Patent Description:
The counting of particles flowing through a restriction, or channel, using the Coulter principle is a well-known technique. In a Coulter counter, the electrical impedance of a fluid along a channel is altered by the presence of a particle, such as a biological cell. Characteristics of the interrogating electrical signal, such as its frequency, may be modulated to provide information about the particle. Devices employing such measurement techniques may be referred to as electrical impedance spectrometry devices or impedance cytometry devices.

<NPL>) describes a microfluidic electrical impedance spectroscopy device (impedance cytometry device). The device comprises metal electrodes and fluidic channels of micro-scale dimensions to enable the analysis of single biological cells.

<NPL>) describes a microfluidic electrical impedance spectroscopy device (impedance cytometry device) in which planar metal electrodes are formed on one or both sides of a micro-channel.

<CIT>) describes a microfluidic electrical impedance spectroscopy device (impedance cytometry device) in which sensing and driving electrodes are formed on opposite sides of a micro-channel and electrically controlled so as to increase the accuracy of the measurement.

<CIT>) describes a microfluidic electrical impedance spectroscopy device (impedance cytometry device) in which electrodes are formed on opposite sides of a micro-channel and electrically controlled so as to reduce the dependency of the measured signal on the position of the particle in the channel.

A disadvantage of conventional microfluidic impedance cytometry devices is that to increase the accuracy of the measurement it is necessary to increase the complexity of the device structure. In particular, to reduce the dependency of the measured impedance signal on the position of the particle in the channel, conventional devices employ electrodes on opposite sides of the channel. Such two-layer electrode structures result in a significant increase in the manufacturing and assembly cost of the device compared to co-planar structures in which electrodes are formed only on a single side of the channel.

<NPL>, a five-electrode coplanar chip enabling accurate particle sizing without the need for focusing. The chip layout is designed to provide a peculiar signal shape from which a new metric correlating with particle trajectory can be extracted.

Aspects of the present invention provide an electrowetting on dielectric (EWOD) device as defined in claim <NUM> or claim <NUM>. The device includes an impedance cytometry device incorporated into its substrate assembly, or into one of its substrate assemblies.

The present invention provides a structure for a microfluidic electrical impedance spectroscopy device, also referred to as an impedance cytometry device, which is suitable for low cost manufacture and assembly, and which provides high accuracy measurements, as compared to conventional configurations. Micro-impedance cytometry is a known technique for counting and analyzing biological cells, for example human blood cells. Different types of blood cells that could be differentiated, for example, include red blood cells (RBCs), and up to the five different types of white blood cells (WBCs), including lymphocytes, neutrophils, monocytes, basophils, and eosinophils. A known issue in micro-impedance cytometry devices is that for a particle of a given size, the measured signal varies as a function of the height of the particle within the microfluidic channel. Reliably determining the cell type based on size is therefore difficult to achieve. Traditionally, sheath flows or hydrodynamic focusing techniques have been used as methods to overcome this variation in the measurement by forcing the particles to move through the center of the microfluidic channel. However, the challenges associated with focusing techniques in microfluidic channels have rendered conventional configurations deficient in accurately measuring particle sizes in a channel.

An enhanced micro-impedance cytometry device as described herein has a co-planar electrode structure. The described arrangement of electrodes enables an accurate measurement of the height of the particle in the microfluidic channel to be obtained. Accordingly, variations in signal magnitude due to particle height may be compensated for and an accurate measurement of the particle size may be obtained. The co-planar structure is significantly cheaper to manufacture and integrate within a sample preparation device as compared to conventional micro-impedance cytometry device structures.

A micro-impedance cytometry device as described herein includes a plurality of individual electrodes, that may be configured as a single drive or signal electrode and multiple sense or measurement electrodes. In one example, the electrode configuration may include a single drive or signal electrode and four sense or measurement electrodes. The sense electrodes may be arranged in two pairs on either side of the drive electrode to form four independently measurable capacitances and resultant impedances. The two sense electrodes located adjacent to the drive electrode may provide a measure of the disturbance in the electric field at the bottom of the microfluidic channel, and the remaining two sense electrodes may provide a measure of the disturbance at the top of the microfluidic channel. Impedance ratios of electrode pairs based on the measured capacitances are found to be substantially independent of cell size, and may therefore be used to compensate or correct for the measured signal magnitude to provide an accurate measurement of particle size regardless of channel height.

An impedance cytometry device is described along with methods of accurately measuring particle size of particles contained in a fluid that is passed through the impedance cytometry device. In exemplary embodiments, the impedance cytometry device includes a substrate, and an electrode arrangement deposited on the substrate in a co-planar fashion. The electrode arrangement includes a drive electrode and a plurality of measurement electrodes located in a same plane as the drive electrode. The plurality of measurement electrodes includes at least two pairs of measurement sub-electrodes, each pair of measurement sub-electrodes including a first measurement sub-electrode positioned adjacent to the drive electrode, and a second measurement sub-electrode separated from the drive electrode by a respective first measurement sub-electrode. The impedance cytometry device is incorporated into a substrate assembly of an electrowetting on dielectric (EWOD) device, such as in a substrate assembly containing electrowetting drive electrodes, or in a substrate assembly containing a common reference electrode. The impedance cytometry device may be incorporated into a fluidic channel of a microfluidic cell counter device, such as a blood cell counter device.

Another aspect of the invention is a method of measuring particle size of particles in a fluid sample as defined in claim <NUM>. The measuring method includes the steps of passing the fluid sample containing particles through the impedance cytometry device incorporated into an electrowetting on dielectric (EWOD) device; supplying a voltage stimulus to the drive electrode; measuring current signals generated on the measurement sub-electrodes to determine impedance changes generated in response to a particle passing through electric fields generated by the measurement electrodes; and determining a particle size based on the impedance changes. Features of the measuring method may include measuring differential current signals between pairs of measurement sub-electrodes. Differential current signals may be used to determine differential impedances within first regions of the microfluidic channel in which electric fields are formed between the first measurement sub-electrodes and the drive electrode, and differential impedances within second regions of the microfluidic channel in which electric fields are formed between the second measurement sub-electrodes and the drive electrode, wherein the first regions are closer to the electrode arrangement than the second regions. A ratio between peaks magnitudes of the differential impedances in the first regions and the second regions may be determined and processed to provide an accurate determination of particle size.

