(398c) Bigger, Cheaper, Faster, More! DEP-Well Electrodes for Cell Electrophysiology

Dielectrophoresis has many advantages for cell electrophysiology.  By fitting the frequency-dependent DEP behavior using the Clausius-Mossotti factor, it is possible to determine the conductivity and permittivity of the membrane and cytoplasm, and thereby understand the static electrophysiology of cells by relating the parameters to membrane morphology, membrane potential, cytoplasmic resistance and ion channel activity. It is simple to use, and cheap to implement; it can give insights into cell behavior that might require very expensive processes to reproduce - such as automated patch clamp, which typically samples only 40 cells but costs hundreds of thousands of dollars. However, whilst we are two years from the 50^th anniversary of Herbert Pohl’s demonstration of dielectrophoretic separation of live and dead yeast, the use of dielectrophoresis beyond “hard-core” dielectrophoreticists – those whose primary research line is dielectrophoresis, rather than those who just use it as a research tool– remains woefully low. 

In order to find ways to bring DEP to the wider scientific community, we have tried to address the perceived shortcomings of the technique.  We have done this by acting converse to academic instinct, by making devices bigger rather than miniaturizing, and simpler rather than more complex, but in such a way as to make them faster to operate and more easy to use.  The resultant 3DEP DEP-well platform consists of multiple laminates of conductors (gold-plated copper) and insulators (polyamide) through which holes are drilled.  Wells are typically 1-2mm deep and 0.5-1.5mm in diameter, containing about 10 electrodes. Since wells are drilled orthogonally to the laminates, many wells can be drilled side-by-side; routing of power lines can enable wells to receive different signals to their neighbors, or all can be wired together. A base can be added to form a closed well for particle analysis, or the well can be left open as a flow-through separator. The simplicity of the devices makes the consumable cost very low; devices can be made  and the familiarity of the well-plate format makes it familiar to biologists; the cells resemble those found on 1536-well plates, and can be interfaced with standard fluid-handling robots.

When the field is applied, the cells move radially – either towards the center or the walls of the well – which can be tracked as a change in light intensity across the well.  Parallel powering allows twenty parallel measurements to be taken; we have developed an integrated reader system containing 20 independent signal generators capable of reaching 50MHz, plus optics and camera.  This allows a 20-point spectrum to be acquired in ten seconds, and the number of cells measured simultaneously (typically 20,000) means that data can be produced and fitted with Pearson coefficients greater than 0.99 with a single pass; data from multiple analyses can be averaged for very high accuracy data in a short period of time. 

Without a base, the chips can be used for DEP separation. The size of the wells increases throughput, whilst the lack of high-aspect-ratio channels reducing pressure problems for chips placed in line with other microfluidic components, and parallelization allows very high throughputs to be achieved.  However, the other great advantage of the device is speed; when used as a separator device, the devices can be built with hundreds of small-bore parallel wells, giving very high throughout (1 ml min^-1 is not infeasible) cell separation with minimal cell loss due to the absence for a requirement of microfluidic tubing.

Applications have already been demonstrated in cancer diagnostics, cell biology, drug discovery and electrophysiology.  For example, when used to analyze cells undergoing apoptosis using the 3DEP system to track proportions of healthy, affected and dead cells, it was found that IC50 measurements derived from DEP results correlated strongly with gold-standard MTT assays with significantly less preparation time and cost.  Other applications include low-cost, point-of-care oral cancer diagnosis; it has been benchmarked against cardiomyocytes for understanding how the cytoplasmic resistance affects cardiac signal conduction; applications including erythrocyte chronobiology, cancer stem cell biology and stratified medicine, will be covered in detail elsewhere.