Patent Description:
Electrowetting on dielectric (EWOD) is a well-known technique for manipulating droplets of fluid by application of an electric field. Active Matrix EWOD (AM-EWOD) refers to implementation of EWOD in an active matrix array incorporating transistors, for example by using thin film transistors (TFTs). It is thus a candidate technology for digital microfluidics for lab-on-a-chip technology. An introduction to the basic principles of the technology can be found in "<NPL>).

<FIG> is a drawing depicting an exemplary EWOD based microfluidic system. In the example of <FIG>, the microfluidic system includes a reader <NUM> and a cartridge <NUM>. The cartridge <NUM> may contain a microfluidic device, such as an AM-EWOD device <NUM>, as well as (not shown) fluid input ports into the device and an electrical connection as are conventional. The fluid input ports may perform the function of inputting fluid into the AM-EWOD device <NUM> and generating droplets within the device, for example by dispensing from input reservoirs as controlled by electrowetting. As further detailed below, the microfluidic device includes an electrode array configured to receive the inputted fluid droplets.

The microfluidic system further may include a control system configured to control actuation voltages applied to the electrode array of the microfluidic device to perform manipulation operations to the fluid droplets. For example, the reader <NUM> may contain such a control system configured as control electronics <NUM> and a storage device <NUM> that may store any application software and any data associated with the system. The control electronics <NUM> may include suitable circuitry and/or processing devices that are configured to carry out various control operations relating to control of the AM-EWOD device <NUM>, such as a CPU, microcontroller or microprocessor.

In the example of <FIG>, an external sensor module <NUM> is provided for sensor droplet properties. For example, optical sensors as are known in the art may be employed as external sensors for sensing droplet properties, which may be incorporated into a probe that can be located in proximity to the EWOD device. Suitable optical sensors include camera devices, light sensors, charged coupled devices (CCD) and similar image sensors, and the like. A sensor additionally or alternatively may be configured as internal sensor circuitry incorporated as part of the drive circuitry in each array element. Such sensor circuitry may sense droplet properties by the detection of an electrical property at the array element, such as impedance or capacitance.

<FIG> is a drawing depicting additional details of the exemplary AM-EWOD device <NUM> in a perspective view. The AM-EWOD device <NUM> has a lower substrate assembly <NUM> with thin film electronics <NUM> disposed upon the lower substrate assembly <NUM>. The thin film electronics <NUM> are arranged to drive array element electrodes <NUM>. A plurality of array element electrodes <NUM> are arranged in an electrode or element two-dimensional array <NUM>, having N rows by M columns of array elements where N and M may be any integer. A liquid droplet <NUM> which may include any polar liquid and which typically may be aqueous, is enclosed between the lower substrate <NUM> and a top substrate <NUM> separated by a spacer <NUM>, although it will be appreciated that multiple liquid droplets <NUM> can be present.

<FIG> is a drawing depicting a cross section through some of the array elements of the exemplary AM-EWOD <NUM> device of <FIG>. In the portion of the AM-EWOD device depicted in <FIG>, the device includes a pair of the array element electrodes 48A and 48B that are shown in cross section that may be utilized in the electrode or element array <NUM> of the AM-EWOD device <NUM> of <FIG>. The AM-EWOD device <NUM> further incorporates the thin-film electronics <NUM> disposed on the lower substrate <NUM>, which is separated from the upper substrate <NUM> by the spacer <NUM>. The uppermost layer of the lower substrate <NUM> (which may be considered a part of the thin film electronics layer <NUM>) is patterned so that a plurality of the array element electrodes <NUM> (e.g. specific examples of array element electrodes are 48A and 48B in <FIG>) are realized. The term element electrode <NUM> may be taken in what follows to refer both to the physical electrode structure <NUM> associated with a particular array element, and also to the node of an electrical circuit directly connected to this physical structure. A reference electrode <NUM> is shown in <FIG> disposed upon the top substrate <NUM>, but the reference electrode alternatively may be disposed upon the lower substrate <NUM> to realize an in-plane reference electrode geometry. The term reference electrode <NUM> may also be taken in what follows to refer to both or either of the physical electrode structure and also to the node of an electrical circuit directly connected to this physical structure.

In the AM-EWOD device <NUM>, a non-polar fluid <NUM> (e.g. oil) may be used to occupy the volume not occupied by the liquid droplet <NUM>. An insulator layer <NUM> may be disposed upon the lower substrate <NUM> that separates the conductive element electrodes 48A and 48B from a first hydrophobic coating <NUM> upon which the liquid droplet <NUM> sits with a contact angle <NUM> represented by θ. The hydrophobic coating is formed from a hydrophobic material (commonly, but not necessarily, a fluoropolymer). On the top substrate <NUM> is a second hydrophobic coating <NUM> with which the liquid droplet <NUM> may come into contact. The reference electrode <NUM> is interposed between the top substrate <NUM> and the second hydrophobic coating <NUM>.

The contact angle θ for the liquid droplet is defined as shown in <FIG>, and is determined by the balancing of the surface tension components between the solid-liquid (γSL), liquid-gas (γLG) and non-ionic fluid (γSG) interfaces, and in the case where no voltages are applied satisfies Young's law, the equation being given by: <MAT>.

In operation, voltages termed the EW drive voltages, (e.g. VT, V<NUM> and V<NUM> in <FIG>) may be externally applied to different electrodes (e.g. reference electrode <NUM>, element electrodes 48A and 48B, respectively). The resulting electrical forces that are set up effectively control the hydrophobicity of the hydrophobic coating <NUM>. By arranging for different EW drive voltages (e.g. V<NUM> and V<NUM>) to be applied to different element electrodes (e.g. 48A and 48B), the liquid droplet <NUM> may be moved in the lateral plane between the two substrates.

<FIG> shows a circuit representation of the electrical load 70A between the element electrode <NUM> and the reference electrode <NUM> in the case when a liquid droplet <NUM> is present. The liquid droplet <NUM> can usually be modeled as a resistor and capacitor in parallel. Typically, the resistance of the droplet will be relatively low (e.g. if the droplet contains ions) and the capacitance of the droplet will be relatively high (e.g. because the relative permittivity of polar liquids is relatively high, e.g. ~<NUM> if the liquid droplet is aqueous). In many situations the droplet resistance is relatively small, such that at the frequencies of interest for electrowetting, the liquid droplet <NUM> may function effectively as an electrical short circuit. The hydrophobic coatings <NUM> and <NUM> have electrical characteristics that may be modelled as capacitors, and the insulator <NUM> may also be modelled as a capacitor. The overall impedance between the element electrode <NUM> and the reference electrode <NUM> may be approximated by a capacitor whose value is typically dominated by the contribution of the insulator <NUM> and hydrophobic coatings <NUM> and <NUM> contributions, and which for typical layer thicknesses and materials may be on the order of a pico-Farad in value.

<FIG> shows a circuit representation of the electrical load 70B between the element electrode <NUM> and the reference electrode <NUM> in the case when no liquid droplet is present. In this case the liquid droplet components are replaced by a capacitor representing the capacitance of the non-polar fluid <NUM> which occupies the space between the top and lower substrates. In this case the overall impedance between the element electrode <NUM> and the reference electrode <NUM> may be approximated by a capacitor whose value is dominated by the capacitance of the non-polar fluid and which is typically small, on the order of femto-Farads.

For the purposes of driving and sensing the array elements, the electrical load 70A/70B overall functions in effect as a capacitor, whose value depends on whether a liquid droplet <NUM> is present or not at a given element electrode <NUM>. In the case where a droplet is present, the capacitance is relatively high (typically of order pico-Farads), whereas if there is no liquid droplet present the capacitance is low (typically of order femto-Farads). If a droplet partially covers a given electrode <NUM> then the capacitance may approximately represent the extent of coverage of the element electrode <NUM> by the liquid droplet <NUM>.