These and further features of the present invention will be apparent with reference to the following description and attached drawings. In the description and drawings, particular embodiments of the invention have been disclosed in detail as being indicative of some of the ways in which the principles of the invention may be employed, but it is understood that the invention is not limited correspondingly in scope. Rather, the invention includes all changes, modifications and equivalents coming within the scope of the claims appended hereto.

In the annexed drawings, like references indicate like parts or features:.

In an exemplary embodiment of a first aspect of the present invention, an electrowetting on dielectric (EWOD) device having a microfluidic impedance cytometry device is provided wherein electrodes for performing the measurement of particles in the microfluidic channel are positioned on a single planar surface and arranged so as to provide a measurement of the particle that is independent of the position of the particle in the microfluidic channel.

<FIG> is a drawing depicting an arrangement of electrodes and microfluidic channel of a microfluidic device <NUM> in accordance with embodiments of the present invention. The microfluidic device <NUM> includes a first substrate <NUM> and a second substrate <NUM> which may be arranged to oppose the first substrate <NUM> and to form a gap or microfluidic channel <NUM> between the substrates. The microfluidic channel <NUM> may be defined by a spacer <NUM>. The first substrate <NUM> and the second substrate <NUM> may be made of a glass or plastic material. The first substrate <NUM> and second substrate <NUM> may be of the same or of different materials. The spacer <NUM> may be a plastic, glue or photoresist material. Alternatively, the spacer <NUM> may be part of the first substrate <NUM> or part of the second substrate <NUM>, for example a feature of the second substrate formed during a manufacturing process such as machining or injection molding.

The spacer <NUM> may be patterned so as to define the microfluidic channel along which a fluid <NUM> containing particles <NUM> to be measured may flow. In such case, a width of the channel may be chosen to be larger than, but of a similar magnitude to, the diameter of the particles <NUM> to be measured. In such case the channel width may, for example, typically be in the range of <NUM>-<NUM>. As is well-known in the art, a height (z) of the gap may be chosen to be larger than, but of a similar magnitude to the diameter of, the particles <NUM> to be measured. For example, the diameter of particles <NUM> to be measured may typically be in the range of <NUM> -<NUM> and the height of the gap may be in the range of <NUM>-<NUM>. The microfluidic channel <NUM>, therefore, may be configured along with control of the particle concentration in the fluid to ensure that generally a single particle is present at any particular (x, y) position in the channel gap and that the channel gap does not become blocked by particles.

Electrodes <NUM> are formed on the first substrate <NUM> in the region of the gap <NUM>. The electrodes may be in direct and therefore resistive electrical contact with the fluid present in the microfluidic channel. In such case the electrodes may be formed from a conductive material that is known to be inert, such as, for example, platinum, gold or the like. Alternatively, an insulator film may be formed over the electrodes such that there is no direct electrical contact between electrodes and the fluid. The electrodes are instead in indirect and therefore capacitive electrical contact with the fluid and deleterious effects, such as leakage resistance between electrodes, may be minimized. In such case the electrodes may be formed from any suitable conductive material and the insulator may be of a well-known type such as silicon dioxide, silicon nitride or aluminum oxide.

Generally, with reference to subsequent figures, examples of an impedance cytometry device suitable for use in an EWOD device of the present invention are described along with methods of accurately measuring particle size of particles contained in a fluid that is passed through the impedance cytometry device. In an example, the impedance cytometry device includes a substrate, and an electrode arrangement deposited on the substrate in a co-planar fashion. The electrode arrangement includes a drive electrode and a plurality of measurement electrodes located in a same plane as the drive electrode. The plurality of measurement electrodes includes at least two pairs of measurement sub-electrodes, each pair of measurement sub-electrodes including a first measurement sub-electrode positioned adjacent to the drive electrode, and a second measurement sub-electrode separated from the drive electrode by a respective first measurement electrode. The impedance cytometry device may be incorporated into a substrate assembly of an electrowetting on dielectric (EWOD) device, such as in a substrate assembly containing electrowetting drive electrodes, or in a substrate assembly containing a common reference electrode.

<FIG> is a drawing depicting a cross-section diagram of the microfluidic device <NUM> illustrating the electrical arrangement of the electrodes <NUM> and the electric field in the gap between the first and second substrates <NUM> and <NUM> in the region of the electrodes. The electrode arrangement <NUM> may be deposited on one of the substrates, e.g., substrate <NUM>, in a coplanar fashion. The electrodes <NUM> may include a drive electrode <NUM> and a plurality of measurement electrodes located in a same plane as the drive electrode. The plurality of measurement electrodes may include at least two pairs of measurement sub-electrodes, such as for example a first set of measurement electrodes <NUM> and a second set of measurement electrodes <NUM> arranged on opposite sides of the drive electrode <NUM>. In the example of <FIG>, each of the two pairs of measurement electrodes <NUM>, <NUM> may further include a first measurement sub-electrode <NUM>, <NUM> and a second measurement sub-electrode <NUM>, <NUM>. Within each set of measurement electrodes <NUM>, <NUM>, the first measurement sub-electrodes <NUM>, <NUM> may be positioned adjacent to the drive electrode <NUM>, and the second measurement sub-electrodes <NUM>, <NUM> may be separated from the drive electrode <NUM> by the respective first measurement sub-electrode <NUM>, <NUM>. In this example arrangement, each of the first measurement sub-electrodes <NUM>, <NUM> forms a first impedance Z1A, Z2A with the drive electrode <NUM>. Each of the second measurement sub-electrodes <NUM>, <NUM> forms a second impedance Z1B, Z2B with the drive electrode <NUM>.

