic beads are completely engulfed in the surfactant. The phase change continues through the 92nd mark at four minutes. The phase change increases at nine minutes.

The horizontal phase change begins to reduce while the structure grows vertically. Whereas at 17 minutes, the phase change has drastically shrunk in size at 25 minutes only a small amount of the phase change remains shown. Here is the initial condition of a water C 12 V five test chamber with magnetic beads in the device before surfactant was transferred.

After six roundtrip transfers, a phase change occurred. Conversely, with no magnetic beads in the device, no phase change was seen proving that the observed results were due to magnetic bead carryover rather than liquid. Sticking to the inner walls of the tubing While attempting this procedure.

It's important to have enough time to allow for the diffusion process to take place when transferring liquids between adjacent reservoirs Following this procedure. Other options like using an electromagnet vegetation can be incorporated into our system in order to answer additional questions like how to speed up the division process and how to minimize the manual manipulation. The, to put this in context, this very simple technique that involves essentially a plastic tube and and magnetic beads and an external magnet has been applied to the separation of different fluids and exploring their phase diagram space.

However, the versatility and simplicity of the technique will pay off in many other applications, primarily in the area of diagnostic analysis of different fluids. By using magnetic bees to transport small volumes either directly or through functionalization of the magnetic beads, we can analyze the components of the fluid sample. And this has applications in any areas.

For example, the separation and analysis of protein from red blood cells in whole blood, similarly in urine, and many other fluids that can be analyzed in this fashion using a simple technique without the complexity and cost associated with conventional microfluidic device fabrication.