Dielectrophoresis (DEP) is a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a non-uniform electric field. The particle is not required to be charged, as all particles exhibit dielectrophoretic activity in the presence of electric fields. The strength of the force depends on the medium, the electrical properties, shape and size of the particle, and the frequency of the electric field. Thus, fields of a particular frequency can manipulate particles with great selectivity. This has allowed for the separation of cells, and the orientation and manipulation of nanoparticles and nanowires. Microfluidic devices can be used to provide a dynamic fluidic environment for studying, manipulating, sorting and counting cells using DEP. In this method, conventional printed circuit boards (PCB) and a multi-channel microfluidic device are used for the non-contact manipulation of cells by employing DEP to manipulate polystyrene beads and cells in laminar flow fields for bioactuation, and to separate beads and cells.
A. Reagents and Preparation
Reagents and Equipment
- Function generator
- RF Power amplifier
- Polystyrene beads
- Cell samples
- Coverslip (80-130 μm thickness)
- Non-conductive cell media: 8.5% (w/v) sucrose, 0.3% (w/v) glucose in ddH20
- PDMS microfluidic channels
- PCB electrodes
- Electrode wires
Preparing PCB Electrodes
- Design PCB electrodes to the desired geometry to generate a non-uniform electrical field.
- Prepare pre-fabricated PCB electrodes by soldering 16-gauge wire to the end of each printed metal electrode. Place the wire onto the end of the electrode and hold the wire in place on the metal area of the PCB with the hot soldering iron to heat the wire. Feed a small amount of solder into the heated wire to fill the wire with solder. After the wire is filled with solder, remove the soldering iron, holding the wire in place while the solder cools.
- Repeat the soldering process for each electrical connection on the PCB.
Preparing Microfluidic Channels
- Prepare the Polydimethylsiloxane (PDMS)-based microfluidic channels are prepared using a PDMS elastomer. The master mould which defines the channels is usually created through standard microfabrication processes using a silicon wafer and SU-8 photoresist.
- Mix base compound to curing agent at a 10:1 ratio for 5 minutes.
- Pour the liquid PDMS onto the prefabricated SU-8 master mould and remove air bubbles by exposing liquid PDMS to vacuum for a few minutes. Repeat vacuum process if needed to completely remove all bubbles.
- Cure the PDMS at 70 °C for 2 hours.
- Remove PDMS slab with microfluidic channels from the wafer with a razor blade, careful not to break wafer.
- Punch holes for introducing fluids and cells into the microfluidic device. (Note: Syringe pumping, gravity or surface-tension based flow can all be used with DEP.)
- Inspect the microfluidic device to ensure it is free of dust and debris. Cleaning the PDMS can be easily achieved using 3M Scotch Magic Tape.
- Plasma bond the PDMS microfluidic channels to a clean no.0 thickness (80-130 μm) coverslip. Heat the coverslip-microfluidic assembly a 100 °C for a minimum of 15 min.
Assembly of Microfluidic Channels onto PCB Electrodes
- Place a small amount (approximately 10 μl) of mineral oil onto the PCB to ensure tight contact between the PCB and the coverslip. Note: An optional step for decreasing electrode visualisation is to coat the PCB surface with a very thin layer of black permanent marker or paint.
- Place the coverslip-microfluidic channel assembly onto the oiled PCB, with coverslip forming contact with the oil (coverslip down). Gently press the coverslip-microfluidic assembly down to ensure a good contact and to minimise air bubbles that can detract from cell and bead visualisation.
- Fill the microfluidic channels with DI water or low-conductivity media; the plasma bonding process facilitates the easy loading of aqueous solutions into the microfluidic channels by temporarily making the normally hydrophobic PDMS surface hydrophilic.
- Introduce cells and/or polystyrene beads into the channel reservoir.
- Connect the output of a function generator to the input of an AC power amplifier, then connect the output of the amplifier to the electrode wires. Cover all electrical wires and surfaces of the setup with electrical tape to protect users from the potential exposure to shock.
- Set the function generator to produce a sine wave output of 1.0-1.5 MHz. The amplitude of the output should be adjusted for the RF power amplifier to produce an output of 80-100V to the PCB. The RF power amplifier in this work requires an input voltage around 220-330 mV. To separate cells and beads, the laminar flow rate must be compliant with the DEP force to move the cells and beads within the main channel (width w = 100 μm, height h = 27 μm) into the target channels (width w = 100 μm, height h = 27 μm). Caution: consult with PCB manufacturer to determine the maximum operating voltages to avoid issues such as PCB overheating or melting. Check the equipment manufacturers' specifications for the function generator and power amplifier manufacturers to determine safe operating settings.
- Initiate DEP to sort cells and beads. Cells experience a positive-DEP whereas polystyrene beads experience a negative-DEP in a nonuniform electric field within the specified frequencies. This enables DEP-force-activated bioactuation and separation of cells and other particles within microfluidic devices using affordable and reusable PCBs as electrodes.
- When DEP is effective at actuating cells or particles, a robust alignment within non-uniform electrical fields is observed for static baths or slow fluid velocities. Under conditions of higher fluid velocities, the cell or particle behavior depends on the relative orientation of the axial flow to the electrical field and the flow velocity. In addition, the cell and particle behaviors are also dependent upon the electrical field strength and the degree of non-uniformity within the electrical field. Typical behaviors of cells or particles include 'pearl chains,' cycling, stalling, or turning.
- Conditions that compromise or abolish DEP include the presence of salts or other ionic molecules in the fluid, weak electric field strength, excessive flow velocities, coverslips that are too thick, or conductive solutions between the coverslip and PCB electrodes (e.g. a cracked coverslip can induce the mixing of water and mineral oil).
- The DEP force can be estimated by measuring the velocity of the particle, manipulated by DEP in static fluid. Due to the small inertia of the particle and highly viscous environment of microfluidic channel, the hydrodynamic drag force will be equal to, but in opposite direction with the DEP force. The hydrodynamic drag force can be calculated with following equation: Fdrag = 6πηRv