Devices, systems and methods for droplet manipulation

Abstract

The present invention relates to an apparatus for droplet manipulation, the apparatus comprising: a drive module configured to receive control signals and operate an electrowetting-on-dielectric (EWOD) chip configured to manipulate droplets; a droplet detection system having a light source and a lens-free imaging device configured to obtain at least one image including a droplet on the electrowetting-on-dielectric chip; and a control unit configured to receive at least one image from the droplet detection system and send control signals to the drive module. The control unit is further configured to adjust at least one of the control signals as a function of the at least one image. The present invention also relates to a system comprising the apparatus and an electrowetting-on-dielectric chip configured to receive signals from the drive module.

Description

[Technical Field]

[0001] The present invention relates generally to devices, systems and methods for droplet manipulation, and more particularly to devices, systems and methods for droplet manipulation via electrowetting-on-dielectric (EWOD). [Background technology]

[0002] Droplet manipulation has significant commercial and scientific potential in many biomedical applications. However, current state-of-the-art droplet manipulation is limited in terms of throughput, cost, and functionality.

[0003] One known technique for tracking droplets on an EWOD chip is described in document WO2016 / 174523 A1 by D. Cyril et al. Summary of the Invention

[0004] The invention is set out in the accompanying claims.

[0005] It is an object of the present invention to at least partially overcome the limitations of the prior art, and in particular to provide an apparatus, system and method for efficient droplet manipulation.

[0006] In a first aspect, the present invention relates to an apparatus for manipulating droplets, the apparatus comprising: a drive module configured to receive control signals and operate an electrowetting-on-dielectric (EWOD) chip, where the EWOD is configured to manipulate droplets; a droplet detection system having a light source and a lens-free imaging (LFI) device configured to obtain at least one image including droplets on the EWOD chip; and a control unit configured to receive at least one image from the droplet detection system and send control signals to the drive module, the control unit further configured to adjust at least one of the control signals as a function of the at least one image. The apparatus of the first aspect has several advantages. First, the lens-free imaging device can capture microscopic images with a large field of view (FOV), allowing for continuous and automatic monitoring and manipulation of multiple droplets in parallel. Second, the images captured by the lens-free imaging device enable adjustment and adaptation of one or more subsequent control signals for manipulating droplets. Third, the device is compatible with a variety of customized EWOD chips.

[0007] Any feature of the first aspect may be described correspondingly in the second aspect.

[0008] In a second aspect, the present invention relates to a system comprising an apparatus, the system further comprising an electrowetting-on-dielectric chip configured to receive a signal from a drive module, preferably in a replaceable cartridge. The system has the advantage of being compatible with various replaceable cartridges. According to exemplary embodiments, the replaceable cartridge is a disposable cartridge. According to exemplary embodiments, the replaceable cartridge is a reusable cartridge.

[0009] According to an exemplary embodiment, at least one of the adjusted control signals is for instructing the drive module to move the droplet to a predetermined position. An advantage of this exemplary embodiment is that the control unit automatically determines a path for moving the droplet to a predetermined position based on an image captured by the lens-free imaging device.

[0010] According to an exemplary embodiment, the control unit is configured to extract the position of the droplet from at least one image, and at least one of the control signals is adjusted based on said position, which has the advantage that not only can the positions of multiple droplets be captured in the image, but also EWOD pixels where no droplet is located.

[0011] According to an exemplary embodiment, the control unit is configured to adjust the control signal such that if the movement of the droplet differs from a first predetermined path after the first control signal, at least a subsequent control signal is sent to move the droplet to the predetermined location along a second path that differs from the first path. An advantage of this exemplary embodiment is that the path along which the droplet moves to the predetermined location can be adjusted to change to a different path during the movement of the droplet.

[0012] According to an exemplary embodiment, the at least one image comprises a holographic image. An advantage of this exemplary embodiment is that the control signals can be adjusted based on raw data, i.e., holograms, from the lens-free imaging device. Because the processing speed of the holograms is not constrained by the computation kernel, this allows the control unit to quickly adjust the control signals based on the holograms captured by the lens-free imaging device.

[0013] According to exemplary embodiments, the at least one image includes a two-dimensional (2D) image or a three-dimensional (3D) image. According to exemplary embodiments, the 2D image or the 3D image may be an image reconstructed from raw data, i.e., a hologram. According to exemplary embodiments, the 2D image or the 3D image may be captured by different imaging devices. Advantageously, the 2D image or the 3D image contains more information than a hologram. Therefore, the control signal may be adjusted as a function of the reconstructed image.

[0014] According to an exemplary embodiment, the control unit is configured to adjust at least one of the control signals as a function of at least one variable that the control unit obtains from the at least one image, each of the at least one variable representing a different characteristic of the droplets selected from one or more of: size, shape, spectral absorbance, spectral transmittance, turbidity, viscosity, and spatial distribution of at least one of the aforementioned characteristics. Advantageously, the characteristics including the morphology of the droplets are captured in the at least one image by the lens-free imaging device. The characteristics captured in the image can further be used as a variable for adjusting the control signal.

[0015] According to an exemplary embodiment, at least one of the control signals is adjusted to instruct the drive module to combine at least two droplets into one droplet.