First regions <NUM> of the gap or microfluidic channel <NUM> (denoted by a first shading pattern in the figure) may be defined as an area in a lower portion of the gap and between the drive electrode (D) <NUM> and the sets of measurement electrodes <NUM>, <NUM>, with some overlap of the sets of measurement electrodes within the channel. Similarly, second regions <NUM> of the gap or microfluidic channel <NUM> (denoted by a second shading pattern in the figure) may be defined as an area in an upper portion of the gap <NUM> and between the drive electrode <NUM> and the set of measurement electrodes <NUM>, <NUM>, also with some overlap of the sets of measurement electrodes within the channel, With the orientation of <FIG>, therefore, the directional indication of the first region <NUM> being in the lower portion of the gap means the first region <NUM> is adjacent to the first substrate <NUM>. Similarly, with the orientation of <FIG>, the directional indication of the second region <NUM> being in the upper portion of the gap means the second region <NUM> is adjacent to the second substrate <NUM>.

The first measurement sub-electrode (M1A, M2A) <NUM>, <NUM> in each set of measurement electrodes is arranged such that the electric field lines associated with the electrical field coupling a respective first measurement sub-electrode and the driving electrode <NUM> are substantially present in only the first regions <NUM>. Accordingly, the associated first impedances, Z1A and Z2A, may be used as a measure of disturbances in the electric field associated with a particle passing through the first regions <NUM>. The second measurement sub-electrode (M1B, M2B) <NUM>, <NUM> in each set of measurement electrodes is arranged such that the electric field lines associated with the electrical field coupling a respective second measurement sub-electrode and the driving electrode <NUM> are substantially present in only the second regions <NUM>. Accordingly, the associated second impedances, Z1B and Z2B, may be used as a measure of disturbances in the electric field associated with a particle passing through the second regions <NUM>.

The effect described above is achieved when the width and separations of the measurement sub-electrodes in the x-dimension along the microfluidic channel <NUM> are chosen to be of a similar magnitude to both the gap <NUM> between the first substrate <NUM> and second substrate <NUM> and the diameter of the particles to be measured. For example, the width and separations of the measurement sub-electrodes may typically be in the range of <NUM>-<NUM>. For the impedances associated with the first and second sets of measurement electrodes on either side of the drive electrode to be independently measured, the size of the drive electrode in the x-dimension may be chosen to be of a magnitude which is a multiple of both the gap <NUM> and the diameter of the particles to be measured.

In operation, a fluid containing particles is forced to flow along the microfluidic channel <NUM> such that the particles pass by the electrodes <NUM>. The concentration of particles in the fluid may be chosen such that only one particle passes by the electrodes at any one time. The resulting changes in the impedances, Z1A, Z1B, Z2A and Z2B, associated with the first and second sets of measurement electrodes may be measured by an impedance measurement unit <NUM> that is configured to measure impedance differences between measurement sub-electrodes of the plurality of measurement electrodes.

The impedance measurement unit <NUM> may include a control unit <NUM> coupled to both a voltage stimulus unit <NUM> and a sensing unit <NUM>. As is well-known in the art, the voltage stimulus unit <NUM> may be arranged to apply a driving voltage signal (Vs), for example a sinusoidal voltage stimulus of fixed amplitude and frequency, to the drive electrode <NUM>. The sensing unit <NUM> may be arranged to measure the current signal generated on the measurement sub-electrodes in response to the voltage stimulus. The current signal of each measurement sub-electrode may be measured independently. Alternatively, a difference signal between pairs of measurement sub-electrodes may be measured using separate differential sensing circuits <NUM> within the sensing unit <NUM> that are configured to measure differential current signals between the different measurement electrodes. For example, as shown in <FIG>, the differential current signals between the first measurement sub-electrodes <NUM>, <NUM> in the first and second sets of measurement electrodes <NUM>, <NUM>, and between the second measurement sub-electrodes <NUM>, <NUM> in the first and second sets of measurement electrodes <NUM>, <NUM> may be measured. Alternatively, the differential current signals between the first and second sub-electrodes within each measurement set may be measured. The control unit <NUM> may be arranged to receive output signals form the sensing unit, and to convert and process the output signals (for example VA and VB) of the sensing unit <NUM> to calculate impedance differences and provide a measurement of particles passing the electrodes that is independent of the particle position within the gap.

In a conventional microfluidic impedance cytometry device with a co-planar electrode arrangement, the magnitude of the measured impedance signal is a function of both the particle diameter and the particle position in the gap in the z-direction that extends between the substrates. Accordingly, it is substantially difficult to accurately discriminate between particles of different diameters. Since fluid samples to be analyzed typically contain particles of many different sizes and types, conventional devices therefore have a significant disadvantage in that conventional devices cannot be used to reliably distinguish and count cells (e.g. blood cells, bacteria etc.) of a particular type. As further described below, an advantage of the present invention is that the device configured as disclosed may be used to reliably distinguish between and count particles of different sizes, or size ranges, corresponding to different types of particles to enable discriminating among different types of particles. For example, the present invention may discriminate between platelets, red blood cells and white blood cells (lymphocytes, neutrophils, monocytes, basophils and eosinophils) in a sample of human or animal blood.

An aspect of the invention is a method of measuring particle size of particles in a fluid sample employing an impedance cytometry device incorporated into an electrowetting on dielectric (EWOD) device. In exemplary embodiments, the measuring method includes the steps of passing the fluid sample containing particles through the impedance cytometry device incorporated into an electrowetting on dielectric (EWOD) device. The method further comprising dynamically switching the electrode arrangement of the impedance cytometry device between a cytometry function, in which a particle size is determined as to a particle in a fluid droplet, and an electrowetting function in which a droplet manipulation is performed on the fluid droplet. Operating the electrode arrangement of the impedance cytometry device in the cytometry function comprises the steps of: supplying a voltage stimulus to the drive electrode; measuring current signals generated on the measurement electrodes to determine impedance changes generated in response to a particle passing through electric fields generated by the measurement electrodes; and determining a particle size based on the impedance changes. Features of the measuring method may include measuring differential current signals between pairs of measurement sub-electrodes. Differential current signals may be used to determine differential impedances within first regions of the microfluidic channel in which electric fields are formed between the first measurement sub-electrodes and the drive electrode, and differential impedances within second regions of the microfluidic channel in which electric fields are formed between the second measurement sub-electrodes and the drive electrode, wherein the first regions are closer to the electrode arrangement than the second regions. A ratio between peaks magnitudes of the differential impedances in the first regions and the second regions may be determined and processed to provide an accurate determination of particle size.