<CIT>) describes how TFT based thin film electronics may be used to control the addressing of voltage pulses to an EWOD array by using circuit arrangements very similar to those employed in active matrix display technologies. The approach of <CIT> may be termed "Active Matrix Electrowetting on Dielectric" (AM-EWOD). There are several advantages in using TFT based thin film electronics to control an EWOD array, namely:.

<FIG> is a drawing depicting an exemplary arrangement of thin film electronics <NUM> in the exemplary AM-EWOD device <NUM> of <FIG>. The thin film electronics <NUM> is located upon the lower substrate <NUM>. Each array element <NUM> of the array of elements <NUM> contains an array element circuit <NUM> for controlling the electrode potential of a corresponding element electrode <NUM>. Integrated row driver <NUM> and column driver <NUM> circuits are also implemented in thin film electronics <NUM> to supply control signals to the array element circuit <NUM>. The array element circuit <NUM> may also contain a sensor capability for detecting the presence or absence of a liquid droplet in the location of the array element. Integrated sensor row addressing <NUM> and column detection circuits <NUM> may further be implemented in thin film electronics for the addressing and readout of the sensor circuitry in each array element.

A serial interface <NUM> may also be provided to process a serial input data stream and facilitate the programming of the required voltages to the element electrodes <NUM> in the array <NUM>. A voltage supply interface <NUM> provides the corresponding supply voltages, top substrate drive voltages, and other requisite voltage inputs as further described herein. A number of connecting wires <NUM> between the lower substrate <NUM> and external control electronics, power supplies and any other components can be made relatively few, even for large array sizes. Optionally, the serial data input may be partially parallelized. For example, if two data input lines are used the first may supply data for columns <NUM> to X/<NUM>, and the second for columns (<NUM>+X/<NUM>) to M with minor modifications to the column driver circuits <NUM>. In this way the rate at which data can be programmed to the array is increased, which is a standard technique used in liquid crystal display driving circuitry.

<FIG> is a drawing depicting an exemplary arrangement of the array element circuit <NUM> present in each array element <NUM>, which may be used as part of the thin film electronics of <FIG>. The array element circuit <NUM> may contain an actuation circuit <NUM>, having inputs ENABLE, DATA and ACTUATE, and an output which is connected to an element electrode <NUM>. The array element circuit <NUM> also may contain a droplet sensing circuit <NUM>, which may be in electrical communication with the element electrode <NUM>. Typically, the read-out of the droplet sensing circuit <NUM> may be controlled by one or more addressing lines (e.g. RW) that may be common to elements in the same row of the array, and may also have one or more outputs, e.g. OUT, which may be common to all elements in the same column of the array.

The array element circuit <NUM> may typically perform the functions of:.

Various methods of controlling an AM-EWOD device to sense droplets and perform desired droplet manipulations have been described. For example, <CIT>) describes the use of capacitance detection to sense dynamic properties of reagents as a way for determining the output of an assay. Such disclosure incorporates an integrated impedance sensor circuit that is incorporated specifically into the array element circuitry of each array element. Accordingly, attempts have been made to optimize integrated impedance sensing circuitry <NUM> of <FIG> into the array element structure, and in particular as part of the array element circuitry <NUM>. Examples of AM-EWOD devices having integrated actuation and sensing circuits are described, for example, in Applicant's commonly assigned patent documents as follows: <CIT>); <CIT>); <CIT>); and <CIT>). The enhanced method of operation described in the current application may be employed in connection with any suitable array element circuitry <NUM> including any suitable integrated impedance sensing circuitry <NUM>.

The above impedance or capacitance sensor element arrays are well-suited for sensing liquid droplets on the element array for the purpose of droplet manipulation operations, but nothing in these disclosures teaches any basis for the sensor arrays to sense objects external to the AM-EWOD cartridge. In the field of touch panel sensor technology, sensing of a remote object such as a stylus can be performed using an active matrix TFT array, as described for example in <CIT>). Such principles, however, have not been applied in the context of a microfluidic device, such as an AM-EWOD device in particular.

In many AM-EWOD device configurations, a disposable AM-EWOD cartridge that includes the element array and thin film electronics is inserted into a broader AM-EWOD instrument that controls and supplies actuation voltages, reads out sensor information and related output signals, inputs and extracts fluid, and provides the operator interface. It is of significant importance, therefore, that the AM-EWOD cartridge be properly docked into and aligned with the AM-EWOD instrument at a position optimized for the desired reaction protocols or scripts. The AM-EWOD instrument and/or AM-EWOD cartridge typically include docking features, but such features may only provide a relatively gross positioning of the AM-EWOD cartridge relative to pertinent AM-EWOD instrument components. For many applications, a high precision of mechanical alignment is required or beneficial, as pixel size of the individual array elements may be on the order of <NUM> or less. In some AM-EWOD cartridges, pixel size may be on the order of <NUM> or <NUM>, which requires even higher alignment precision. As one example, the requirement for precise cartridge alignment can relate to the alignment of instrument magnets located in the AM-EWOD instrument relative to the electrode array, such as for example in connection with magnetic bead-based washing operations. High precision alignment of the magnets relative to the electrode array is required for bead-based washing to be reliable and effective. In addition to alignment to the magnets, as another example cartridge alignment to optical and/or thermal components in the AM-EWOD instrument also may be important.

Precise mechanical cartridge alignment, however, particularly for the smaller ranges of array element (pixel) sizes, may be difficult to achieve given the cumulative tolerance stack of the mechanical components. For example, tolerance contributions from glass cutting of the cartridge substrates, alignment of the glass substrates within the plastic housing of the AM-EWOD instrument, alignment of instrument magnets relative to mechanical docking features for the cartridge, and the like can combine to accumulate an overall tolerance range that may not preclude significant misalignment for certain high-precision applications. Conventional configurations do not address the potential for such misalignment of the AM-EWOD cartridge relative to the AM-EWOD instrument components.

<CIT> proposes an electrode drive and sensing circuit and method for a fluidics droplet actuator apparatus. The circuit comprises a droplet operations electrode. An electrowetting (EW) driver is connected to the droplet operations electrode by a signal path. The EW driver is to supply an electrowetting drive signal component to the droplet operations electrode. A capacitance measurement (CM) device is connected to the droplet operations electrode by the signal path. The CM device is to sense a sensing signal component indicative of at least one of a presence or absence of a droplet at the droplet operations electrode. A first coupling circuit is positioned between the EW driver and the droplet operations electrode along the signal path. A second coupling circuit is positioned between the CM device and the same droplet operations electrode along the signal path.

<CIT> proposes a multiplex fluid processing cartridge includes a sample well, a deformable fluid chamber, a mixing well with a mixer disposed therein, a lysis chamber including a lysis mixer, an electrowetting grid for microdroplet manipulation, and electrosensor arrays configured to detect analytes of interest. An instrument for processing the cartridge is configured to receive the cartridge and to selectively apply thermal energy, magnetic force, and electrical connections to one or more discrete locations on the cartridge and is further configured to compress the deformable chamber(s) in a specified sequence.