[0016] According to exemplary embodiments, the control unit is configured to adjust at least one of the control signals to continue or stop mixing of the droplets as a function of at least one variable that the control unit obtains from the at least one image, the at least one variable representing the spatial distribution of at least one property or combination of properties of the droplets selected from one or more of: size, shape, spectral absorbance, spectral transmittance, turbidity, and viscosity. According to exemplary embodiments, the image is a 3D image. Advantageously, the use of the image allows the droplets to be mixed or merged and unified automatically.

[0017] According to an exemplary embodiment, the control unit is configured to adjust at least one of the control signals as a function of at least one variable that the control unit obtains from at least one image, each of said at least one variable being representative of an object in the droplet. Advantageously, features in the droplet image, i.e. the object, are captured in at least one image by a lens-free imaging device. Features in the image can further be used as a variable for adjusting the control signal.

[0018] According to an exemplary embodiment, the objects are cells. Advantageously, particles such as cells in a droplet can be captured in the image. Such particles in the image can further be used as variables for adjusting the control signal.

[0019] According to an exemplary embodiment, the object is an air bubble. Advantageously, defects such as air bubbles can be captured in the image. Such defects in the image can further be used as variables for adjusting the control signal.

[0020] According to an exemplary embodiment, the object is a different non-dissolving droplet, i.e., a further droplet that does not dissolve in the droplet. Advantageously, the characteristics of the droplet and the different droplets therein can be extracted. Advantageously, the difference between at least two droplets can be captured in at least one image by the lens-free imaging device. Such difference in the image can further be used as a variable for adjusting the control signal.

[0021] According to an exemplary embodiment, the control unit is configured to adjust at least one of the control signals as a function of at least one variable obtained by the control unit from at least one image, each of the at least one variable representing a different defect on the electrowetting-on-dielectric chip. An advantage of this exemplary embodiment is that the image also captures defects on the EWOD chip. These defects indicate fault locations on the EWOD chip. Therefore, the control signal can be adjusted to avoid such locations when manipulating the droplet.

[0022] According to an exemplary embodiment, the electrowetting-on-dielectric chip comprises multiple electrowetting-on-dielectric layers, and the control unit is configured to adjust at least one of the control signals as a function of at least one variable obtained by the control unit from multiple holographic images using focus adjustment, each of the at least one variable representing the presence or absence of droplets on a different electrowetting-on-dielectric. Advantageously, droplets can be manipulated simultaneously in parallel on multiple stacked EWOD layers, which allows for more efficient and rapid droplet manipulation.

[0023] Any feature of the second aspect may be described as corresponding to the first aspect.

[0024] In a third aspect, the present invention relates to a method for manipulating droplets in an apparatus according to embodiments herein, the method comprising the steps of: a. causing a control unit (104) to receive at least one image comprising a plurality of droplets; and b. causing the control unit (104) to generate control signals for simultaneously manipulating the droplets by electrowetting as a function of the received at least one image. Using such a method, it is advantageous to be able to simultaneously manipulate multiple droplets. According to exemplary embodiments, using such a method, at least 1,000 droplets are simultaneously manipulated. According to exemplary embodiments, using such a method, at least 10,000 droplets are simultaneously manipulated.

[0025] Any feature of the third aspect may be described as corresponding to the first or second aspect.

[0026] In a fourth aspect, the present invention relates to a computer program comprising instructions for causing an apparatus according to the first aspect to perform the steps of the method according to the third aspect. In a fifth aspect, the present invention relates to a computer readable medium having stored thereon the computer program of the fourth aspect. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows a block diagram of a first embodiment of an instrument for droplet manipulation, combined with a top view of an EWOD chip. [Figure 2] 1 shows a block diagram of a second embodiment of a system for droplet manipulation and a side view of an EWOD chip. [Figure 3] 10 shows a block diagram of a third embodiment of a system for droplet manipulation and examples of images of droplets in a hologram image and a reconstructed image. [Figure 4] 10 shows a block diagram of a fourth embodiment of a system for droplet manipulation and an example image from which droplet properties have been extracted. [Figure 5] 10 shows a block diagram of a fifth embodiment of a system for droplet manipulation and example images captured at time intervals to extract droplet characteristics. [Figure 6] 10 shows a block diagram of a sixth embodiment of a system for droplet manipulation and an example image of a droplet with an object. [Figure 7] 10 shows a block diagram of a seventh embodiment of a system for droplet manipulation and an example image of a droplet with an air bubble as a defect. [Figure 8] 10 shows a block diagram of an eighth embodiment of a system for droplet manipulation and a side view of an EWOD chip having two EWOD layers. [Figure 9] 13 shows an enlarged view of the droplet detection system and EWOD chip of a ninth embodiment of a system for droplet manipulation. [Figure 10] A close-up view of the solution being loaded onto the EWOD chip is shown. [Figure 11] 1 shows a close-up view of the droplet generation process. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present disclosure will be further clarified by the following description and the accompanying drawings. Various exemplary embodiments are described herein with reference to the following figures, in which like numerals indicate like entities. The described figures are schematic and non-limiting. Furthermore, reference numerals in the claims should not be construed as limiting the scope of the present disclosure. Furthermore, the same reference numerals in different figures refer to the same or similar elements.