<FIG> shows an example of measured impedance signals generated in response to a particle passing through the first regions <NUM> of the gap <NUM> of the microfluidic device <NUM> illustrated in <FIG>. As denoted in <FIG>, the x-axis direction is the direction along the microfluidic channel or gap <NUM>, and the x-axis zero position is designated to be the position coincident with a center line of the drive electrode <NUM>. The solid line represents the imaginary component of the differential impedance measured between the first measurement sub-electrodes <NUM>, <NUM> in the first and second sets of measurement electrodes <NUM>, <NUM> (M1AM2A). Said differential impedance may be, for example, derived from the output of the differential sensing unit <NUM> as a function of the voltage output signals VA and VB. The dotted line represents the imaginary component of the differential impedance measured between the second measurement sub-electrodes <NUM>, <NUM> in the first and second sets of measurement electrodes <NUM>, <NUM> (M18-M28). <FIG> shows an example of measured impedance signals generated in response to a particle passing through the second regions <NUM> of the gap <NUM>. The solid line and dotted lines represent similar measurements as described above for <FIG>.

As shown by comparison of the relative peak magnitudes of the solid and dotted lines in <FIG> and <FIG>, particles passing by the electrodes in the lower portion of the gap cause a relatively larger maximum change in the first measured impedances, Z1A and than the second measured impedances, Z1B and Z2B. Conversely, particles passing by the electrodes in the upper portion of the gap may cause a relatively larger maximum change in the second impedances than the first impedances. The inventors have found that such measurements may be employed for enhanced particle discrimination. As shown in <FIG>, it is observed that a ratio of the peak magnitudes of the two measured differential impedances is approximately constant across a wide range of particle diameters. In <FIG>, said ratio is plotted as a function of position within the microfluidic channel or gap. As denoted in <FIG>, the z-axis direction is the direction perpendicular to the electrode plane through the microfluidic channel or gap <NUM>, and the z-axis zero position is designated to be the position coincident with the surface of the substrate <NUM>. As seen in <FIG>, the ratio curves among the representative particle sizes are largely comparable, with the differences being insignificant for determining particle size. The graph of <FIG> shows that the differential peak magnitude ratio is essentially the same regardless of particle size at a given position within the gap <NUM> in the z-direction.

Accordingly, the measured impedance signals may be processed to produce an accurate count of particles of a particular diameter, or particular range of diameters, passing by the electrodes that is independent of particle position in the gap in the z-direction. A processed impedance value can be calculated as a function of output voltages based on the measured differential impedances, including using the referenced ratio of the peak magnitudes of the two measured differential impedances. The processed impedance value can then be associated with particle size in a manner that is accurate and is independent of a particle's position within the microfluidic channel. For the example arrangement shown in <FIG>, the output signals VA, VB or the sensor unit <NUM> may be processed to achieve this effect by application of an equation of the following form: <MAT> where:.

An example of the effect of applying such a processing method to the measured impedance signals is illustrated in <FIG> and <FIG>. <FIG> is a graph that depicts typical raw uncorrected impedance measurement results as a function of particle size and z-position in the microfluidic channel. The y-axis data represents the peak of the sum of the differential measurements i.e. max(VA(t) + VB(t)). The scaling factor is determined by taking the data used to plot <FIG> and setting 'n' as a variable parameter. One then changes 'n' until the plot changes from a curve (<FIG>) to the approximately parallel lines seen in <FIG>. Ideally, there will be distinct "bands" where Z (the processed output value) for a given particle diameter, e.g. <NUM>, does not overlap with the Z (processed output value) for a second e.g. <NUM> diameter particle. The scaling factor 'n' can be changed manually or a program can be written to automatically determine the 'n' value that provides the best distinction between the processed output values for different particle sizes, e.g., aiming to minimize the chance of there being overlapping max/min values for the two or more different particle sizes that have to be differentiated with unique output values.

It is noted that this uncorrected plot is equivalent to that generated from a conventional co-planar electrode arrangement. As shown by the plot, particles of different sizes and at different positions in the gap may generate similar uncorrected measurement results and therefore be indistinguishable from each other. Accordingly, conventional configurations are unable to distinguish among different particles based on particle size.

On the other hand, <FIG> is a graph that depicts corrected impedance measurement results arising from processing the uncorrected results using the above equation. As can be seen, a particle of a particular size results in a unique output value, or range of output values, for all positions in the gap in the z-direction, which permits distinguishing among different particles based on particle size.

Subsequent <FIG> show alternative configurations or optional features of an impedance cytometry device and related EWOD devices based on comparable principles as above. It should be noted that for simplicity, the impedance measurement unit <NUM> and its associated components are omitted in the subsequent figures. It will be appreciated that comparable measurement and control structures may be employed in combination with any of the embodiments of <FIG>.

<FIG> is a drawing depicting an alternative arrangement of electrodes for an impedance cytometry device suitable for incorporation into an EWOD device in accordance with another embodiment of the present invention. Similarly to the previous electrode arrangement, an electrode arrangement <NUM> for a microfluidic device may include a drive electrode (D) <NUM>, a first set of measurement electrodes <NUM>, and second set of measurement electrodes <NUM> formed on a substrate <NUM>. In this example arrangement, the first measurement sub-electrodes of each set of measurement electrodes <NUM>, <NUM> (M1A, M2A) may be chosen to have a different width than the second measurement sub-electrodes of each set of measurement electrodes <NUM>, <NUM> (M1B, M2B) in the x-direction along the substrate. In the example arrangement of <FIG>, the first sub-electrodes <NUM>, <NUM> may, for example, be less wide than the second sub-electrodes <NUM>, <NUM>. Adjustment of the sub-electrode lengths enables the relative magnitude of the impedance measurements associated with the first and second measurement sub-electrodes to be adjusted. Advantageously, this may allow the dynamic range and signal-to-noise ratio of the impedance measurement circuits to be optimized.