There is a need in the art, therefore, for an improved system and method for AM-EWOD device operation that can account for a mechanical tolerance range that may not be suitable for certain high-precision device operations. The present invention addresses such deficiencies of conventional configurations by sensing components of the AM-EWOD instrument that are external to the AM-EWOD cartridge, and modifying a reaction protocol or script of droplet manipulations in a manner that accounts for mechanical tolerances of the AM-EWOD system. To achieve such result, sensor circuitry within the array element circuitry of the array elements can perform dual functions of both sensing liquid droplets that are positioned internally within the AM-EWOD cartridge, and sensing external locators that may be positioned within the broader AM-EWOD instrument but external to the AM-EWOD cartridge. Outputs from the sensor circuitry is used for adjusting the position of droplet manipulation operations as part of the reaction protocol or script to account for mechanical misalignment within the AM-EWOD system, and particularly misalignment of the AM-EWOD cartridge relative to components of the AM-EWOD instrument.

As referenced above, each AM-EWOD array element contains an impedance or capacitance sensor function integrated into the array element circuitry. Typically, this sensing circuitry is used for sensing droplet position in relation to droplet manipulation operations, but in accordance with embodiments of the present invention, the sensing circuity also is configured to sense the position of a conductive locator positioned within the AM-EWOD instrument, when such locator is brought close to, or into contact with, the outer surfaces of the glass substrates of the AM-EWOD cartridge. For example, the conductive locator may be one of the magnets in the instrument used for magnetic bead-based washing. The conductivity of the locator needs only to be sufficient to conduct electrical signals through the locator to perform the described sensing function. Because the electrical current conducted through the locator in the described embodiments is small, the locator only needs to be slightly conductive, for example having a resistance of <NUM> Mohm or less.

In an exemplary embodiment, a voltage signal is applied to a magnet element in the instrument. A sensor image is generated based on the outputs of the array element sensing circuitry, and the sensor image shows the transduced position of the magnet element. In an alternative embodiment, a passive arrangement may be implemented in which there is no electrical signal applied to the locator, whereby highly sensitive sensing circuity is capable of detecting the locator without application of a voltage signal to the locator.

The sensing circuitry of the array elements is thus capable of detecting the position of locators within the AM-EWOD instrument, for example the instrument magnets referenced above, relative to the electrode array. Two or more locator points may be detected to account for rotational as well as translational misalignment within a plane of the element array. The misalignment is determined by the AM-EWOD control system, and based on the extent of misalignment, the control system compensates for the misalignment by adjusting the reaction protocol or script. For example, the control system may control actuation voltages to nudge or slightly move the centroid of a droplet relative to an array element so the centroid becomes optimally located relative to the instrument magnet or other desired instrument component.

An advantage of the present invention is that accounting for misalignment by adjusting the reaction protocol or script relaxes the mechanical tolerance requirements on cartridge/instrument alignment, which may make the cartridge and/or instrument easier and cheaper to produce. A further advantage is that the efficacy of the wash operation or other droplet operations may be improved by more accurate alignment by precise droplet position adjustments, which may reduce the volume of supernatant fluid surrounding a bead pellet, or otherwise may reduce the amounts of sample or reagent fluids that are required for a reaction protocol.

An aspect of the invention, therefore, is a microfluidic system and related method of operation that accounts for misalignment of an AM-EWOD cartridge relative to a microfluidic instrument (i.e. an instrument that controls the microfluidic cartridge) by determining a position of a locator component of the microfluidic instrument, and modifying a reaction protocol or script of droplet manipulation operations in a manner that compensates for the misalignment. In exemplary embodiments, the microfluidic system includes: an electro-wetting on dielectric (EWOD) cartridge comprising an element array configured to receive liquid droplets, the element array comprising a plurality of individual array elements each including array element circuity comprising sensing circuitry that is integrated into the array element circuitry; a microfluidic instrument that is configured to receive the EWOD cartridge and having an electrically conductive locator that is external to the EWOD cartridge; and a control system configured perform electrowetting operations by controlling actuation voltages applied to the element array to perform manipulation operations as to liquid droplets present on the element array. The control system further is configured to: read an output from the sensing circuitry, determine a position of the locator relative to the element array based on the output, and determine a misalignment of the EWOD cartridge relative to the microfluidic instrument based on the position of the locator. The microfluidic system further may include a voltage supply that applies a voltage perturbation to the locator, and the control system reads the output from the sensing circuitry in response to the voltage perturbation applied to the locator. The control system further is configured to adjust a droplet manipulation operation to compensate for the determined misalignment. The control system may perform such operations by executing program code stored on a non-transitory computer readable medium.

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.

The present invention pertains to an improved system and method for AM-EWOD device operation that can account for component misalignment from a mechanical tolerance range that may not be suitable for certain high-precision device operations. The present invention operates by sensing components of the AM-EWOD instrument that are external to the AM-EWOD cartridge, and modifying a reaction protocol or script of droplet manipulations in a manner that accounts for mechanical tolerances of the AM-EWOD system. To achieve such result, sensor circuitry within the array element circuitry of the array elements can perform dual functions of both sensing liquid droplets that are positioned internally within the AM-EWOD cartridge, and sensing external locators that may be positioned within the broader AM-EWOD instrument but external to the AM-EWOD cartridge. Outputs from the sensor circuitry may be used for adjusting the position of droplet manipulation operations as part of the reaction protocol or script to account for mechanical misalignment within the AM-EWOD system, and particularly misalignment of the AM-EWOD cartridge relative to components of the AM-EWOD instrument.

<FIG> is a drawing depicting a perspective view of an exemplary AM-EWOD based microfluidic system <NUM> in accordance with embodiments of the present invention. <FIG> is a schematic drawing depicting a cross-sectional view of the microfluidic system <NUM> of <FIG>. The microfluidic system <NUM> includes a microfluidic cartridge <NUM>, which typically is disposable and intended for one-time use, and a microfluidic instrument <NUM> into which the microfluidic cartridge <NUM> is docked. As used herein, the term microfluidic instrument generally refers to a control device or control unit that controls the microfluidic cartridge. The microfluidic cartridge <NUM> is configured for EWOD or AM-EWOD operation and thus typically includes a thin film transistor (TFT) glass substrate <NUM>, a top substrate <NUM>, and a plastic housing <NUM> into which the glass substrates are embedded. The plastic housing may incorporate adhesives for securing the components in place, and internal spacer elements for spacing and sealing the two glass substrates. The microfluidic cartridge <NUM> also includes a first electrical connector <NUM> for mating to the microfluidic instrument <NUM> in a manner that permits electrical signals to be exchanged between the microfluidic cartridge <NUM> and the microfluidic instrument <NUM>. As referenced above, the microfluidic cartridge <NUM> is configured for EWOD or AM-EWOD operation, and thus the TFT substrate <NUM> and related components may include array elements, array element circuitry, and control signal lines as described above with reference to <FIG>.

The microfluidic instrument <NUM> is configured to receive the microfluidic cartridge <NUM> and is designed to make insertion and removal of a microfluidic cartridge straightforward for the user. The microfluidic instrument <NUM> includes a second electrical connector <NUM> that mates with the first electrical connector <NUM> to permit the electrical signals to be exchanged between the microfluidic cartridge <NUM> and the microfluidic instrument <NUM>. The microfluidic instrument <NUM> further includes docking features 116a and 116b for mechanically supporting and positioning the microfluidic cartridge <NUM> during insertion and removal. The docking features may interact with housing features <NUM> of the microfluidic cartridge <NUM> to aid in the insertion, removal, and positioning of the microfluidic cartridge <NUM> within the microfluidic instrument <NUM>. It will be appreciated that any suitable configuration of docking features and cooperating housing features may be employed. Docking may be achieved by sliding insertion, clamping, or any other mechanical means suitable for positioning the microfluidic cartridge within the instrument.