[0029] The term "comprising" used in the claims should not be interpreted as being limited to the means listed thereafter, nor as excluding other elements or steps. Thus, it should be interpreted as specifying the presence of a stated feature, integer, step, or component, but not as excluding the presence or addition of one or more other features, integers, steps, components, or groups thereof. Thus, the term "comprising" covers both the presence of only the stated features (and thus can always be replaced with "consisting of" to limit the scope to the stated features) and the presence of these features together with one or more other features. Therefore, the term "comprising" in the present invention also includes, as an embodiment, the absence of additional components. Therefore, the scope of the expression "a device comprising means A and B" should not be interpreted as being limited to a device consisting only of components A and B. In the context of the present invention, the only relevant components of the device are A and B.

[0030] The term "a" should be interpreted as a function before a mass noun to indicate a particular kind or example, not as a function before a singular noun referring to one object.

[0031] The term "instrument" refers to a tool or device designed for droplet manipulation.

[0032] A droplet refers to a volume of a first fluid between 30 picoliters and 100 microliters in a second fluid when the first and second fluids are immiscible. According to an exemplary embodiment, the first fluid is aqueous and the second fluid is oil-based, such as silicone oil. According to an exemplary embodiment, a droplet can further include particles and / or objects, such as biological cells, gas bubbles, other droplets, etc. Droplet manipulation generally refers to actions applied to droplets, including, but not limited to, changing the position of droplets, interacting with droplets, and causing changes in droplet properties.

[0033] As shown in Figure 1, the present invention relates to an apparatus (100) comprising a drive module (101), a droplet detection system (103), and a control unit (104). The apparatus (100) is compatible for use with an EWOD chip (102).

[0034] An example EWOD chip, as shown in FIG. 2, includes an EWOD layer (25), a transparent cover (24), and a thin-film transistor (TFT) backplane (26). The EWOD layer (25) and the transparent cover (24) are spaced apart to form a manipulation space where droplets can be manipulated and where liquid is disposed around the droplets. The manipulation space may be referred to as an electrowetting layer. The manipulation space typically has a height of 10-1000 μm to enable droplet manipulation. According to an example embodiment, the transparent cover (24) is a glass cover. According to an example embodiment, the EWOD chip further includes a fluidic device, such as a reservoir, for providing droplets and fluid. According to an example embodiment, the reservoir contains biochemicals, such as cell culture media, buffer solutions, oil, or water. According to an example embodiment, the biochemicals can be supplied by a fluid pump attached to the reservoir, or can be supplied manually or automatically via a pipette. Such biochemicals may be contained, for example, in tubes, containers, plates, syringes that are attached to fluid pumps.

[0035] The EWOD chip further comprises an electronic interface (21) configured to receive control signals from a driving module (101). The EWOD layer (25) comprises an electrode array. The TFT backplane provides electrical connections between the driving module (101) and each electrode in the electrode array. The TFT backplane has electrode pins for establishing electrical connections with devices. The droplets are manipulated by the electrodes, which alter the wetting properties of the droplets through electrostatic effects controlled by the electrode array, changing the contact angle of the droplets on the EWOD chip surface. The EWOD chip may further comprise a carrier for support purposes.

[0036] According to an exemplary embodiment, the driving module (101) has an electronic interface compatible with the corresponding electronic interface of the EWOD chip. Such a driving module (101) receives control signals from the control unit. According to an exemplary embodiment, the driving module (101) is a general computer, an ASIC chip, or an FPGA module.

[0037] The apparatus (100) includes a droplet detection system (103). The droplet detection system (103) includes a lens-free imaging device (31). The term "lens-free imaging device" is interchangeable with "lens-free holographic imaging device." According to an exemplary embodiment, the droplet detection system (103) further includes one or more other imaging devices, such as a CCD camera and / or a fluorescence detector. The droplet detection system includes a light source (32) for illuminating an illumination area of ​​the EWOD chip. According to an exemplary embodiment, the light source is configured to illuminate at least a portion of the top surface of the EWOD chip. The top surface generally refers to the surface of the EWOD chip that receives light from the light source. According to an exemplary embodiment, the top surface is a transparent cover. According to another exemplary embodiment, the top surface is a TFT backplane. Because the EWOD chip is substantially transparent to light, the terms "top surface" and "bottom surface" are interchangeable depending on the context. The EWOD chip is positioned at a midpoint along the optical path between the light source and the lens-free imaging device (31). The glass cover 24, the operational space, the EWOD layer 25, and the TFT backplane 26 are substantially transparent so that light emitted from the light source can be detected by the lens-free imaging device 31. The lens-free imaging device 31 includes an imaging chip 312 and an imaging PCB 311. According to an exemplary embodiment, the lens-free imaging device can have a time resolution of 1 millisecond or less.

[0038] The exemplary lens-free imaging device (31) has a field of view (FOV) of 20 mm. 2 The illumination area is greater than or equal to the FOV of the lens-free imaging device 31. According to an exemplary embodiment, the drop detection system 103 comprises multiple lens-free imaging devices.