Alternatively or additionally, the same effect and advantage may be achieved by choosing the spacing between the first sub-electrodes, second sub-electrodes and drive electrode to be different. <FIG> is a drawing depicting an alternative arrangement of electrodes in which a different spacing is provided as between different sub-electrodes. Generally, within each pair of measurement sub-electrodes, a first spacing <NUM> between the first measurement sub-electrode <NUM>, <NUM> and the drive electrode <NUM> differs from a second spacing <NUM> between the first measurement sub-electrode <NUM>, <NUM> and the respective second measurement sub-electrode <NUM>, <NUM>. In this particular example arrangement of <FIG>, the first spacing is wider than the second spacing. Other differences of spacing may be employed to optimize performance.

<FIG> is a drawing depicting a further alternative arrangement of electrodes for an impedance cytometry device suitable for incorporation into an EWOD device in accordance with another embodiment of the present invention. In this example arrangement, an electrode arrangement <NUM> for a microfluidic device may include a first drive electrode (D1) <NUM> and a second drive electrode (D2) <NUM> that are formed on a substrate <NUM> alongside a first set of measurement electrodes <NUM> and a second set of measurement electrodes <NUM>. The first measurement sub-electrodes of each set of measurement electrodes <NUM>, <NUM> (M1A, M2A) are adjacent to the corresponding drive electrodes <NUM>, <NUM>, and the second measurement sub-electrodes of each set of measurement electrodes <NUM>, <NUM> (M1B, M2B) are separated from the corresponding drive electrodes <NUM>, <NUM> by the first sub-electrodes <NUM>, <NUM>. The first drive electrode <NUM> and first set of measurement electrodes <NUM> may be sufficiently separated from the second drive electrode <NUM> and second set of measurement electrodes <NUM> that they are not in electrical communication, i.e. there are no electric field lines that couple between the electrodes <NUM>, <NUM> and the electrodes <NUM>, <NUM>. In operation the drive electrodes <NUM>, <NUM> may be connected to the same voltage stimulus. An advantage of this arrangement is that it may provide improved matching between the first measured impedances, Z1A and and between the second measured impedances, Z1B and Z2B. Accordingly, the arrangement of <FIG> may allow a more accurate discrimination between particles of different particular diameters, and hence a more accurate particle count, to be made.

In the invention, a microfluidic impedance cytometry device having any of the above electrode arrangements is integrated or incorporated within an electrowetting-on-dielectric (EWOD) device. As is well known in the art, EWOD devices may be used to manipulate droplets of fluid by electronic means using electrowetting techniques. Integration of a microfluidic impedance cytometry device within an EWOD device allows the number of particles within a droplet to be counted. With an impedance cytometry device of sufficient accuracy, for example as described in this disclosure, the number of particles of a particular size and/or diameter may additionally be counted.

In an exemplary embodiment of an EWOD device, electrodes for impedance cytometry are formed on the same substrate as the electrodes that are used to apply the electrowetting force for the actuation of droplets on the surface of the substrate. <FIG> is a drawing depicting a cross-section of an exemplary EWOD device <NUM>. As illustrated in the cross-section diagram of the example device of <FIG>, cytometry electrodes <NUM> and electrowetting electrodes <NUM> are formed on the same plane on the surface of a substrate <NUM>. Alternatively, the cytometry electrodes and electrowetting electrodes may be formed in different layers on the same substrate. An insulating layer <NUM> and a hydrophobic coating layer <NUM> are formed on top of the cytometry electrodes and electrowetting electrodes, and the insulating layer separates the electrodes from the fluid droplet <NUM>. The cytometry electrodes <NUM> and electrowetting electrodes <NUM> may be connected to circuits that control their respective functions. Such circuits may be external to the device or may be formed on the substrate by, for example, thin film transistors. In operation, the electrowetting electrodes <NUM> may cause the fluid droplet to pass over the cytometry electrodes <NUM>. Accordingly, particles within the droplet will be caused to pass over the cytometry electrodes and may be measured and counted. The concentration of particles within the droplet may be controlled and the size and shape of the droplet may be controlled to ensure that generally only one particle passes over the cytometry electrodes <NUM> at any one time. An accurate count of particles and determination of particle size and/or diameter may therefore be made.

<FIG> is a drawing depicting in cross-section an exemplary microfluidic impedance cytometry device integrated within an EWOD device <NUM> comprising two substrates in accordance with embodiments of the present invention. In the example of <FIG>, similarly to that of <FIG>, the impedance cytometry electrodes are formed on a first substrate of the electrowetting device alongside the electrowetting electrodes. Cytometry electrodes <NUM> and electrowetting electrodes <NUM> are formed on the same plane on the surface of a first substrate <NUM>. Alternatively, the cytometry electrodes and electrowetting electrodes may be formed in different layers on the same substrate. An insulating layer <NUM> and a first hydrophobic coating layer <NUM> are formed on top of the cytometry electrodes and electrowetting electrodes, and the insulating layer separates the electrodes from the fluid droplet <NUM>. The cytometry electrodes <NUM> and electrowetting electrodes <NUM> may be connected to circuits that control their respective functions. In the two-substrate configuration, the EWOD device <NUM> further includes a second substrate <NUM> opposite from the substrate <NUM> relative to the microfluidic channel where the droplet <NUM> resides. A common reference electrode <NUM> is formed on the second substrate <NUM>, and a second hydrophobic coating layer <NUM> is formed on the reference electrode <NUM>. In operation, an electrowetting drive voltage is generated as a voltage difference between the electrowetting electrode <NUM> and the reference electrode <NUM>.

