Elementor #360 Dielectrophoresis

Dielectrophoresis, the DEP-well and the 3DEP

Dielectrophoresis (DEP) is a phenomenon related to electrophoresis, but with some important differences.

Electrophoresis, as its name suggests, measures the electric properties of a material – that is, its net charge.  It uses a DC voltage to attract anions and cations to the relevant counter-electrode, and the speed and direction of movement can be used to determine the electrical properties.

Dielectrophoresis is similar but uses a shaped AC field to determine the dielectric properties.  These affect the particle’s dipole, and allow us to determine the dielectric properties (such as resistance and capacitance).  We do this by observing particle motion at several AC frequencies, rather than at DC, and inferring the properties from analysing how the dipole changes at these different frequencies.

Complex particles such as cells exhibit different behaviours at different frequencies.  The measure of this is referred to as the DEP spectrum (plural: spectra) or, sometimes, the DEP fingerprint.  From this, it is possible (either manually or automatically) to fit a best-fit curve and hence infer the properties of the particles from that best-fit model.

Cell and Particle Characterisation

For particle characterisation, DEP-Well devices are closed at the base to form wells like those on a standard well plate. The samples are contained inside the wells and monitored by a reader from above or below. Compared to conventional micro-fabricated devices, extracting results from DEP-Wells is significantly easier. The sample cells are either attracted or repelled from the well side by an amount proportional to their polarisability. If the well is probed with a light beam, positive DEP pulls the cells to the wall and the well lightens (less absorbance) while negative DEP pushes the cells into the centre and the well darkens (more absorbance). The strength of the force is proportional to the rate at which the light intensity changes. By monitoring the changes in light intensity, several wells can be used in parallel to increase throughput. To increase flexibility further, DEP-well chips have addressable wells that allow the user to apply different frequencies to wells on the same chip. 

This flexible approach could also allows the manufacturing of a wide range of devices, from small DEP-Spectra Chips that analyse only one sample to DEP-Well plates up to a 1536 well-plate format. 

Dielectrophoretic multi-well plates are assay formats for cell characterisation in modern laboratories. The possibility of performing and analysing a large number of experiments in parallel has become an important tool in drug discovery and high-throughput screening. With DEP-Well technology, dielectrophoretic analysis can be integrated and combined with conventional and emerging well-plate systems to perform label-free dielectrophoretic high-throughput screening.

Mechanism

DEP-Wells are filled with bacterial or cell suspension.

  • Positive dielectrophoresis removes the cell from the bulk liquid and reduces light scattering.
  • Negative dielectrophoresis focuses particles in the centre of the well and increases light scattering.
  • The strength and direction of the force can be quantified by measuring the amount of transmitted light.
  • DEP-Well plates can be used to characterise particles and thereby identify changes in their electrophysiology during interactions with drugs.
  • This can rapidly identify processes such as drug resistance or apoptosis. Probing the DEP-Well at different field frequencies and comparing the amount of transmitted light allows measurement properties of the cell wall, cell membrane or cytoplasm. Applying different frequencies in parallel to a number of wells allows the measurement of the spectral response in only one step to measure interactions between a drug and cells against time. Mixtures of different cells can be quantified and separated by applying a defined sequence of field frequencies to the well.

 The 3DEP is designed to measure the DEP spectrum accurately and quickly using DEPwell technology. Experiments usually give results in the form of a curve with calculated characteristics in about 10 seconds.

The curve below demonstrates the effectiveness of the technique.Blue dots are obtained from an analysis of normal epithelial cells taken by a mouth swap. The red curve is of cells from a cancerous lesion in a patient’s mouth, again taken using a normal mouth swab. The differences are very apparent and it is hoped to exploit these to give a fast but effective test.

 

 

  1.  The starting level level relates to the conductance of the cell membrane
  2.  The frequency where the curve goes up  (called the lower dispersion) relates to the membrane capacitance,
  3.  The frequency where the curve goes back down (the upper dispersion) shows the intracellular conductivity,
  4.  The final level at high frequencies gives the intracellular permittivity.

Where data are quite noisy it is useful to average several experimental spectra together to improve the fit.