[0039] The lens-free imaging device (31) captures at least one raw hologram image of at least a portion of the illumination area. The hologram image is captured for the entire FOV. The hologram image includes a diffraction pattern over the FOV and a corresponding diffraction pattern for a droplet of interest on the EWOD chip (102). The hologram image can be further reconstructed into a two-dimensional (2D) image and / or a three-dimensional (3D) image. According to an exemplary embodiment, the droplet detection system comprises a processing unit configured to reconstruct the hologram image into a 2D image and / or a 3D image.

[0040] The control unit (104) receives at least one image from the drop detection system. According to an exemplary embodiment, the image is a hologram image. According to an exemplary embodiment, the image is a reconstructed 2D image or a 3D image. According to an exemplary embodiment, the control unit (104) comprises a central processing unit for processing the images received from the drop detection system. According to an exemplary embodiment, the central processing unit is configured to reconstruct the hologram image into a 2D image and / or a 3D image. According to an exemplary embodiment, the central processing unit is in a general-purpose computer.

[0041] The control unit (104) sends control signals to the drive module (101) to instruct the electrodes of the EWOD chip. The control signals instruct the electrodes to modify the contact angle of the droplet on the EWOD layer, so that the droplet is controlled by the electrostatic effect exerted by the electrodes. The control signals can be, but are not limited to, displacing the droplet, splitting the droplet, dispensing the droplet, merging the droplet, etc.

[0042] At least one of these control signals is adjusted as a function of at least one image.

[0043] A feature captured in the image is used as a variable of a function for adjusting the control signal. According to an exemplary embodiment, the feature is the position of the droplet captured in the image. According to an exemplary embodiment, such a feature can be at least one parameter of the relationship between at least two droplets captured in the image, such as a distance. According to an exemplary embodiment, such a feature can be at least one parameter of a droplet property extracted from the received hologram image corresponding to the diffraction pattern of the droplet of interest. According to an exemplary embodiment, multiple features are considered as variables for adjusting the control signal.

[0044] According to an exemplary embodiment, the control unit (104) is configured to extract a position of the droplet from at least one image, and at least one of the control signals is adjusted based on said position.

[0045] Electrodes can be damaged during fabrication or operation. The damage can be partial destruction of the hydrophobic coating or dielectric layer, or complete destruction of the dielectric. According to an exemplary embodiment, during operation, the control unit (104) sends control signals to instruct a droplet to be displaced to a predetermined location along a first path. If the droplet's movement differs from the predetermined path defined by the control unit, an electrode failure is detected. The control unit (104) designs a second path to the predetermined location that is different from the first path and adjusts subsequent control signals to guide the droplet along the second path. By doing so, the droplet is still sent to the predetermined location but avoids using the path via the failed electrode. A failed electrode refers to an electrode that cannot adequately modify the wetting properties of a droplet of interest to move on the electrode. Electrode failure can be caused by damage to the electrode itself or damage to the electrical circuitry coupled to the electrode.

[0046] According to an exemplary embodiment, during operation, the control unit (104) sends a control signal to instruct the droplet to be displaced to a predetermined position along a first path, but the last electrode, i.e., the predetermined position, fails. The control unit (104) assigns the droplet to a second position different from the predetermined position. According to an exemplary embodiment, the predetermined position is in a predetermined region, and the second position can be another position in the same predetermined region that has not yet been assigned to another droplet.

[0047] According to an exemplary embodiment, the control unit (104) is configured to perform an EWOD inspection for defects. The control unit (104) adjusts at least one of the control signals as a function of at least one variable obtained by the control unit (104) from at least one image, each of the at least one variable representing a different defect in the electrowetting-on-dielectric chip (102). According to an exemplary embodiment, the defect is damage to the dielectric layer. For example, if a pinhole occurs in the device due to dielectric breakdown, the damaged area, including the electrode, is visible in the captured image, such as the edge of the missing portion of the dielectric layer or a different grayscale of the missing portion of the dielectric layer compared to the surrounding undamaged layer. In another example, if a small area of ​​the hydrophobic or dielectric layer peels off, the resulting thickness change can be visualized in the captured image, showing a different local optical density compared to the functioning electrode. The optical density difference at or around the electrode can be a variable for adjusting at least one subsequent control signal. According to an exemplary embodiment, such an EWOD inspection can be performed before a droplet is loaded onto the EWOD chip. In this way, all control signals are adjusted during operation to avoid the use of damaged areas and failed electrodes. According to an exemplary embodiment, such EWOD testing can be performed during operation so that any breakdown of the electrodes can be observed in time. The control signals are adjusted during operation in response to defects detected during the EWOD testing.

[0048] As shown in Figure 3, the light source (32) of the droplet detection system (103) illuminates an illumination area on the EWOD chip (102). The lens-free imaging device (32) captures a hologram image from the interference of light. The control unit (104) receives the hologram image and / or the reconstructed 2D / 3D image from the droplet detection system (103). At least one control signal is adjusted as a function of the at least one image. The control signal is sent to the EWOD chip (102). Examples of a hologram image and a reconstructed 2D image of a droplet of interest are shown.

[0049] According to an exemplary embodiment, the image is a hologram image. Features of the droplet, such as its position, size, shape, refractive index, turbidity, etc., are captured in the raw hologram image. These features can be found through supervised or analytical models developed on the hologram. These features can be used directly as variables to adjust the control signals. This allows for rapid adjustment of the control signals, as the processing speed is not limited by the computational kernel required for the reconstruction process.