<FIG> is a drawing depicting in cross-section another exemplary microfluidic impedance cytometry device integrated within an EWOD device <NUM> comprising two substrates in accordance with embodiments of the present invention. The configuration of <FIG> bears similarity to that of <FIG>, with the difference that in the embodiment of <FIG>, the cytometry electrodes of a microfluidic impedance cytometry device are formed alongside the common reference electrode on the second substrate of an EWOD device. An advantage of this embodiment is that droplet manipulation is not hindered by the presence of the cytometry electrodes. Electrowetting electrodes <NUM> are formed on the surface of a first substrate <NUM>. An insulating layer <NUM> and a first hydrophobic coating layer <NUM> are formed on top of the electrowetting electrodes, and the insulating layer separates the electrodes from the fluid droplet <NUM>. In the two-substrate configuration, the EWOD device <NUM> further includes a second substrate <NUM> opposite from the substrate <NUM> relative to the microfluidic channel where the droplet <NUM> resides. Common reference electrodes <NUM> are formed on the second substrate <NUM>, and a second hydrophobic coating layer <NUM> is formed on the reference electrodes <NUM>. In operation, an electrowetting drive voltage is generated as a voltage difference between the electrowetting electrode <NUM> and the reference electrode <NUM>. In the example of <FIG>, cytometry electrodes <NUM> and common reference electrodes <NUM> are formed on the same plane on the surface of the second substrate <NUM>. Alternatively, the cytometry electrodes and common reference electrodes may be formed in different layers on the same substrate. As in the previous embodiment, the cytometry electrodes <NUM> and electrowetting electrodes <NUM> may be connected to circuits that control their respective functions.

In an exemplary mode of operation of an EWOD device including an impedance cytometry device, a control circuit may be connected to the cytometry electrodes that enables the electrode function to be switched dynamically between a cytometry function in which a particle size is determined as to a particle in a fluid droplet as described above, and an electrowetting function in which a droplet manipulation is performed on the fluid droplet. Droplet manipulations, for example, may include forming droplets, moving droplets along the electrowetting array, splitting droplets, mixing droplets, determining or sensing droplet properties, and other droplet manipulations as may be known in the art of EWOD devices. The control circuit may be external to the device or may be formed on the substrate by, for example, thin film transistors.

<FIG> teaches one method of incorporating an impedance cytometer <NUM> into a microfluidic device <NUM> for counting blood cells. The microfluidic device <NUM> includes a first chamber <NUM> partially filled with a solution <NUM>, a mixing chamber <NUM>, a fluidic channel <NUM> with an integrated impedance cytometer <NUM> in fluid communication with a rotating valve <NUM>, and a waste chamber <NUM>. A whole blood sample is input through aperture <NUM> and into the rotating valve <NUM> with an internal channel that can meter a predetermined small volume of blood <NUM>. Generally, cells are counted based on impedance measurements taken at the impedance cytometry device. Components common to previous embodiments are denoted by reference numerals index by <NUM>.

Typically, <NUM>µl of whole human blood contains ~<NUM> million red blood cells (RBCs), ~<NUM>,<NUM> white blood cells (WBCs), and ~<NUM>,<NUM> platelets. To accurately count the number of WBCs, the RBCs must be removed, i.e., lysed within the mixing chamber <NUM>. To enable the RBCs to be counted accurately, a whole blood sample is diluted, for example in a <NUM>:<NUM>,<NUM> to <NUM>:<NUM>,<NUM> ratio. The whole blood sample may be diluted in a <NUM>:<NUM>,<NUM> to <NUM>:<NUM>,<NUM> ratio, or diluted in a <NUM>:<NUM>,<NUM> ratio. Platelets are counted preferably alongside the RBCs. It follows that there are two different sample preparation protocols -- one for WBCs, and the other for RBCs and platelets combined.

In one method of counting blood cells, the solution <NUM> may be a lysis solution to lyse RBCs so that the WBCs can be differentiated and counted (up to a <NUM>-part differential). The lysis solution is mixed with the sample input within the mixing chamber <NUM>, and the product of the mixing is then communicated through the fluidic channel <NUM> with the integrated impedance cytometer <NUM> for cell counting. The lysis reagent is any reagent mixture containing a chemical known to lyse RBCs, such as for example saponins, quarternary ammonium salts, or the like. Preferably, the lysis reagent used contains saponin. The lysis reagent may be <NUM>% v/v formic acid and <NUM>% w/v saponin.

Optionally and/or additionally, a quench reagent may be further added after a pre-determined length of time for optimal lysis, wherein the quench reagent is any mixture known to halt or substantially reduce the rate of RBC lysis. The quench reagent may be <NUM>% w/v sodium carbonate and <NUM>% w/v sodium chloride. In exemplary methods of counting blood cells, the blood:lysis:quench reagents are mixed in a ratio of <NUM>:<NUM>:<NUM>. (See embodiments in <CIT>, or <CIT>).

Alternatively, the solution <NUM> could be a diluent solution so that RBCs and platelets can be sufficiently diluted to enable accurate quantification of RBCs and platelets. The diluent reagent is one compatible with RBCs and platelets. Compatible implies that the reagent in question does not cause serious degradation to the sample, nor promotes clotting of either the RBCs or the platelets. Dilutent reagents may include, but are not limited to, PBS, running buffer (comprising PBS, <NUM> EDTA, <NUM>% BSA), and the like.

The method of counting blood cells may further include performing a haemoglobin measurement in a haemoglobin measurement chamber, such is in the chamber <NUM> holding the solution <NUM>. For haemoglobin to be measured accurately, the RBCs must be lysed. It follows that the most preferable position for the haemoglobin measurement is after the RBCs have been lysed in the WBC counting prototcol. It is further preferable to convert the haemoglobin into a stable oxidized form (methemoglobin) by adding chemical haemoglobin reagent mixtures. Suitable reagents include, but are not limited to, Drabkins's reagent (which contains sodium bicarbonate, potassium ferricyanide and potassium cyanide and converts haemoglobin into cyanmethemoglobin), ferrocyanide, or the like. Such haemoglobin reagents could be pre-dried in the haemoglobin measurement chamber. In practice, haemoglobin is converted to cyanmethemoglobin (e.g. by reacting the blood with a Drabkin's reagent) and measured spectrophotometrically (the reacted Drabkin's reagent and haemoglobin form a stable, coloured end-product). A simple LED/photodiode combination can be employed for the quantitative, colourimetric determination of blood haemoglobin using absorbance in accordance with Beer's law, as is known in the art.