The microfluidic instrument <NUM> may have a benchtop format, that for example is designed for use in an analytical laboratory. The microfluidic instrument <NUM> also may be miniaturized into a hand-held format that for example is appropriate for point-of-care applications in medical treatment facilities. The microfluidic instrument <NUM> includes components that permit control of the microfluidic cartridge <NUM> to perform a variety of chemical and biochemical reaction protocols and scripts by AM-EWOD operation. The microfluidic instrument <NUM>, therefore, may include the following components: control electronics for supplying voltage supplies and timing signals for controlling actuation and de-actuation of the AM-EWOD array elements; heater elements <NUM> for heating portions of the AM-EWOD array elements to control the temperature of the liquid droplets, which is desired or required for certain reaction protocols; optical components or sensors <NUM> that measure optical properties of droplets on the AM-EWOD element array; magnet elements <NUM> for applying magnetic fields to the liquid droplets and the AM-EWOD element array; and features for liquid input or extraction, such as for example pipettes incorporated into the microfluidic instrument. The optical components <NUM> may include both light sources, such as for example light-emitting diodes (LEDs) or laser diodes, for illuminating liquid droplets, and also detection elements, such as for example photodiodes or other image sensors for detecting the optical signals returned from the liquid droplet. Optical measurements of liquid droplets may employ sensing techniques such as absorbance, fluorescence, chemiluminescence, and the like. As to the magnets <NUM>, many reaction protocols employ the use of magnetic beads within liquid droplets to perform purification or "washing" steps. By using magnetic fields applied from magnets in the microfluidic instrument, magnetic beads may be clumped together or released and be moved through the body of the liquid droplet to perform such washing steps.

The microfluidic cartridge <NUM> includes a two-dimensional active matrix array of array elements having electrodes on which the droplets are manipulated, such as described above with respect to <FIG>. Actuation patterns applied to individual electrodes are controlled to perform various droplet manipulations as described above in connection with <FIG>. Typical electrode widths are <NUM>, <NUM>, or may be as small as <NUM>. The liquid droplets may be of corresponding size and may be positioned in x-y space to array-element size precision for performing droplet manipulation operations.

<FIG> is a drawing depicting a block diagram of operative portions of the exemplary microfluidic system <NUM> of <FIG> and <FIG>. Similarly as described with respect to <FIG>, the microfluidic instrument <NUM> may include a computer-based control system <NUM> that controls instrument electronics <NUM> via a data link <NUM>. Under such control, the instrument electronics supplies actuation data signals <NUM>, and reads out sensor data signals <NUM>, via an instrument/cartridge electrical connector interface <NUM> (e.g., including the electrical connectors <NUM> and <NUM> of <FIG>). The control system <NUM> may include a storage device <NUM> that may store any application software and any data associated with the system. The control system <NUM> and instrument electronics <NUM> may include suitable circuitry and/or processing devices that are configured to carry out various control operations relating to control of the microfluidic cartridge <NUM>, such as a CPU, microcontroller or microprocessor. The microfluidic cartridge <NUM> includes an element array <NUM> of individual array elements <NUM> comparably as described above, upon which liquid droplets <NUM> may be dispensed to perform droplet manipulation operations by actuating and de-actuating one or more array elements in accordance with the actuation data signals <NUM>. The sensor data signals <NUM> further may be outputted by circuitry of the microfluidic cartridge <NUM> to the instrument electronics <NUM>.

Accordingly, the control system <NUM> may execute program code embodied as a control application stored within the storage device <NUM>. It will be apparent to a person having ordinary skill in the art of computer programming, and specifically in application programming for electronic control devices, how to program the control system to operate and carry out logical functions associated with the stored control application. Accordingly, details as to specific programming code have been left out for the sake of brevity. The storage device <NUM> may be configured as a non-transitory computer readable medium, such as a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), or any other suitable medium. Also, while the code may be executed by control system <NUM> in accordance with an exemplary embodiment, such control system functionality could also be carried out via dedicated hardware, firmware, software, or combinations thereof, without departing from the scope of the invention.

The control system may be configured to perform some or all of the following functions:.

The control system <NUM>, such as via the instrument electronics <NUM>, may supply and control the actuation voltages applied to the electrode array of the microfluidic cartridge <NUM>, such as required voltage and timing signals to perform droplet manipulation operations and sense liquid droplets on the AM-EWOD element array. The control system further may execute the application software to generate and output control voltages for droplet sensing and performing sensing operations.

The various methods described herein pertaining to enhanced microfluidic operation may be performed using AM-EWOD structures and devices described with respect to <FIG>, including for example any control electronics and circuitry, sensing capabilities, and control systems including any processing device that executes computer application code stored on a non-transitory computer readable medium. A reaction protocol including series and/or parallel combinations of droplet manipulation operations are typically conducted in accordance with software instructions that form a script, which may include a script specific to the particular reaction protocol being executed by the droplets. The reaction protocol also is typically conducted using feedback, whereby information from the sensors of droplet sizes and droplet positions is fed back to the software, and the sequence of droplet manipulation operations in time and/or space is adjusted.

To achieve high precision in droplet manipulation operations requiring interaction with the microfluidic instrument components, such as the magnets, optical components, or heaters, it is necessary for the microfluidic cartridge to be well aligned within the microfluidic instrument. Accordingly, the magnets, heaters, or optical components need to be reliably and reproducibly positioned with respect to the array of element electrodes, so that typically an instrument feature (e.g. position of a magnet) is co-located to within one pixel precision relative to a liquid droplet. As referenced above, however, precise mechanical cartridge alignment, particularly for the smaller ranges of array element (pixel) sizes, may be difficult to achieve given the cumulative tolerance stack of the mechanical components. For example, tolerance contributions from glass cutting of the cartridge substrates, alignment of the glass substrates within the plastic housing of the AM-EWOD instrument, alignment of instrument magnets relative to mechanical docking features for the cartridge, and the like can combine to accumulate an overall tolerance range that may not preclude significant misalignment for certain high-precision applications.

The need for precise alignment may be particularly important with relation to the microfluidic instrument magnets. Magnetic bead-based operations, such as for example droplet washing operations, require the actuation patterns aligned with the array element electrodes to be correlated with the magnet positions so as to achieve the best results. An example of such a washing method is described in Applicant's <CIT>). Droplets are manipulated into precise shapes and magnetic beads are moved through a narrow neck formed in the liquid droplet. If the magnets are misaligned relative to the liquid droplet, the washing operation may not be successful. High precision alignment becomes even more significant for reaction protocols, and in particular washing operations, using relatively small droplets, such as for example of diameter on the order of one array element (pixel).

For example, <FIG> is a drawing depicting an exemplary washing operation with bead separation based on the use of instrument magnets. In this example, a liquid droplet <NUM> is dispensed onto the element array <NUM> of individual array elements <NUM>. Magnetic beads are incorporated into the liquid droplet <NUM>, which form a magnetic bead clump <NUM> under the magnetic field generated by one or more instrument magnets (not shown). As shown in the progression of the portions of <FIG>, due to the magnet position relative to the liquid droplet <NUM>, following the washing process a separation operation can be performed whereby the clumped magnetic beads <NUM> is split away from the liquid droplet <NUM>. As seen in the progression of <FIG>, the droplet <NUM> containing the magnetic beads <NUM> is moved as indicated by the arrow in the figure by electrowetting forces across the element array <NUM>. A region of high magnetic field is created by positioning an instrument magnet close the element array. The magnetic beads within the liquid droplet clump together as shown in the region of a high magnetic field gradient. By continuing to move the droplet, a split occurs with the magnetic beads <NUM> (and a small surrounding shell of liquid) becoming separated from the liquid droplet <NUM>. In this example, if the instrument magnet is misaligned relative to the position and movement of the liquid droplet, the separation operation does not work as intended as shown in <FIG>.