[0050] According to an exemplary embodiment, the image is reconstructed from a holographic image. The reconstruction process involves numerical calculations on the two-dimensional image obtained from the imaging sensor. Such numerical calculations may include, for example, deconvolution, wave or beam propagation, transformations (such as Fourier transforms), two-dimensional filtering operations such as noise removal, etc. The reconstructed image reveals physical characteristics of the droplet.

[0051] According to an exemplary embodiment, the control unit (104) receives both the hologram image and the reconstructed image.

[0052] According to an exemplary embodiment, the image can be processed as a whole. According to an exemplary embodiment, the image is divided into sub-regions and the sub-regions are processed separately, so that processing can be performed on different sub-regions in parallel.

[0053] The instrument 100 is compatible for use with EWOD chips. According to exemplary embodiments, the EWOD chips are in replaceable cartridges. The cartridges can be reusable or disposable. If the cartridges are disposable devices, the instrument 100 can be used with multiple cartridges in multiple experiments, one after the other. If the cartridges are reusable, the cartridges can optionally be primed between experiments. The prime step ensures that the surface properties of the EWOD chips are restored. According to exemplary embodiments, the instrument 100 provides a mechanical slot that allows a user to attach the cartridge to the instrument. The EWOD chips are configured to receive signals from the drive module 101 of the instrument 100. The EWOD chips have a corresponding electronic interface for communicating with the drive module 101.

[0054] According to an exemplary embodiment, at least one of the adjusted control signals is for instructing the drive module (101) to move the droplets to a predetermined location. Detection of a feature, or a change in a feature, can be used as a trigger event to send the droplets to a predetermined location and group the droplets in a predetermined area.

[0055] As shown in FIG. 4, the light source (32) of the droplet detection system (103) illuminates an illumination area on the EWOD chip (102). The lens-free imaging device (32) captures a hologram image from the interference of light. The control unit (104) receives the hologram image and / or the reconstructed 2D / 3D image from the droplet detection system (103). At least one control signal is adjusted as a function of at least one image. According to an exemplary embodiment, the entire image containing multiple droplets can be processed to adjust subsequent control signals. According to an exemplary embodiment, the raw hologram image can be divided into predefined sub-areas where droplets are present. These sub-areas can be processed independently. The control unit (104) can send control signals to the sub-areas.

[0056] The control signal is sent to the EWOD chip 102. Examples of droplet properties of interest, such as size, shape, spectral transmittance, spectral absorbance, turbidity, etc., and the spatial distribution of some of these properties within the droplet are shown. Any of these characteristics, or a combination thereof, can be used as a variable to adjust the control signal.

[0057] According to an exemplary embodiment, the control unit (104) is configured to adjust at least one of the control signals as a function of at least one variable that the control unit (104) obtains from the at least one image, each of the at least one variable representing a different characteristic of the droplet selected from size, shape, spectral absorbance, spectral transmittance, turbidity, and viscosity.

[0058] According to an exemplary embodiment, at least one of the adjusted control signals is adjusted to instruct the drive module (101) to combine at least two droplets into one droplet.

[0059] According to an exemplary embodiment, the control unit (104) is configured to adjust at least one of the control signals to continue or stop mixing of the droplets as a function of at least one variable that the control unit (104) obtains from the at least one image, the at least one variable representing the spatial distribution of at least one property or combination of properties of the droplets selected from one or more of size, shape, spectral absorbance, spectral transmittance, turbidity, and viscosity. According to an exemplary embodiment, "mixing of droplets" refers to the process of mixing the droplets by electrowetting, thereby causing the droplets to merge. In certain contexts, "mixing of droplets" is interchangeable with "merging of droplets."

[0060] According to an exemplary embodiment, the dimensions of the droplet of interest can be extracted from the image. According to an exemplary embodiment, predetermined values, optionally including margin values, can be stored in the control unit and compared to the measured dimensions of the droplet. If the dimensions of the droplet are not as desired, the droplet can be moved to a predetermined location for further investigation.

[0061] According to example embodiments, the shape of the droplet of interest can be extracted from the image. According to example embodiments, the volume of the droplet can be estimated. According to example embodiments, the surface shape of the droplet can be estimated from a diffraction pattern captured in a 3D image of the droplet. The estimated surface shape provides an indication of the surface tension of the droplet, and therefore wetting properties can be estimated from the 3D image. Any of these features, or a combination thereof, can be used as variables to adjust the control signal.

[0062] According to exemplary embodiments, the spectral absorbance or transmittance of the droplets can be revealed from the images. Because the droplets scatter and / or absorb light that is detected by the lens-free imaging device (31), diffraction effects can be quantified by reconstructing the holographic fringe pattern. Thus, the attenuation coefficient of the droplet solution can be estimated and used as a variable to adjust the control signal.

[0063] The light absorption of the droplet solution can be spectrally dependent. Light absorption characteristics can vary at different wavelengths in the visible and near-infrared spectral range (the sensitivity of the image sensor). According to an exemplary embodiment, the light source (32) can apply multiple different wavelengths, and the lens-free imaging device (31) captures holographic images at the different wavelengths. Reconstructed images of the droplet at different wavelengths reveal the droplet's spectral absorption. According to an exemplary embodiment, the dilution of a substance inside the droplet is a feature. According to an exemplary embodiment, the presence or absence of a specific soluble substance with a chemical property that absorbs light in a specific spectral window ranging from 1 nm to 500 nm can be inferred as a feature. According to an exemplary embodiment, changes in absorption over a given time period can be observed as a feature to monitor chemical or biological reactions inside the droplet. Any of these features, or a combination thereof, can be used as a variable to adjust a control signal.