After cell counting, and the haemoglobin measurement, the processed blood samples are collected in a waste chamber.

The two different sample processing protocols for WBCs and RBCs/platelets may be carried out on separate cartridges, and the cells of interest counted on separate impedance sensors. An alternative method would carry out the processing protocols for WBCs and RBCs/platelets in parallel, and then count e.g. the WBCs then the RBCs/platelets on a single impedance chip in series. In a further alternative method, the two different sample processing protocols could be carried out in series and the e.g. WBCs followed by the RBCs/platelets counted on a single impedance sensor.

Alternatively and/or additionally, co-planar impedance cytometers as described in this patent can be integrated into microfluidic blood cell counters of the types described in the following patents; <CIT>, <CIT>, or <CIT>.

<FIG>teaches an EWOD device <NUM> with an integrated impedance cytometer <NUM>. A droplet of whole blood <NUM> is merged with a solution droplet <NUM> using electrowetting to produce a mixed droplet <NUM>. A daughter droplet <NUM> is split from the mixed droplet <NUM> using electrowetting and moved, via electrowetting forces, through the integrated impedance cytometer <NUM>. The solution droplet <NUM> can be either a lysis solution to lyse RBCs so that the WBCs can be differentiated and counted, or it can be a diluent solution so that RBCs and platelets can be sufficiently diluted to enable accurate quantification of RBCs and platelets.

EWOD technology is well suited to merging, mixing, splitting and moving droplets, offering several ease-of-workflow benefits compared to mechanical microfluidic cartridges.

<FIG> describes an EWOD device <NUM> for counting blood cells where the impedance cytometer <NUM> is integrated into the top plate <NUM> of the EWOD device <NUM>. <FIG> teaches an EWOD device <NUM> for counting blood cells where the impedance cytometer <NUM> is integrated into the bottom plate <NUM> of the EWOD device <NUM>. Components common to previous embodiments are denoted by reference numerals index by <NUM>.

The two different sample processing protocols for WBCs and RBCs/platelets may be carried out simultaneously and the cells of interest counted on separate impedance sensors. An alternative method would carry out the processing protocols for WBCs and RBCs/platelets in parallel, and then count e.g. the WBCs then the RBCs/platelets on a single impedance chip in series. In a further alternative method, the two different sample processing protocols could be carried out in series and the e.g. WBCs followed by the RBCs/platelets counted on a single impedance sensor.

An aspect of the invention, therefore, is an an electrowetting on dielectric (EWOD) device including a substrate assembly and includes electrowetting electrodes; and an impedance cytometry device incorporated into the substrate assembly. The impedance cytometry device comprises an electrode arrangement provided in the substrate assembly in a co-planar fashion, the electrode arrangement comprising a drive electrode and a plurality of measurement electrodes located in a same plane as the drive electrode. The plurality of measurement electrodes includes at least two pairs of measurement sub-electrodes, each pair of measurement sub-electrodes including a first measurement sub-electrode positioned adjacent to the drive electrode, and a second measurement sub-electrode separated from the drive electrode by a respective first measurement electrode. The EWOD device further comprises a control circuit connected to the drive electrode and the measurement electrodes, the control circuit configured to dynamically switch operation of the drive electrode and the measurement electrodes between a cytometry function and an electrowetting function. The EWOD device may include one or more of the following features, either individually or in combination.

In an exemplary embodiment of the EWOD device, the substrate assembly comprises a substrate onto which is deposited the electrode arrangement of the impedance cytometry device and the electrowetting electrodes, an insulator layer deposited on the substrate, and a hydrophobic coating deposited on insulator layer to define the microfluidic channel.

In an exemplary embodiment of the EWOD device, the electrode arrangement of the impedance cytometry device and the electrowetting electrodes are formed on a same plane on a surface of the substrate.

Another aspect of the invention is an electrowetting on dielectric (EWOD) device comprising: a first substrate assembly and a second substrate assembly; wherein one of the first or second substrate assemblies includes electrowetting electrodes and the other of the first or second substrate assembly includes a common reference electrode, and the first substrate assembly and the second substrate assembly are spaced apart to define a microfluidic channel between the first and second substrate assemblies; and an impedance cytometry device incorporated into one of the first substrate assembly or the second substrate assembly. The impedance cytometry device comprises an electrode arrangement provided in the first or the second substrate assembly in a co-planar fashion, the electrode arrangement comprising a drive electrode and a plurality of measurement electrodes located in a same plane as the drive electrode. The plurality of measurement electrodes includes at least two pairs of measurement sub-electrodes, each pair of measurement sub-electrodes including a first measurement sub-electrode positioned adjacent to the drive electrode, and a second measurement sub-electrode separated from the drive electrode by a respective first measurement electrode. The EWOD device further comprises a control circuit connected to the drive electrode and the measurement electrodes, the control circuit configured to dynamically switch operation of the drive electrode and the measurement electrodes between a cytometry function and an electrowetting function.

The EWOD device of this aspect may include one or more of the following features, either individually or in combination.

In an exemplary embodiment of the EWOD device, the first substrate assembly comprises a first substrate onto which is deposited the electrode arrangement of the impedance cytometry device and the electrowetting electrodes, an insulator layer deposited on the first substrate, and a hydrophobic coating deposited on insulator layer to define the microfluidic channel; and the second substrate assembly comprises a second substrate onto which is deposited the common reference electrode, and a second hydrophobic coating deposited on common reference electrode to define the microfluidic channel.