The present invention enhances the implementation of droplet manipulation operations on the AM-EWOD element array of the microfluidic cartridge. This is achieved by ensuring the manipulation operations are performed at a position on the element array that is optimally aligned to a component of the microfluidic instrument. Embodiments of the enhanced system and method operate to measure the alignment of the microfluidic cartridge relative to the microfluidic instrument based on the position of a locator component of the microfluidic instrument, and adjust the software reaction protocol or script to control the position of the liquid droplet manipulation operations accordingly.

An aspect of the invention, therefore, is a microfluidic system and related method of operation that accounts for misalignment of an AM-EWOD cartridge relative to a microfluidic instrument by determining a position of a locator component of the microfluidic instrument, and modifying a reaction protocol or script of droplet manipulation operations in a manner that compensates for the misalignment. In exemplary embodiments, the microfluidic system includes: an electro-wetting on dielectric (EWOD) cartridge comprising an element array configured to receive liquid droplets, the element array comprising a plurality of individual array elements each including array element circuity comprising sensing circuitry that is integrated into the array element circuitry; a microfluidic instrument that is configured to receive the EWOD cartridge and having an electrically conductive locator that is external to the EWOD cartridge; and a control system configured perform electrowetting operations by controlling actuation voltages applied to the element array to perform manipulation operations as to liquid droplets present on the element array. The control system further is configured to: read an output from the sensing circuitry, determine a position of the locator relative to the element array based on the output, and determine a misalignment of the EWOD cartridge relative to the microfluidic instrument based on the position of the locator. The microfluidic system further may include a voltage supply that applies a voltage perturbation to the locator, and the control system reads the output from the sensing circuitry in response to the voltage perturbation applied to the locator. The control system further is configured to adjust a droplet manipulation operation to compensate for the determined misalignment. The control system may perform such operations by executing program code stored on a non-transitory computer readable medium.

<FIG> is a cross-sectional schematic drawing depicting a portion of an exemplary microfluidic system <NUM> in accordance with embodiments of the present invention. The microfluidic system <NUM> includes a microfluidic cartridge <NUM> having an element array <NUM> that is inserted into a microfluidic instrument <NUM> as previously described. For simplicity, instrument docking features <NUM> are shown with other instrument components being omitted from the figure for illustration purposes. In this exemplary embodiment, the microfluidic instrument <NUM> includes a conductive locator <NUM> to which an electrical signal is applied by a voltage supply <NUM> via any suitable electrical connection <NUM>. The conductive locator <NUM> is positioned, or can be moved within the microfluidic instrument so as to be positioned, within close proximity to or touching the lower surface of the microfluidic cartridge <NUM> corresponding to the TFT substrate.

The locator <NUM> may be an existing feature in the microfluidic instrument <NUM>, for example a magnet, optical component, heater, or the like. In a preferred implementation, accurate results are best achieved when the locator <NUM> is the same instrument component as to which precise alignment is desired to perform a droplet manipulation operation, e.g., an instrument magnet for a washing operation, a heater for a temperature-controlled reaction step, an optical component for an optical illumination or optical sensing operation, and so on. Accordingly, the locator <NUM> is a component of the microfluidic instrument <NUM> that is external from the microfluidic cartridge <NUM>. In addition, different instrument components potentially may act as a locator, with the specific locator being selected by the control system from among the potential locators depending upon the operation to be performed at a given point or step in a reaction protocol or script.

In operation, the microfluidic cartridge <NUM> is inserted into the microfluidic instrument <NUM> at an inserted position relative to the locator <NUM>, as for example may be dictated by the instrument docking features <NUM>. As referenced above, each array element in the element array <NUM> has integrated impedance or capacitance sensing circuity that is integrated into the array element circuitry. During droplet manipulation operations, the sensing circuitry is used for sensing droplet position, and in accordance with embodiments of the present invention the sensing circuity also is configured to sense the position of the locator <NUM> when such locator is brought close to, or into contact with, the outer surface of one of the glass substrates of the AM-EWOD cartridge <NUM> as positioned within the microfluidic instrument <NUM>. In this embodiment, a voltage signal is applied to the locator <NUM> by the electrically connected voltage supply <NUM>. Based on output signals from the sensing circuitry of the associated array elements, a sensor image is read out by the instrument electronics and control system, which shows the transduced position of the locator <NUM> relative to the element array <NUM> of the microfluidic cartridge <NUM>. A misalignment is determined by the control system analysis of the sensor image. Typically, two or more locator points may be detected to account for rotational as well as two-dimensional translational misalignment along the element array. Based on the extent and nature of any misalignment, the control system compensates for the misalignment by adjusting the reaction protocol or script. For example, the control system may control actuation voltages to nudge or slightly move the centroid of a liquid droplet relative to the element array so the centroid becomes optimally located relative to the conductive locator in the microfluidic instrument for performing the desired operation.

The above compensation for misalignment may be performed using any suitable array element circuitry. For example, <FIG> is a drawing depicting a two-dimensional array of elements including exemplary array element circuitry for an AM-EWOD device that embodies a previous design of Applicant. <FIG> depicts a <NUM> x <NUM> element array, although it will be appreciated that comparable principles are applicable to any array size. This basic array element circuitry has three thin film transistors (TFTs T1, T2, and T3) and two capacitors (C1 and C2), and is associated with seven addressing lines. The boundaries of a pixel are denoted by the dotted line. Row and column addressing lines are shown passing through the pixel in the horizontal and vertical directions respectively. Power supply connections, which could in principle be supplied either in row or column lines, are shown by short horizontal lines (e.g. VCCA in <FIG>). Connecting wires are shown with a solder dot, and without the dot, crossing lines do not connect. The Rdrop and Cdrop represent the resistance and capacitance across the device from the reference (top) electrode TP to the hydrophobic coating on which the liquid droplet may sit, and any other insulator layers incorporated into the device are represented by the capacitance CI. The values of Rdrop and Cdrop will vary based on the presence or absence of a liquid droplet, as described above with respect to <FIG>. Example AM-EWOD devices having this basic circuit design are described in Applicant's commonly assigned <CIT>) and <CIT>).

As described in such previous patent documents, the circuitry of <FIG> generally is operated as follows as to each array element. To program an array element by writing voltage data to said array element, the voltage to be programmed is loaded onto addressing line SL, and a pulse is applied to the gate line GL appropriate for the row being programmed. This turns on a drive transistor T1, and the circuit node connected to the electrode is charged to the programmed voltage. When GL is taken low, this voltage is preserved, stored on a storage capacitor C1. Typically, C1 is larger than the second or sensor capacitor C2 by at least about an order of magnitude. To perform sensing, in a reset step a reset transistor T2 is turned on by an RST signal, so the gate of a sensor readout transistor T3 charges to VCCA. In conventional configurations, VCCA is a reset potential chosen below the threshold voltage of T3 such that T3 remains off and any previous voltage is cleared. In a sensing step, the RST signal is set low so that the gate of sensor readout transistor T3 is not driven and an addressing line RWS is pulsed. For the duration of the RWS pulse, the electrode potential is perturbed to a higher voltage. The change in voltage achieved is principally a function of the ratio of capacitor C1 to the total capacitance at the electrode, which includes the load associated with whether the droplet is present or absent. The perturbation is coupled through the sensor capacitor C2 to the gate of T3, and transistor T3 is accordingly turned on to an extent determined by the amplitude of the pulse as coupled. A pixel voltage supply VPIX provides a voltage input so as to generate an output current through T3, which again will be dependent upon the voltage coupled to the gate of T3. The resultant current passes through T3 and is sunk down a sensor output column line COL, which may then be sensed by detection circuitry at the bottom of the column (not shown).