[0064] According to an exemplary embodiment, the light source 32 includes multiple sub-light sources that illuminate the droplets at different, unique wavelengths. According to an exemplary embodiment, a single light source is used to illuminate the droplets at multiple different wavelengths.

[0065] According to exemplary embodiments, turbidity characteristics can be evaluated from the image. Because light from the droplet is scattered, which results in a diffraction pattern, turbidity and haze caused by the first fluid in the droplet can be detected. The degree of turbidity can be correlated to a holographic image of the optical characteristics or a reconstructed image of the holographic image. The degree of turbidity can be defined, for example, in formazin nephelometric units (FNU) or nephelometric turbidity units (NTU). According to exemplary embodiments, turbidity characteristics can be used to estimate the non-dissolved components within the droplet, such as the cleanliness of the solution, particle concentration, etc. According to exemplary embodiments, they can also be used to estimate the time evolution of the non-dissolved components of the droplet. According to exemplary embodiments, statistical data obtained from turbidity can be used to track the progress of chemical or biological reactions within the droplet. Any of these characteristics, or a combination thereof, can be used as a variable to adjust a control signal.

[0066] According to an example embodiment, a set of predetermined feature values ​​related to droplet characteristics, such as droplet volume and viscosity, are stored as reference values ​​with a margin. During operation, if a feature value exceeds the margin, the feature can be used to adjust a subsequent control signal to adjust an EWOD parameter, such as voltage or frequency applied to an electrode.

[0067] According to an exemplary embodiment, several properties or characteristics can be measured over a time interval, as shown in Figure 5. Properties can be the viscosity, shape, volume, etc. of the droplets. Properties can also be interactions between droplets, such as droplet stability. Thus, for example, merging of similar or different media in droplet form or droplet splitting can be analyzed with high temporal and spatial resolution.

[0068] According to exemplary embodiments, features within the droplet, such as an object or objects, may also be captured in the image. According to exemplary embodiments, the object may be a biological entity, such as, but not limited to, a cell, a molecule, or a molecular fragment. A biological cell refers to a structural and functional unit of an organism, such as a cell formed of a cytoplasm enclosed within a membrane and which may contain many biological molecules, such as proteins and nucleic acids. A molecule refers to a biological molecule, such as RNA, DNA, or a protein. A molecular fragment may be an RNA fragment, a DNA fragment, a peptide, or the like. According to exemplary embodiments, characteristics of a biological cell may be captured in the image, such as size, shape, spectral absorbance, or spectral transmittance. According to exemplary embodiments, characteristics of a biological cell may be captured in the image over a time interval, such as cell growth and development. According to exemplary embodiments, characteristics of a biological cell may be captured in the image as it responds to a biological entity, such as a cell responding to a particular chemical, an intercellular reaction, or the like.

[0069] According to an exemplary embodiment, as shown in FIG. 7, unexpected objects, such as bubbles, micelles, or other liquid phase irregularities, are captured in the image. The unexpected objects may indicate that the electrode in contact with the droplet is not functioning properly. The electrode may fail in the future. According to an exemplary embodiment, the control signal is adjusted to direct the droplet currently in contact with the electrode to a neighboring electrode. According to an exemplary embodiment, the control signal is adjusted to avoid using this electrode to displace other droplets. Any paths designed for other droplets are recalculated and redesigned. Additionally, the electrodes may be switched off by the control unit (104).

[0070] According to example embodiments, the object may be another droplet. According to example embodiments, the control signal may be adjusted to manipulate the droplet based on a difference in a characteristic of two droplets captured in an image. According to example embodiments, the control signal may be adjusted to manipulate the droplet based on a change in a characteristic of two droplets over a time interval captured in an image.

[0071] According to an exemplary embodiment, the EWOD chip comprises multiple EWOD layers, each functioning independently and capable of being controlled independently and in parallel by a control unit (104).

[0072] According to an exemplary embodiment, the TFT backplane, the EWOD layer, and the glass cover form an EWOD sub-operation unit, and multiple EWOD sub-operation units are stacked on top of each other. According to an exemplary embodiment, the control unit (104) may have a sub-control module for controlling each EWOD sub-operation unit. The lens-free imaging device (31) can image through multiple stacked EWOD sub-operation units without mechanical focusing or movement. Focusing is performed digitally from the recorded hologram to focus on multiple layers simultaneously. Control signals are individually adjusted to the droplets of each EWOD layer.

