Development of a 3D Graphene Electrode Dielectrophoretic Device

This protocol demonstrates procedures to fabricate a 3D laminated graphene paper, electrode micro device, and experimental methods to manipulate poly starring beads or biological cells by DI electrophoresis or DEP within the device. From this point on, we will refer to dielectrophoresis as DEP. The fabrication of devices accomplished by first alternately stacking in layers of 50 micron graphing paper and N minus one layers of 100 micron pressure sensitive polymer tape, yielding half signal and half ground electrodes.

Layers are pressed together, trimmed, then the micro wellis drilled through the structure. Finally, two copper leads are attached to the two ends of the device Using silver epoxy. The second step is to prepare DEP media with desired conductivity.

Poly starring beads are then suspended in this medium solution at certain Volume ratio. The third and last step is loading the device with the prepared bead suspension, mounting it onto the microscope stage, connecting the device to a function generator, and eventually initiating the DEP. In the end use microscope to record the DEP responses and study the cell or particle properties by further analysis.

Graphene is A versatile material utilized for its strength as well as its thermal and electrical properties. This protocol utilizes an inexpensive graphene paper as a conductive electrode layer in a three dimensional di electrophoretic micro device that is capable of discerning particles and cells for applications such as blood typing or medical diagnostics of disease cells. Advantages of this graphing paper electrode micro device include easier fabrication, higher sample therapeutic, and the geometrical versatility.

The 3D non-uniform election field can manipulate large sample volumes with DP forces that are unique in their ability to discern polarizable particles and cells based on their intrinsic chemical and morphological properties. Assemble the laminated device with graphene paper And tape. Here we use 75 micron thick hydrophobic graphene paper from a commercial manufacturer and 100 micron thick pressure sensitive double-sided tape.

First cut graphene paper with a surgical blade and straight edge ruler into 6.7 centimeters by 1.5 centimeter rectangles and use scissors to cut double-sided pressure sensitive tape into five 1.3 centimeters by approximately five centimeter strips. Lay the first layer of graphene paper on a clean glass slide. Slowly cover one end of the graphene paper with one strip of tape, leaving approximately two millimeter margarine to ensure an insulation between any two adjacent graphene paper layers.

Place the second layer of graphene paper on the top of the tape. Offset to the first layer of graphene paper. Apply moderate pressure on top to assure good ceiling between layers.

Repeat above two steps for the remaining layers, ensuring the top and bottom layers are both graphene paper. Use scissors to remove the excess tape from the device edges leaving a small approximately one millimeter margarine to ensure sealed insulation between graphing paper layers. Perform a quick insulation test using a multimeter position the positive and negative probes on the electrical leads on opposite sides of the device.

High resistance of approximately hundreds of kilo ohms indicates good insulation between layers. Now the laminated structure is Ready for micro well drilling. We Use a micro and mill to drill a micro well in the laminated structure.

Here we use a micro and nil with diameter 700 micron and flute length close to 2.1 millimeter. The choice of micro nil is according to the diameter of the micro well and the device thickness Immobilize the laminated structure on the micro drilling stage with clamps position the end mill closely on top of the structure with computerized controls start and point pressurized air over the device. Start spinning the end mill and then slowly lower the end mill into and through the center of the laminated structure.

Stop at a distance slightly greater than the laminated structure depth to ensure thorough removal of debris from the new hole. Move the end mill up and down several times through the micro. Well to smooth the inside wall.

The inner surface of micro well should be as vertical and clean as possible For optimal electric field gradients and light passage through the Micro well cut standard 32 gauge copper wires Into two three centimeter long pieces and fold two right angles at two centimeters. Manually mix approximately 0.75 millimeters of part A and approximately 0.75 millimeters. Part B of silver conductive epoxy.

In a small container, attach the wires by carefully applying silver epoxy to the tips of and in between all three graphene paper layers to ensure good contact between layers. Next, place the one centimeter wire and in between any two layers, softly squeeze the layers to remove excess epoxy and ensure good electrical contact. Repeat for the other side of the laminated structure.

Place the whole device on a rack and oven to dry overnight at 70 degrees Celsius and one atmospheric pressure. Remove the device from the oven and verify electrical insulation between the electrode Leads. Here we make solution media Of a spectrum of conductivities by mixing different ratios of pre-made 290 milli osmolar per liter minol solution and PBS.

These are simple solutions chosen to crudely mimic cell culture buffer. A linear correlation exists between the volume ratio of these two solutions and the resulting solution conductivity, mixed polystar beads or biological cells with prepare conductivity media at one to 100, volume by volume ratio by pipetting. 10 microliters of polys starring beads and one milliliter a media solution into a sample vial.

Mix the sample thoroughly by Vortexing. Clamp the device onto a glass Slide with moderate pressure using modified paper clamps or equivalent. The footing should be close enough to the micro well to seal the laminated structure to the glass slide preventing any sample leakage.

The clamp tightness should be controlled to not deform the device negatively altering the well geometry and light path score 0.5 millimeter thick covered glass slide with a diamond tip glass cutter wide enough to just cover the micro well crack covered glass to size and set aside. Using this micro syringe, slowly inject approximately one microliter of the polystyrene bead suspension into the micro well. To avoid introducing any bubbles, repeat injection if bubbles appear and use care to not damage micro well walls with the sharp needle slightly overfill the micro well and immediately slide prepared glass cover over the micro well to remove excess fluid preventive evaporation and ensure reproducible volumes.

For each experiment, secure the complete laminated micro device to the microscope stage and attach a function generator. Alligator clips to the devices to copper leads and the microscope software adjust focus to clearly view the micro well. In the center of screen here, a hundred hertz to 10 megahertz and 15 volts.

Peak tope signal were applied to the device. Beads were observed in the microwell at 10 x magnification at one to 200 microns above the glass slide surface. Click button to start camera recording and acquisition.

Initiate function generator signal two seconds after starting camera recording. Images are digitally saved at one to five frames per second for approximately five minutes for later intensity analysis. Upon experiment completion, remove the device and dismantle the clamps.

Clean both the glass slide and device in soapy water. Then rinse well In the absence of an electrical field, particles Solely sediment to the device bottom due to gravity. An electric fields around one kilohertz positive DEP behavior signified by movement up the field gradient closer to the graphene paper.

Electrodes at the micro well edges is observed at electric fields above 100 kilohertz and into the megahertz range. Negative DEP signified by bead movement down electric field gradient to the micro well center is observed. This 3D graphene paper electrode micro device yielded experimental results in a close agreement with both theoretically predictive behavior and the previously reported experimental results.

Device fabrication is extremely Versatile. The protocols provided can be easily adapted for devices with more layers or other materials. This video details a batch wise 3D DEP micro device.

However, in future applications, particle or cell suspensions could be continuously flowed through the micro well of the device to achieve higher throughput. DEP sorting, one example of versatility is that water absorbing graphene paper could simultaneously function as an electrode and filter for particle or cell concentrators or even as electro kinetic biochemical sensors. After watching this video, the viewers should have a good understanding of how to fabricate a multi-layer graphene paper, electrode micro device, as well as how to operate the device to conduct particle or cell Experiments.