This driving scheme may be modified for sensing a conductive locator in accordance with embodiments of the present invention. <FIG> is a timing diagram for operation of the circuitry of <FIG> to sense a locator in accordance with embodiments of the present invention. Generally, in a droplet sensing operation, a perturbation voltage is applied to an addressing line such as RWS. For sensing the conductive locator, as shown in <FIG> the RWS line instead is maintained at a DC voltage and a perturbing voltage is applied directly to the locator (LOC), which is illustrated schematically in <FIG> by the electrical connection of the voltage supply <NUM> to the locator <NUM>. The resultant perturbation is coupled to the element electrodes of array elements in proximity to the locator. As further shown in <FIG>, the magnitude of the perturbation of an element electrode potential varies in accordance with the degree of proximity to the locator. The magnitude of the perturbation of an element electrode potential in close proximity to the locator is greater as compared to the magnitude of the perturbation of an element electrode potential farther away from the locator. The magnitude of the electrode perturbation may be measured by measuring the resultant output current down the output column line COL. Commensurately, the magnitude of the output current measured from an element electrode in close proximity to the locator is greater as compared to the magnitude of the output current measured from an element electrode farther away from the locator.

It will be appreciated that a similar modification to the driving scheme can be applied to any suitable array element circuitry configuration, including the various circuit configurations described in the patent documents referenced in the background section of the current application.

The above operation is further illustrated in <FIG>, which is a drawing depicting an exemplary portion of an AM-EWOD cartridge <NUM> in relation to a locator <NUM> of a microfluidic instrument. Similarly as described above in connection with other figures, the microfluid cartridge <NUM> includes a first hydrophobic coating <NUM> and a second hydrophobic coating <NUM> that define a channel <NUM> into which liquid droplets and a filler fluid (e.g., oil) may be dispensed. The cartridge <NUM> further may include a TFT glass substrate <NUM> onto which there is patterned an array of element electrodes <NUM>. Four element electrodes 172a-d are shown in this example, although comparable principles apply to any size electrode array. The element electrodes 172a-d are spaced apart from the first hydrophobic coating <NUM> by an ion barrier <NUM>, and a reference electrode <NUM> may be deposited on the second hydrophobic coating <NUM> opposite from the channel <NUM>.

<FIG> depicts a state in which a voltage is applied to the locator <NUM>, which is conductive. The voltage perturbation applied to the locator <NUM> couples to the electrode array <NUM> capacitively through the glass substrate, as illustrated by representative field lines <NUM>. The resultant electric field is strongest at the element electrode in closest proximity to the locator <NUM>, which in this example is element electrode 172b. The electric field is weaker at element electrodes 172a and 172c, and essentially is negligible at element electrode 172d. In this manner, the modified method of driving causes the element array to function as a capacitive array sensor that can detect the position and proximity of a conductive locator <NUM> that is external to the microfluidic cartridge <NUM>.

<FIG> is a drawing depicting an output image <NUM> that is derived from output currents measured from the element array <NUM> when a voltage perturbation is applied to the locator <NUM>. The electrical interaction of the locator with the element array is indicated by the output image, with the shading in this example representing the degree of proximity of array elements to the locator with the darkest image portion <NUM> corresponding to the array element closest to the locator. Image portions that correspond to array elements farther form the locator are illustrated with less dark shading, with the shading darkness decreasing with distance from the locator. In this manner, the position of the locator relative to the element array is detectable to a resolution of around one array element (pixel). Such resolution is achieved with any common sized pixel in an AM-EWOD device, such as for example electrode widths of <NUM>, <NUM><NUM> or <NUM>. The output image <NUM> may be generated by the control system, and based on the output image <NUM>, the control system can modify the reaction protocol or script to adjust droplet positioning as warranted for optimal positioning of the liquid droplet for a droplet manipulation operation.

In the example of <FIG>, the locator sensing is considered active sensing in that the output image is derived from measuring the output current in response to a voltage perturbation applied to the locator. For array element circuitry of high sensitivity, passive sensing of a conductive locator can be sufficient provided such circuitry is sufficiently sensitive to detect a passive conductive locator to which no electrical signal or perturbation is applied. An example of such a high-sensitive circuit is described in Applicant's Appl Serial No. <CIT>. In such example, the sensing circuitry is improved by enhancing the sensitivity to very small capacitance variations, which for the present invention can be associated with locator positioning even without applying a voltage perturbation to the locator. As a non-limiting example of a high-sensitive circuit, to accomplish such enhanced sensitivity in the circuit design of the '<NUM> application, a pre-charging effect is applied whereby the sensor readout transistor in an array element is altered to turn on the sensor readout transistor during a sensing phase. For example, a positive pre-charging voltage may be applied across the gate and source of the sensor readout transistor to turn said transistor on, or a negative voltage may be applied across the gate and source of a p-type sensor readout transistor to turn on the sensor readout transistor. The element array may be operated in either a self or mutual capacitance mode as described in the '<NUM> Application. The positioning of the locator in near proximity to the element array results in interaction with the electric field distribution in a similar way as shown in <FIG>, which results in a change in the capacitance measured as "present" at an electrode within the array.

The present invention thus provides a way of measuring the microfluidic instrument/microfluidic cartridge alignment. The alignment information subsequently is utilized by the control system to perform a compensation operation that includes feedback of the measured position information to align droplet manipulation operations, and adjustment of the reaction protocol or script to optimally align the droplet manipulation operations relative to pertinent microfluidic instrument components that are external to the microfluidic cartridge, such as for example instrument magnets, heater, or optical components (which also may act as the locators in the compensation method). In practice, this may be accomplished by the control system controlling actuation voltages to nudge or slightly move the centroid of a liquid droplet relative to the element array so the droplet centroid becomes optimally located relative to the pertinent instrument component.

An advantage of the present invention is that accounting for misalignment by adjusting the reaction protocol or script relaxes the mechanical tolerance requirements on cartridge/instrument alignment, which may make the cartridge and/or instrument easier and cheaper to produce. A further advantage is that the efficacy of a wash operation or other droplet manipulation operation may be improved by more accurate alignment by precise droplet position adjustments, which in the context of a wash operation in particular may reduce the volume of supernatant fluid surrounding a bead pellet, or otherwise may reduce the amounts of sample or reagent fluids that are required for a reaction protocol. The result is improved performance of droplet manipulation operations requiring critical or precise alignment, such as for example magnetic bead-based droplet operations (requiring precision alignment to instrument magnets), optical illumination and sensing (requiring precision alignment to optical components), and thermal control (requiring precision alignment to heater elements).

<FIG> is a cross-sectional drawing depicting a variation on the embodiment of <FIG>, in which the exemplary portion on of the AM-EWOD cartridge <NUM> is positioned in relation to multiple locators, e.g., a first locator <NUM> and a second locator <NUM> of a microfluidic instrument <NUM>. <FIG> is a drawing depicting a top view of the array of element electrodes <NUM> of <FIG> in relation to the locators <NUM> and <NUM>. The locators <NUM> and <NUM> are positioned respectively at Positions A and B relative to the element array <NUM>. By measuring the position of the element array <NUM> relative to multiple locators, the position of the cartridge <NUM> relative to the microfluidic instrument may be measured in two dimensions relative to the x-y plane of the element array. With such measurements, compensation of the reaction protocol or script for errors in position may be implemented accounting for planar x-y and rotational misalignment.