[0073] According to an exemplary embodiment, a preferred arrangement of a dual-layer EWOD chip is shown in FIG. 8. The EWOD chip includes a glass cover (24) and a first EWOD layer (251) forming a first operating space therebetween. A first TFT backplane (261) is coupled to the first EWOD layer (251). The EWOD chip further includes a second EWOD layer (252) forming a second operating space between the second EWOD layer (252) and the glass cover (24). The EWOD chip further includes first and second carriers (221, 222) for supporting the corresponding first and second TFT backplanes (261, 262) and first and second electronic interfaces (211, 212) for receiving control signals from a driving module of the device (100). The first and second electronic interfaces (211, 212) are electrically coupled to the corresponding first and second TFT backplanes. The fluidic device (25) can be shared to supply fluid to the first and second operational spaces. During operation, the light source (32) shines light onto the EWOD chip, forming an illuminated area on the EWOD chip. The lens-free imaging device of the droplet detection system (103) includes an imaging chip (312) and an imaging PCB (311) that detect light and capture a holographic image. The control unit (104) receives images from the droplet detection system (103) and adjusts the control signals sent to the drive module as a function of the received images. When an image is taken of a droplet in the first operational space, the adjusted control signals based on the image are sent to the first interface (211). When an image is taken of a droplet in the second operational space, the adjusted control signals based on the image are sent to the second interface (211). According to an exemplary embodiment, the two EWOD layers share a common ground electrode. According to an exemplary embodiment, the TFT backplanes corresponding to the two EWOD layers are driven from two different controllers of the control unit (104), i.e., in parallel and independently. According to an alternative exemplary embodiment, they can be driven in a time-multiplexed manner, i.e., in a time-switched manner, from a single controller. This can be advantageous for reducing costs.

[0074] According to an exemplary embodiment, the control unit (104) sends control signals to the droplet detection system (103) to instruct the lens-free imaging device (31) to clean an area or sub-area in the illumination field.

[0075] According to an exemplary embodiment, the droplet detection system (103) further comprises fluorescence detectors (FLO1 and FLO2), as shown in FIG. 9. Fluorescence detection requires a fluorescence light source. According to an exemplary embodiment, the light source (32) comprises a first sub-light source for lens-free imaging and a second sub-light source for fluorescence detection. The light source (32) can further comprise a one-way mirror, where the first sub-light source is directed to the EWOD chip through a first surface of the mirror and the second sub-light source is reflected by a second surface of the mirror and directed to the EWOD chip.

[0076] In a third aspect, the present invention relates to a method of droplet manipulation. According to exemplary embodiments, at least two droplets with adjusted control signals are manipulated simultaneously. The control signal for each of the at least two droplets is adjusted as a function of at least one received image. According to exemplary embodiments, at least 100 droplets are manipulated with control signals adjusted as a function of the image. According to exemplary embodiments, at least 1000 droplets are manipulated with control signals adjusted as a function of the image. According to exemplary embodiments, at least 1 million droplets are manipulated with control signals adjusted as a function of the image.

[0077] According to an exemplary embodiment, an assay for each droplet is assigned by the control unit (104). Images are taken at each step of the assay. According to an exemplary embodiment, multiple droplets are displaced to predetermined positions. The path of each droplet is individually and collectively designed. According to an exemplary embodiment, the paths of multiple droplets are calculated to 1) minimize the number of reused electrodes and 2) minimize the overall time it takes to displace multiple droplets. The efficiency of droplet displacement is maximized while avoiding premature electrode degradation due to short-term overuse, i.e., hysteresis and permanent charging.

[0078] According to an exemplary embodiment, in a first step, the position of each droplet is captured in an image, and a target position of the droplet is predetermined. In a second step, a movement path of each droplet in the droplet group is designed. In a third step, the total movement time, the number of reused electrodes, and the number of reuses of each electrode are calculated. The second and third steps are repeated as different movement paths of each droplet are iterated, and as a result, the time information and the electrode reuse information may change in each iteration. The iteration ends when a desired path strategy is achieved, such as when the time and electrode reuse status no longer improve.

[0079] According to an exemplary embodiment, the control unit (104) is configured to generate droplets containing the objects. After a solution containing hundreds of thousands of objects, ranging from approximately 1 to 100 microliters, is loaded into the cartridge fluidic device, a predefined region of the operating space of the EWOD chip is activated to further load several columns of the electrode array with the solution, as shown in FIG. 10. Thus, a certain amount of solution is present on the EWOD chip. As shown in FIG. 11, a unit cell of m × n electrodes is activated to generate droplets from the predetermined region where the solution containing the objects is present. According to an exemplary embodiment, n is 3. The unit cells can be repeatedly activated in columns on the EWOD layer to generate droplets in parallel. Rows of electrodes are present between the unit cells. After each such activation sequence, the column of droplets generated moves to the next column of electrodes, and new droplets are generated in the column. Thus, multiple droplets can be generated in parallel. According to an exemplary embodiment, to prevent electrode wear due to repeated droplet generation activation, the droplet generation unit cell can be changed to a different electrode. With a realistic design with an electrode row size of 400 and a frame rate of 10 Hz, it is possible to generate hundreds of single droplets per second from a single inlet of the cartridge, thus generating hundreds of thousands of single droplets on the EWOD chip in a matter of minutes.