Applying the data to Electrophysiological analysis

The 3DEP enables the electrophysiology of cells to be better understood:

  • Membrane conductance describes the ability of a membrane to conduct charge, either through it or around it. It can be affected by the charges on the membrane surface, ion channel activity, and membrane morphology; the latter reason can be identified where there is a similar change in effective membrane capacitance, which is generally considered to be affected by membrane morphology almost exclusively. Multiplied by cell surface area, this gives the common whole-cell conductance parameter often used in cell electrophysiology.
    • Effective membrane capacitance describes the ability of the membrane to store charge. Although in general the membrane composition changes little (and therefore the amount of charge a small patch of membrane can store remains approximately constant), the model assumes the surface is billiard-ball smooth and spherical. Deviation from this – such as when the cell surface is covered in blebs, microvilli or invaginations – means that there is actually more membrane than expected, meaning more charge is stored. This appears in the model as an elevated value of effective membrane capacitance per unit area of cell. Naturally, this makes the parameter a very good indicator of cell surface morphology; if a cell actually were perfectly spherical we would anticipate a capacitance per unit area of 8mFm-2, and a proportional increase indicates a similar proportional increase in area. Recent work has suggested that certain cell mechanisms altering the composition of the membrane (such as glycosylation) can have an effect without a morphology change, but the most common explanation is morphological. This has been a very effecting method of discriminating between cells – including observing changes in differentiating stem cells, or differences in cancerous vs. healthy cells. It is also useful for identifying morphology changes when interpreting changes in membrane conductance. As with conductance, multiplying by area gives the whole-cell capacitance commonly used in cell electrophysiology
    •  

Intracellular conductivity is a measure of the ionic charge in the cytoplasm that can move freely and hence contribute to conduction. It is related to membrane potential; the cytoplasm conductivity is related to the sum of the positive and negative charges in the cell, whereas the membrane potential derives from the difference in numbers of positive and negative charge. As such it is a useful marker for change in ion concentrations, or ion channel activity. It is also a very useful marker for cell permeabilisation; when a cell membrane becomes permeable, the pores that appear allow connection between cell interior and exterior, causing a rapid drop in intracellular conductivity. It is also a useful marker for apoptosis, as one of the first events in the apoptotic cascade is ion efflux and water efflux, both of which can change the conductivity significantly (down in the former case, up in the latter). The actual change depends on the cell type and apoptosis-inducing agent, but the change is usually significant. If you divide 1 by the intracellular conductivity it gives the cytoplasm resistivity parameters commonly used in cell electrophysiology.

  • Multiple populations. Sometimes a sample will contain a mixture of two or more cell types with radically different electrical characteristics. Where two or three such populations exist it is possible to model the two of them. For example, a common even in the study of apoptosis is the formation of apoptotic bodies, which typically appear as a second population with a radius of 0.5-1µm and a lower-than-normal intracellular conductivity. The ratio of the two populations is indicative of their relative concentration, although this is not a direct comparative measure (as the populations have different sizes and absorbances). We have successfully modelled up to three populations in the same sample, though this requires very high quality spectra, which can be obtained by averaging together many different spectra using the software provided. Where populations are very heterogeneous, such as in clinical samples taken from oral swabs, it is not possible to determine the properties of all of the populations; however, the 3DEP software can be used to discriminate between different samples on the basis of different dispersion characteristics. This technique has been used to classify clinical samples as cancerous or non-cancerous, where determination of the actual electrophysiological parameters is not required.
  1.  The starting level level relates to the conductance of the cell membrane
  2.  The frequency where the curve goes up  (called the lower dispersion) relates to the membrane capacitance,
  3.  The frequency where the curve goes back down (the upper dispersion) shows the intracellular conductivity,
  4.  The final level at high frequencies gives the intracellular permittivity.

Where data are quite noisy it is useful to average several experimental spectra together to improve the fit.

Applying the data to Electrophysiological analysis

The 3DEP enables the electrophysiology of cells to be better understood:

  • Membrane conductance describes the ability of a membrane to conduct charge, either through it or around it. It can be affected by the charges on the membrane surface, ion channel activity, and membrane morphology; the latter reason can be identified where there is a similar change in effective membrane capacitance, which is generally considered to be affected by membrane morphology almost exclusively. Multiplied by cell surface area, this gives the common whole-cell conductance parameter often used in cell electrophysiology.
    • Effective membrane capacitance describes the ability of the membrane to store charge. Although in general the membrane composition changes little (and therefore the amount of charge a small patch of membrane can store remains approximately constant), the model assumes the surface is billiard-ball smooth and spherical. Deviation from this – such as when the cell surface is covered in blebs, microvilli or invaginations – means that there is actually more membrane than expected, meaning more charge is stored. This appears in the model as an elevated value of effective membrane capacitance per unit area of cell. Naturally, this makes the parameter a very good indicator of cell surface morphology; if a cell actually were perfectly spherical we would anticipate a capacitance per unit area of 8mFm-2, and a proportional increase indicates a similar proportional increase in area. Recent work has suggested that certain cell mechanisms altering the composition of the membrane (such as glycosylation) can have an effect without a morphology change, but the most common explanation is morphological. This has been a very effecting method of discriminating between cells – including observing changes in differentiating stem cells, or differences in cancerous vs. healthy cells. It is also useful for identifying morphology changes when interpreting changes in membrane conductance. As with conductance, multiplying by area gives the whole-cell capacitance commonly used in cell electrophysiology
    •  

