Our research is focused on the study of cell biomechanics of circulating cells, such as human red blood cells. We leverage electrokinetics, microfluidics and material science to understand the mechanical origins of damage in cell membranes, as well as the mechanisms underlying the shortened lifespan of blood cells in certain diseases. Fatigue study of biological cells is challenging.
It requires application of cyclic loads to cell membranes and tracking the deformation in individual cells. Using amplitude-shift keying, or ASK, to modulate electro deformation behavior of red blood cells, we could quantify how cell deformability changes align with the loading cycles. We demonstrated for the first time that red blood cells'membranes can be degraded by cyclic stretching alone.
Our protocol utilizes dielectrophoresis to move cells to the electrode edges for electrode deformation measurement. It does not require any stabilization technique and can be used for cell suspension directly. Using interdigitated micro electrodes allows us to measure tens of cells in a single field of view.
To prepare the dielectrophoresis, or DEP medium, weigh 12.75 grams of sucrose and 0.45 grams of dextrose using a scale. Dissolve both powders in a solution of 150 milliliters of deionized water and 3.5 milliliters of PBS. Using a low range conductivity tester, measure the conductivity to ensure it is 0.04 Siemens per meter.
Using an osmometer, confirm that the osmolarity is within the normal range of the blood plasma. Store the prepared DEP medium at four degrees Celsius. In a 15 milliliter tube, dissolve 0.5 grams of bovine serum albumin in 10 milliliters of the DEP medium and mix thoroughly using a vortex mixer to prepare the devices'prime medium.
To begin tape the SU-8 master silicon wafer for the microfluidic channel design on the inside of a plastic 14 centimeter Petri dish and clean it with nitrogen gas. Weigh appropriate amounts of polydimethylsiloxane, or PDMS base and PDMS curing agent, in a paper cup. Using a wooden spatula, mix the two until the mixture becomes cloudy white in color.
Pour the PDMS mixture into the plastic Petri dish containing the silicon wafer. Then place the Petri dish into a vacuum desiccator equipped with a three-way stopcock. Turn the valve of the stopcock to connect the vacuum to the desiccate chamber to remove air bubbles from the mixture.
Once all air bubbles are removed from the features of the channels, place the Petri dish inside an oven at 70 degrees Celsius for four hours. After the Petri dish cools to room temperature, place it on a cutting mat. Using a scalpel, cut out the portion of the PDMS above the silicon wafer.
Place the cutout PDMS between two sheets of the lab wrapping film. The gap between the indent of the microchannel and the film helps to locate the inlet and outlet of the microfluidic channel. Next, using a razor blade, cut out an individual channel from the large PDMS.
And with respective biopsy punches, make appropriate inlet and outlet holes in the channel. Place the whole punched channel with the channel side facing up onto a clean glass slide. Place a glass substrate containing thin film indium tin oxide, or ITO, interdigitated electrodes on the same slide with the electrodes facing up.
Then gently place the glass slide into a plasma cleaner. After closing the gas valve and switching on the pump, wait for two minutes to obtain a sensor reading of 600 to 800 millitorr. Next, turn on the power switch and wait 30 seconds before turning the RF power knob from low to high and waiting for one minute.
Then to switch off the set, follow the reverse sequence. Immediately after opening the chamber of the plasma cleaner, lift and rotate the PDMS 180 degrees so that the channel side is facing down. Place the channel on the top of the ITO substrate to initiate the bonding process.
Using tweezers, gently press down on the corners of the PDMS for about three seconds. Load the priming medium into a one milliliter syringe with a 23 gauge needle. Slowly and carefully wet the channel by inserting the needle straight into the inlet well before releasing the medium without introducing air bubbles.
After incubating for at least three minutes, remove the prime medium using a 10 microliter pipette tip. Finally, wash the channel with dielectrophoresis medium three times by inserting the medium into the channel. To begin wash 20 microliters of the whole blood by centrifuging it with one milliliter of PBS at 268G for three minutes.
Then discard the supernatant. Resuspend the resultant red blood cells, or RBCs, in one milliliter of PBS by gentle pipetting. Wash the RBCs again and discard the supernatant as demonstrated previously.
Using a 10 microliter micro pipette tip, extract five microliters of RBC pellet and fully dispense it into one milliliter of the dielectrophoresis or DEP medium. Wash the cells and discard the supernatant as demonstrated. Resuspend the pelleted RBCs in one milliliter of DEP medium by gentle pipetting.
After washing the cells and discarding the supernatant, pipette two microliters of RBC pellet into 500 microliters of DEP medium. Confirm the concentration of the resulting cell suspension using a standard cell counting slide. To begin, place the microfluidic device into the bottom part of the test fixture.
Align the top part of the fixture onto the device, and using two sets of nylon screws and nuts, assemble the two parts. Place the test fixture on the microscope stage. Locate one desired set of electrodes under the microscope, connect the corresponding pair of electrode wires that match the located electrode set to the output terminal of the function generator.
Remove five microliters of the dielectrophoresis or DEP medium from the three millimeter inlet of the microfluidic channel. Using a 10 microliter pipette tip, slowly load five microliters of the prepared red blood cell or RBC suspension into the inlet and allow the cells to settle for one minute. Observe the channel under 20X magnification.
Press the sine button and define a sine wave with an amplitude of two volts root mean square at a frequency of three megahertz. Press the mod button to enable modulation. Press the type option to change the wave mode into amplitude-shift keying or ASK.
Set the modulation frequency to 250 millihertz corresponding to a four second loading/unloading period. Turn on the output of the function generator and record a one minute video every 10 minutes at 30 frames per second. The RBCs spontaneously responded to the electrical excitation by moving towards edges of electrodes with higher field strength.
Under the on-keying phase, RBCs were stretched due to electro deformation, while under the off-keying phase RBCs were relaxed. When tracking individual RBCs during the one hour fatigue testing, a gradual decrease in cellular deformation was observed.