[0080] Some defects on EWOD chips are not directly visible in the captured image. The system can be used to check the quality of EWOD chips, particularly for invisible defects, by exploiting the electro-optic effect of the droplets. According to an exemplary embodiment, at least one droplet containing an electro-optic material sensitive to an electric field is introduced onto an operating EWOD chip. When an EWOD electrode in contact with the droplet is activated, a change in the contact angle between the EWOD chip and the droplet occurs. The change in the effective dielectric constant results in a change in the holographic pattern of the droplet as seen by the lens-free imaging device. The relative change in the dielectric constant can be used as a function to test whether the EWOD electrodes are functioning within specifications. If the relative change in the dielectric constant is not within a predetermined margin, subsequent control signals for all droplets are adjusted to avoid the use of the electrodes. An advantage is that it eliminates the need for additional electrical sensing circuitry to detect hardware problems. The gain in circuit area can be used for other useful functions, such as a larger capacitor to activate the droplet more quickly. Another advantage is that such tests can be performed periodically to check the progress of circuit reliability with one or a few images. A third advantage is that readouts are quick and data is easily obtained without the need for additional complex hardware. Exemplary embodiments allow for quality checks to be performed before manipulating a droplet of interest. Exemplary embodiments allow for quality checks to be performed during manipulation of a droplet of interest.

[0081] In fourth and fifth aspects, the present invention also relates to a computer program and a computer-readable medium having the program stored thereon. The computer program executes the described method in an instrument (100) or system. The instrument (100) stores all experimental data and analysis results of the experimental droplet operation on a local disk or uploads them to cloud storage. According to exemplary embodiments, the instrument (100) further comprises a user interface that informs the user of the progress of the experimental operation, instructs the user to follow specific steps, and receives input from the user related to the details of the experiment. The user has timely access to all available information for each experiment.

Claims

1. An apparatus (100) for droplet manipulation, comprising: a driving module (101) configured to receive control signals and operate an electrowetting-on-dielectric chip (102), the electrowetting-on-dielectric chip (102) being configured to manipulate droplets (1); a droplet detection system (103) having a light source (32) and a lens-free imaging device (31) configured to obtain at least one image including the droplet (1) on the electrowetting-on-dielectric chip (102); a control unit (104) configured to receive the at least one image from the droplet detection system (103) and to send control signals to the drive module (101); Equipped with The apparatus (100), wherein the control unit (104) is further configured to adjust at least one of the control signals as a function of the at least one image.

2. 10. A system comprising the device (100) of claim 1, further comprising an electrowetting-on-dielectric chip (102) configured to receive signals from the drive module (101), preferably in a replaceable cartridge.

3. the at least one adjusted control signal is for instructing the drive module (101) to move the droplet to a predetermined position; The system of claim 2 .

4. the control unit (104) is configured to extract the position of the droplet from the at least one image, and the at least one control signal is adjusted based on the position.

4. The system according to claim 2 or 3.

5. the control unit (104) is configured to adjust the control signal such that if the movement of the droplet differs from a predetermined first path after a first control signal, at least a subsequent control signal is sent to move the droplet to the predetermined position along a second path different from the first path. The system of claim 4.

6. the at least one image includes a holographic image; A system according to any one of claims 2 to 5.

7. the at least one image comprises a two-dimensional image or a three-dimensional image; A system according to any one of claims 2 to 6.

8. the control unit (104) is configured to adjust at least one of the control signals as a function of at least one variable that the control unit (104) obtains from the at least one image, each of the at least one variable representing a different characteristic of the droplet selected from one or more of: size, shape, spectral absorbance, spectral transmittance, turbidity, viscosity, and a spatial distribution of at least one of the aforementioned characteristics; A system according to any one of claims 2 to 7.

9. the at least one control signal is adapted to instruct the drive module (101) to mix at least two droplets into one droplet, and optionally the control unit (104) is configured to adjust the at least one control signal to continue or stop the mixing of droplets as a function of at least one variable that the control unit (104) obtains from the at least one image, the at least one variable being representative of a spatial distribution of at least one property or combination of properties of the droplets selected from one or more of: size, shape, spectral absorbance, spectral transmittance, turbidity, and viscosity. A system according to any one of claims 2 to 8.

10. the control unit (104) is configured to adjust at least one of the control signals as a function of at least one variable that the control unit (104) obtains from the at least one image, each of the at least one variable being indicative of an object in the droplet; A system according to any one of claims 2 to 9.

11. The object is a cell, a bubble, or a different non-dissolving droplet. The system of claim 10.

12. the control unit (104) is configured to adjust at least one of the control signals as a function of at least one variable that the control unit (104) obtains from the at least one image, each of the at least one variable representing a different defect in the electrowetting-on-dielectric tip (102). A system according to any one of claims 2 to 11.

13. the electrowetting-on-dielectric chip (102) has a plurality of electrowetting-on-dielectric layers, and the control unit (104) is configured to adjust at least one of the control signals as a function of at least one variable that the control unit (104) obtains from a plurality of holographic images by using focus adjustment, each of the at least one variable representing whether droplets are present on a different electrowetting-on-dielectric layer; The system of claim 6.

14. 10. A method of manipulating droplets in the device of claim 1, comprising: a. causing the control unit (104) to receive at least one image comprising a plurality of droplets; b) causing said control unit (104) to generate control signals for simultaneously manipulating said droplets by electrowetting as a function of said at least one image received; A method comprising:

15. A computer program comprising instructions for causing an apparatus according to claim 1 to carry out the steps of the method according to claim 14.

16. A computer readable medium having stored thereon the computer program of claim 15.

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