Intracellular conductivity is a measure of the ionic charge in the cytoplasm that can move freely and hence contribute to conduction. It is related to membrane potential; the cytoplasm conductivity is related to the sum of the positive and negative charges in the cell, whereas the membrane potential derives from the difference in numbers of positive and negative charge. As such it is a useful marker for change in ion concentrations, or ion channel activity. It is also a very useful marker for cell permeabilisation; when a cell membrane becomes permeable, the pores that appear allow connection between cell interior and exterior, causing a rapid drop in intracellular conductivity. It is also a useful marker for apoptosis, as one of the first events in the apoptotic cascade is ion efflux and water efflux, both of which can change the conductivity significantly (down in the former case, up in the latter). The actual change depends on the cell type and apoptosis-inducing agent, but the change is usually significant. If you divide 1 by the intracellular conductivity it gives the cytoplasm resistivity parameters commonly used in cell electrophysiology.

  • Multiple populations. Sometimes a sample will contain a mixture of two or more cell types with radically different electrical characteristics. Where two or three such populations exist it is possible to model the two of them. For example, a common even in the study of apoptosis is the formation of apoptotic bodies, which typically appear as a second population with a radius of 0.5-1µm and a lower-than-normal intracellular conductivity. The ratio of the two populations is indicative of their relative concentration, although this is not a direct comparative measure (as the populations have different sizes and absorbances). We have successfully modelled up to three populations in the same sample, though this requires very high quality spectra, which can be obtained by averaging together many different spectra using the software provided. Where populations are very heterogeneous, such as in clinical samples taken from oral swabs, it is not possible to determine the properties of all of the populations; however, the 3DEP software can be used to discriminate between different samples on the basis of different dispersion characteristics. This technique has been used to classify clinical samples as cancerous or non-cancerous, where determination of the actual electrophysiological parameters is not required.

The DEParator

Referring back to the graph above, the axis on the left denotes whether the cells are experiencing positive DEP attracting them to the sides of the well (a positive value) or negative DEP when they are repelled into the centre of the well.

 The DEParator uses this effect using a DEPwell device to separate the cell types. In the example above, setting the DEP frequency between 10kHz and 20kHz results in the normal cells being taken out of the cell media flow by positive DEP, with the cancerous ones being repelled from the structure and taken away by the flow.

This illustrates the power of the combination of both units. The 3DEP analyses the characteristics of the wanted and unwanted cells, and the DEParator can then be used to select out those wanted for future work, with no need of labelling or other invasive or disruptive techniques

The DEP-Well Separator system within the DEParator is made of electrically-enabled, perforated sheets of electrode laminate. The perforations allow a suspension of particles to flow through the sheet, similar to the function of filter paper. However, unlike filter paper, particles are separated by different electrical properties and not size. This allows for the separation of particles of similar size and surface properties, but with different internal properties.

Within the laminate, the high field gradient generated by the electrodes at the wall attracts one type of particle and retains it. Other particles are repelled by the field gradient and are focused into the centre of the electrode channel and continue to flow through the well. After a collection period, the medium can be switched to a recovery medium by a switching off the field or a change in the field-frequency. The particles that were initially trapped are released into the recovery medium and can be collected. This regenerates the filter for re-use.

DEP-Well technology has a number of applications and can perform operations that are not possible with conventional filters.

A dielectrophoretic filter can separate particles based on their different dielectric properties. DEP-Well technology can be used in biotech applications to separate mixtures of cells or bacteria that have similar sizes and cannot be separated by conventional filters.

  • Examples include separation of live and dead cells.
  • DEP-Well technology enables a defined type of particle to be efficiently separated out of a mixture of particles and separated into a new batch of increased purity and concentration. This capability has numerous clinical applications, for example, in the separation of cancer cells from blood or other biological fluids.
  • Since particles are only trapped by the electric field, DEP-Well filters can be easily regenerated by switching the field off. This offers scope for replacing approaches relying upon conventional disposable filters, thereby reducing cost and waste management issues for operators.
  • Several DEP-Well filters in series allow a number of separations. Such a stack of filters allows the separation of highly heterogeneous particle mixtures into their individual particle components by sequential particle trapping and particle release phase actions.
  • The DEP-Well laminate enables filter fabrication at scales of size and throughput that are comparable to industrial scale filtration systems.

If you are interested in potentially using DEPwelll structures within your process please refer to our CONTRACT DEVELOPMENT page for further information