<FIG> is a drawing depicting a variation on the embodiment of <FIG> and <FIG>, in which the exemplary portion on of the AM-EWOD cartridge <NUM> is positioned in relation to multiple locators e.g., a first locator <NUM> and a second locator <NUM> of a microfluidic instrument, and the locators may be positioned at different distances spaced apart from or relative to the element array <NUM>. As referenced above, locators may correspond to instrument components utilized for certain operations, such as magnets for bead-based washing, heaters for thermal control, optical components for illumination and making optical measurements, and the like. Particularly as to magnets, it is desirable to move the magnets to be adjacent to or nearly touching the cartridge substrate when bead-based washing is performed, and moved farther away from the cartridge substrate when washing is not being performed to preclude any undesirable effects from magnetic fields. With such a system, location measurements with respect to multiple locators located at different distances with respect to the element array, and particularly instrument magnets, may provide an enhanced measurement of cartridge position relative to the locators to aid in determining planar x-y or rotational misalignment.

In this regard, <FIG> is a drawing depicting an output image <NUM> that is derived from output currents measured from the element array <NUM> when a voltage perturbation is applied to the first and second locators <NUM> and <NUM>. As shown in <FIG>, first locator <NUM> is essentially touching or near touching the cartridge substrate and second locator <NUM> is spaced farther apart from the cartridge substrate. Similarly as illustrated in the output image <NUM> of <FIG>, as to the output image <NUM> of <FIG>, the electrical interaction of the locators with the element array is illustrated with shading representing the degree of proximity of array elements to the locator, with the darker image portions corresponding to the array elements closest to the locator. In this example, image portion <NUM> is darkest as corresponding to the array element that is nearest to the first locator <NUM> that is essentially touching or near touching the cartridge substrate. Image portion <NUM> corresponds to an array element nearest to the second locator <NUM>. Image portion <NUM> thus appears less darkly shaded as compared to image portion <NUM> because the second locator <NUM> is farther from the cartridge substrate than the first locator <NUM>. Such measurements based on locators at different distances relative to the cartridge substrate may be used to detect the position of the cartridge in a "z" direction perpendicular to a plane of the electrode array <NUM>. Accordingly, such measurements may be used to compensate functions performed by the instrument for which z-alignment is significant, such as for example intensity of a light source (e.g., when the cartridge is further away, light intensity is increased), positioning of pipettes for fluid input or extraction, or for focus adjustment of detection optics such as a camera.

<FIG> is a cross-sectional drawing depicting a variation on the embodiment of <FIG>, in which the exemplary portion on of the AM-EWOD cartridge <NUM> is positioned in relation to a magnet array <NUM> of a microfluidic instrument. In this embodiment, the magnet array <NUM> includes multiple magnet elements <NUM> that may act as multiple locators. As seen in such figure, some of the magnet elements may be located essentially touching or near touching the cartridge substrate, while some of the magnet elements may be spaced apart from the substrate and maybe at different distances. As a matter of manufacturing, the magnet elements are movable toward and away from the cartridge substrate as needed for fluidic operations, and it desirable that all magnet elements be essentially touching the cartridge substrate when in the "up" position for use. Based on mechanical tolerances, however, there may be a slight variation of positioning when the magnet elements are moved into the position of use. In this example, the spacing is exaggerated, as the difference in spacing resulting from mechanical tolerances is slight (on the order of fractions of a millimeter), but is still measurable.

Accordingly, the magnet elements <NUM> that form the magnet array <NUM> are to be moved up or down as a single component with a common drive mechanism. When brought into the "up" position adjacent to the cartridge substrate, however, not all magnet elements will be precisely touching the cartridge substrate due to the mechanical tolerances. Measurement of the intensity of coupling from each magnet position can be used to determine which magnets are touching, how far away non-touching magnets are, and whether this configuration is within a requisite specification. If not, an error can be reported. In this manner, using the magnet elements as locators for measuring cartridge positioning may be used as a quality control function to ensure that any mechanical misalignments or deviations of magnet elements or other instrument components fall within acceptable specification ranges.

As referenced above, by measuring the precise positioning of the one or more locators, misalignment of the microfluidic cartridge relative to the microfluidic instrument can be compensated by adjustments to a reaction protocol or script. <FIG> is a drawing depicting an exemplary compensation operation performed in accordance with embodiments of the present invention. In <FIG>, an element array <NUM> of individual array elements <NUM> has dispensed thereon a liquid droplet <NUM> as to which a manipulation operation is to be performed as denoted by the arrow <NUM> (e.g., moving the position of the droplet). This operation is to be performed in relation to a locator <NUM> located within the microfluidic instrument, for example a magnet element.

The top portion of <FIG> illustrates the expected position of the locator <NUM> relative to the element array <NUM> based on mechanical insertion of the microfluidic cartridge relative to the microfluidic instrument. Based on simple mechanical assertion, the locator is expected to be adjacent to array element <NUM>, and thus the initial reaction protocol or script calls for electrowetting forces to move the liquid droplet <NUM> to be positioned at the array element <NUM>. As shown in the bottom potion of <FIG>, however, due to mechanical tolerances, the actual position of the locator <NUM> as measured using any of the embodiments above is adjacent to array element <NUM>. In this example, therefore, there is a misalignment of one pixel down and one pixel right from the expected position (based on the orientation of the figure). If the initial reaction protocol were to be followed, therefore, the droplet <NUM> would not be placed optimally to perform the desired droplet manipulation operation, e.g., a bead-based washing using a magnet <NUM> followed by magnet separation such as shown in <FIG> when moving the droplet. To compensate for the misalignment, therefore, the initial reaction protocol or script is modified to an adjusted reaction protocol or script, whereby the position of the liquid droplet <NUM> is modified from array element <NUM> to array element <NUM> to compensate for the misalignment.

More generally, such compensation methods may be employed for any suitable reaction protocol in which certain droplet manipulation operations should be performed at fixed positions. Examples (without limitation) of such operations include fluid input and extraction, magnetic bead-based washing, thermal control, optical illumination and sensing, and the like. In the example of <FIG>, a single droplet operation is positionally adjusted. In an alternative compensation method, the position of the whole reaction protocol is adjusted. Comprehensive positional adjustment may be advantageous, for example, when fluid loading and/or extraction is automatically performed by the microfluidic instrument whereby it is advantageous to align the fluid ports with the loading/extracting instrument, and it would thus be beneficial that all the droplet operations in the reaction protocol be adjusted in accordance with fluid input and/or extraction positions.

<FIG> is a cross-sectional schematic drawing depicting a variation on the exemplary microfluidic system <NUM> of <FIG>, wherein the microfluidic instrument <NUM> includes an automated input/extraction component <NUM>. The automated input/extraction component <NUM> may be an automated pipetting device integrated into the microfluidic instrument <NUM>, or may be a component separate from the portion of the microfluidic instrument that receives the microfluidic cartridge. Using an automated input/extraction component <NUM>, the position of an input/extraction element <NUM>, such as for example a pipette, in the x-y plane above the plane of the element array of the microfluidic cartridge <NUM> may be adjusted in accordance with the measured position of the locators, which in this example are the magnets <NUM> although any suitable locators may be used. The position of the locators in turn is used to measure the position of the microfluidic cartridge <NUM>. The microfluidic cartridge <NUM> includes a fluid port <NUM> that may be used for fluid input and/or extraction, and thus the position of the microfluidic cartridge is indicative of the position of the fluid port <NUM> relative to the pipette <NUM>. If there is a misalignment of the microfluidic cartridge relative to the microfluidic instrument based on the measurements of the locator positions, the position of the automated input/extraction component <NUM> may be adjusted to optimally align the pipette <NUM> with the fluid port <NUM>.