In an exemplary embodiment of the EWOD device, the first substrate assembly comprises a first substrate onto which is deposited the electrowetting electrodes, an insulator layer deposited on the first substrate, and a hydrophobic coating deposited on insulator layer to define the microfluidic channel; and the second substrate assembly comprises a second substrate onto which is deposited the electrode arrangement of the impedance cytometry device and the common reference electrode, and a second hydrophobic coating deposited on common reference electrode to define the microfluidic channel.

Another aspect of the invention is a method of measuring particle size in a fluid sample. The measuring method includes the steps of: passing the fluid sample containing particles through an impedance cytometry device incorporated into an electrowetting on dielectric (EWOD) device, wherein the EWOD device comprises: a first substrate assembly and a second substrate assembly; wherein one of the first or second substrate assemblies includes electrowetting electrodes and the other of the first or second substrate assembly includes a common reference electrode, and the first substrate assembly and the second substrate assembly are spaced apart to define a microfluidic channel between the first and second substrate assemblies; and the impedance cytometry device is incorporated into one of the first substrate assembly or the second substrate assembly; the impedance cytometry device comprising: an electrode arrangement deposited on the one of the first substrate assembly or the second substrate assembly in a co-planar fashion, the electrode arrangement comprising a drive electrode and a plurality of measurement electrodes located in a same plane as the drive electrode; wherein the plurality of measurement electrodes includes at least two pairs of measurement sub-electrodes, each pair of measurement sub-electrodes including a first measurement sub-electrode positioned adjacent to the drive electrode, and a second measurement sub-electrode separated from the drive electrode (by a respective first measurement electrode. The method further comprises dynamically switching the electrode arrangement of the impedance cytometry device between a cytometry function, in which a particle size is determined as to a particle in a fluid droplet, and an electrowetting function in which a droplet manipulation is performed on the fluid droplet. Operating the electrode arrangement of the impedance cytometry device in the cytometry function comprises: supplying a voltage stimulus to the drive electrode; measuring current signals generated on the measurement sub-electrodes to determine impedance changes generated in response to a particle passing through electric fields generated by the measurement electrodes; and determining a particle size based on the impedance changes. The measuring method may include one or more of the following features, either individually or in combination.

In an exemplary embodiment of the measuring method, measuring current signals generated on the measurement sub-electrodes comprises measuring differential current signals between pairs of measurement sub-electrodes.

In an exemplary embodiment of the measuring method, measuring current signals generated on the measurement sub-electrodes comprises measuring differential current signals between the first measurement sub-electrodes in first and second pairs of measurement electrodes, and between the second measurement sub-electrodes in the first and second pairs of measurement electrodes.

In an exemplary embodiment of the measuring method, determining the impedance changes comprises: determining impedance differentials within first regions of the microfluidic channel in which electric fields are formed between the first measurement sub-electrodes and the drive electrode; and determining impedance differentials within second regions of the microfluidic channel in which electric fields are formed between the second measurement sub-electrodes and the drive electrode; wherein the first regions are closer to the electrode arrangement than the second regions.

In an exemplary embodiment of the measuring method, the method further includes determining a ratio between peak magnitudes of the differential impedances in the first regions and the second regions; calculating a processed impedance value as a function of output voltages based on the measured differential impedances, including using the ratio of the peak magnitudes of the differential impedances; and determining particle size based on the processed impedance value.

In an exemplary embodiment of the measuring method, the method further includes sizing the microfluidic channel and controlling a concentration of particles in the fluid such that only one particle passes through the impedance cytometry device at a time.

In an exemplary embodiment of the measuring method, the sample input is a blood sample, and the method further comprises counting one or more red blood cells, white blood cells, and platelets in the sample input.

Although the invention has been shown and described with respect to a certain embodiment or embodiments, equivalent alterations and modifications may occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In particular regard to the various functions performed by the above described elements (components, assemblies, devices, compositions, etc.), the terms (including a reference to a "means") used to describe such elements are intended to correspond, unless otherwise indicated, to any element which performs the specified function of the described element (i.e., that is functionally equivalent), even though not structurally equivalent to the disclosed structure which performs the function in the herein exemplary embodiment or embodiments of the invention. In addition, while a particular feature of the invention may have been described above with respect to only one or more of several embodiments, such feature may be combined with one or more other features of the other embodiments, as may be desired and advantageous for any given or particular application.

Claim 1:
An electrowetting on dielectric (EWOD) device (<NUM>) comprising:
a substrate assembly that includes electrowetting electrodes (<NUM>); and
an impedance cytometry device incorporated into the substrate assembly;
wherein the impedance cytometry device comprises an electrode arrangement (<NUM>) provided in the substrate assembly in a co-planar fashion, the electrode arrangement comprising a drive electrode (D;D1,D2) and a plurality of measurement electrodes (<NUM>,<NUM>,<NUM>,<NUM>; <NUM>,<NUM>,<NUM>,<NUM>;<NUM>,<NUM>,<NUM>,<NUM>; M1A,M1B,M2A,M2B) located in a same plane as the drive electrode (D;D1,D2);
wherein the plurality of measurement electrodes includes at least two pairs (<NUM>,<NUM>;<NUM>,<NUM>;<NUM>,<NUM>) of measurement sub-electrodes, each pair of measurement sub-electrodes including a first measurement sub-electrode (<NUM>,<NUM>;<NUM>,<NUM>:<NUM>,<NUM>; M1A, M2A) positioned adjacent to the drive electrode (D;D1,D2), and a second measurement sub-electrode (<NUM>,<NUM>; <NUM>,<NUM>;<NUM>,<NUM>;M1B, M2B) separated from the drive electrode (D;D1,D2) by a respective first measurement electrode (<NUM>,<NUM>; <NUM>,<NUM>;<NUM>;<NUM>; M1A, M2A);
wherein the EWOD device further comprises a control circuit connected to the drive electrode and the measurement electrodes, the control circuit configured to dynamically switch operation of the drive electrode and the measurement electrodes between a cytometry function and an electrowetting function.