<FIG> is a schematic drawing depicting a variation on the exemplary microfluidic system <NUM> of <FIG>, wherein the microfluidic instrument <NUM> includes an optical instrument <NUM>. The optical instrument <NUM> implements optical illumination and/or sensing using an optics system <NUM>, which may include for example an illumination light source (e.g., laser, LED) and/or an optical sensor (e.g. photodiode, avalanche photodiode), with the position of the optical instrument <NUM> being precisely determined relative to the element array. The optical instrument <NUM> may be integrated as part of the microfluidic instrument or a component separate from the portion of the microfluidic instrument that receives the microfluidic cartridge. The position of the optical instrument <NUM> relative to the element array of the microfluidic cartridge <NUM> may be adjusted in accordance with the measured position of the locators, which in this example again are the magnets <NUM> although any suitable locators may be used. If there is a misalignment of the microfluidic cartridge relative to the optical instrument <NUM> based on the measurements of the locator positions, the position of the optical instrument <NUM> may be adjusted to optimally align the optics system <NUM> with any array element that has a liquid droplet that is to be optically illuminated and/or sensed.

An aspect of the invention, therefore, is a microfluidic system and related method of operation that accounts for misalignment of an AM-EWOD cartridge relative to a microfluidic instrument (i.e. an instrument that controls the microfluidic cartridge) by determining a position of a locator component of the microfluidic instrument, and modifying a reaction protocol or script of droplet manipulation operations in a manner that compensates for the misalignment. In exemplary embodiments, the microfluidic system includes: an electro-wetting on dielectric (EWOD) cartridge comprising an element array configured to receive liquid droplets, the element array comprising a plurality of individual array elements each including array element circuity comprising sensing circuitry that is integrated into the array element circuitry; a microfluidic instrument that is configured to receive the EWOD cartridge and having an electrically conductive locator that is external to the EWOD cartridge; and a control system configured to perform electrowetting operations by controlling actuation voltages applied to the element array to perform manipulation operations as to liquid droplets present on the element array. The control system further is configured to: read an output from the sensing circuitry; determine a position of the locator relative to the element array based on the output; and determine a misalignment of the EWOD cartridge relative to the microfluidic instrument based on the determined position of the locator. The microfluidic system may include one or more of the following features, either individually or in combination.

In an exemplary embodiment of the microfluidic system, the system further includes a voltage supply that applies a voltage perturbation to the locator, and the control system reads the output from the sensing circuitry in response to the voltage perturbation applied to the locator.

In an exemplary embodiment of the microfluidic system, the locator includes a magnet element.

In an exemplary embodiment of the microfluidic system, the locator comprises a magnet array comprising a plurality of magnet elements that are movable relative to the EWOD cartridge, and the controller is configured to determine the position of each magnet element relative to the element array.

In an exemplary embodiment of the microfluidic system, the locator comprises a plurality of locator elements and the controller is configured to determine the position of each locator element relative to the element array.

In an exemplary embodiment of the microfluidic system, the locator includes one or more of a magnet element, a heater, and an optical component.

The controller further is configured to adjust a manipulation operation of a liquid droplet to compensate for the determined misalignment of the EWOD cartridge relative to the microfluidic instrument.

In an exemplary embodiment of the microfluidic system, the controller is configured to adjust the manipulation operation by performing an electrowetting operation to move a liquid droplet to a position that is optimal relative to a component of the microfluidic instrument that is employed during the manipulation operation.

In an exemplary embodiment of the microfluidic system, the component of the microfluidic instrument that is employed during the manipulation operation includes the locator.

Another aspect of the invention is a related method of operating a microfluidic system according to any of the embodiments. In exemplary embodiments, the method of operating includes the steps of: reading an output from the sensing circuitry; determining a position of the locator relative to the element array based on the output; determining a misalignment of the EWOD cartridge relative to the microfluidic instrument based on the position of the locator; and adjusting a manipulation operation of a liquid droplet to compensate for the determined misalignment of the EWOD cartridge relative to the microfluidic instrument. The method of operating may include one or more of the following features, either individually or in combination.

In an exemplary embodiment of the method of operating, the method further includes applying a voltage perturbation to the locator, and reading the output from the sensing circuitry in response to the voltage perturbation applied to the locator.

In an exemplary embodiment of the method of operating, the method further includes determining a misalignment of the EWOD cartridge relative to a component of the microfluidic instrument that is employed during the manipulation operation; and adjusting the manipulation operation by performing an electrowetting operation to move a liquid droplet to a position that is optimal relative to the component of the microfluidic instrument that is employed during the manipulation operation.

In an exemplary embodiment of the method of operating, the component of the microfluidic instrument that is employed during the manipulation operation includes the locator.

In an exemplary embodiment of the method of operating, the locator is a magnet element and the manipulation operation is a magnetic bead-based washing and separation operation.

In an exemplary embodiment of the method of operating, a reaction protocol comprises a plurality of manipulation operations, and the method further comprises performing an electrowetting operation to move one or more liquid droplets to respective positions that are optimal relative to components of the microfluidic instrument that are employed during a plurality of manipulation operations that are part of the reaction protocol.

In an exemplary embodiment of the method of operating, the locator includes multiple locator elements, and the method further comprises determining a misalignment of the EWOD cartridge relative to the microfluidic instrument in the two-dimensional x-y plane corresponding to the element array, and/or determining a rotational misalignment of the EWOD cartridge relative to the microfluidic instrument.

In an exemplary embodiment of the method of operating, the locator comprises a magnet array having plurality of magnet elements that is movable between a first position closest to the EWOD cartridge and a second position farthest from the EWOD cartridge, the method comprising the steps of: moving the magnet array to the first position; determining a distance of each magnet element from the EWOD cartridge; determining whether the distances satisfy a specification; and outputting the result of the determination of whether the distances satisfy the specification.

In an exemplary embodiment of the method of operating, the method further includes generating an output image based on the output from the sensing circuitry that indicates the position of the locator relative to the element array.

Another aspect of the invention is a non-transitory computer-readable medium storing program code which is executed by a processing device for controlling a microfluidic system, the processing device being configured to perform electrowetting operations by controlling actuation voltages applied to the element array to perform manipulation operations as to liquid droplets present on the element array. The program code is executable by the processing device to perform the method steps accordingly to any of the embodiments.

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.

Claim 1:
A microfluidic system comprising:
an electro-wetting on dielectric (EWOD) cartridge (<NUM>,<NUM>,<NUM>) comprising an element array (<NUM>,<NUM>, <NUM>) configured to receive liquid droplets, the element array comprising a plurality of individual array elements (<NUM>, <NUM>, <NUM>, <NUM>) each including array element circuity comprising sensing circuitry (<NUM>) that is integrated into the array element circuitry;
a microfluidic instrument (<NUM>, <NUM>) that is configured to receive the EWOD cartridge (<NUM>,<NUM>,<NUM>) and having an electrically conductive locator (<NUM>,<NUM>,<NUM>, <NUM>, LOC) that is external to the EWOD cartridge; and
a control system (<NUM>) configured to perform electrowetting operations by controlling actuation voltages applied to the element array (<NUM>,<NUM>, <NUM>) to perform manipulation operations as to liquid droplets (<NUM>,<NUM>) present on the element array;
wherein the control system (<NUM>) further is configured to:
read an output from the sensing circuitry (<NUM>);
determine a position of the locator (<NUM>,<NUM>,<NUM>, <NUM>) relative to the element array (<NUM>,<NUM>, <NUM>, <NUM>) based on the output;
determine a misalignment of the EWOD cartridge (<NUM>,<NUM>,<NUM>) relative to the microfluidic instrument (<NUM>, <NUM>) based on the determined position of the locator (<NUM>,<NUM>,<NUM>, <NUM>); and
adjust a manipulation operation of a liquid droplet (<NUM>,<NUM>) to compensate for the determined misalignment of the EWOD cartridge (<NUM>,<NUM>,<NUM>) relative to the microfluidic instrument (<NUM>, <NUM>).