A fluid processing device for processing droplets

Abstract

The present invention relates to a fluid processing device for processing droplets. The device comprises a plate-like structure comprising a plurality of functional pixels. Each functional pixel is adapted to perform multiple operations on a droplet located on the pixel. Specifically, each pixel can selectively carry out at least two of the following processing operations: manipulating the droplet (e.g., its position), applying heat to the droplet, and subjecting the droplet to a magnetic field. These different operations can be selectively performed at different times on the same pixel, allowing for complex, multi-step protocols to be executed in a single, compact device. This solution enables the integration of various microfluidic functions, typically requiring separate instruments, into one unified platform. The invention can be applied to biochemical analysis, molecular diagnostics, and drug discovery, where precise control over small volumes of fluids is crucial.

Description

[0001] IMEC VZW

[0002] PA 2024 / 160 PCT1

[0003] P626O5 / WO

[0004] A FLUID PROCESSING DEVICE FOR PROCESSING DROPLETS

[0005] TECHNICAL FIELD

[0006] The present disclosure relates to a device for processing one or more droplets of a fluid or liquid. The disclosure provides such a device, which is adapted to selectively perform different processing operations to one or more droplets located on a same area at different times. The droplet processing device may be used for implementing a workflow, for instance, library preparation for next-gen gene sequencing, nucleic acid synthesis.

[0007] BACKGROUND

[0008] Synthetic DNA manufacturing and next -generation sequencing (NGS) are cutting- edge fields that have revolutionized genetic research, biotechnology, and medicine. These technologies have enabled rapid advancements in our understanding of genetic variation, disease mechanisms, and biological systems, with wide-ranging applications in clinical research, diagnostics, reproductive health, and environmental studies.

[0009] Synthetic DNA manufacturing involves creating artificial DNA sequences in the laboratory. The process begins with oligonucleotide synthesis, where short DNA fragments are chemically synthesized. These fragments are then assembled into longer sequences, known as genes, through automated processes. Techniques like Gibson Assembly and Golden Gate Assembly are employed to join multiple DNA fragments seamlessly, enabling the construction of complex genetic circuits and synthetic genomes. Synthetic DNA has numerous applications, including the development of mRNA vaccines, gene therapies, and precision medicines, as well as the creation of new biological systems and organisms in synthetic biology.

[0010] Next-generation sequencing, on the other hand, allows for the rapid and cost- effective sequencing of DNA and RNA. Unlike traditional Sanger sequencing, which sequences one DNA fragment at a time, NGS enables the simultaneous sequencing of millions of fragments, providing ultra-high throughput, scalability, IMEC VZW

[0011] PA 2024 / 160 PCT1

[0012] P626O5 / WO and speed. The NGS process begins with the preparation of a sequencing library, where DNA or RNA samples are fragmented into shorter pieces and tagged with unique molecular barcodes. These fragments are then amplified and sequenced in parallel, generating massive amounts of data in a single run.

[0013] Both synthetic DNA manufacturing and NGS rely heavily on the preparation of DNA libraries. Library preparation is an indispensable step that entails the adaptation of samples to make them compatible with downstream processes. This step typically involves multiple processing stages, including DNA fragmentation, end repair, adapter ligation, and amplification. These processes require precise manipulation of small volumes of fluids, careful temperature control, and often the use of magnetic beads for purification steps.

[0014] SUMMARY

[0015] As the demand for synthetic nucleic acid synthesis and sequencing services continues to rise, several challenges have become increasingly prominent, including:

[0016] - Complexity of workflows: Protocols often consist of many steps, each requiring precise control over various parameters such as temperature, reaction time, and reagent concentrations. In an example of nucleic acid synthesis, the reagent of monomers or nucleic acid fragments are delivered in precise volumes with minimized dead volume requirements. Multiple chemistry reagents, buffers and other liquid elements are involved with varying physical properties. Accuracy and selectivity of reactions play critical roles to prevent errors and impact on follow-up steps in the protocols. In many cases, quality control steps are required to prevent low yields and to ensure repeatability of the protocols.

[0017] - Sample quantity and quality: Many applications require processing of numerous samples, often with limited quantity and variable quality, necessitating highly efficient and flexible preparation methods.

[0018] - Accuracy and purity: Achieving the necessary length, accuracy, and purity of the DNA produced remains a primary challenge, particularly in synthetic DNA manufacturing. IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO

[0019] - Time and cost: Current methods can be time-consuming and expensive, with library preparation accounting for a significant portion of the total time and cost in many genomic studies.

[0020] - Scalability: As demand increases, there is a growing need for systems that can handle higher throughput without compromising on quality or flexibility.

[0021] - Automation: While some steps have been automated, many workflows still require significant manual intervention, leading to potential errors and inconsistencies.

[0022] Current laboratory workflow implementations, particularly in the fields of synthetic DNA manufacturing and next-generation sequencing, face several significant challenges.

[0023] Existing lab workflow implementations are cumbersome, error-prone, and expensive. They typically require multiple dedicated instruments and a large number of consumables, as well as substantial manual labor to execute complex workflows. This approach necessitates the use of numerous types of consumables such as pipette tips, well-plates, and tubes, leading to increased costs and potential for errors.

[0024] The bulkiness of current systems in terms of form factor significantly limits the capacity of end-users to deploy multiple systems in limited lab spaces. This limitation leads to a loss of capacity or inability to parallelize workflows effectively. Current systems are disadvantaged by their bulky nature, as each instrument typically has its own redundant package, electronics, and data interface adapted to work standalone. These systems occupy valuable lab space, reducing overall laboratory efficiency.

[0025] Existing solutions often lack sufficient programmability and flexibility, leading to increased waiting times for sample processing. Users are frequently required to batch large quantities of samples that require identical workflow execution, leading to organizational difficulties. Current solutions offer limited programmability and often lack flexibility in adapting workflow parameters for IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO multiplexed samples. This rigidity can result in inefficient use of resources and time.

[0026] Current solutions suffer from a lack of scalability, leading to the processing of samples in rigid quantities. This limitation creates operational difficulties for endusers, as it prevents them from easily adapting their processes to varying sample loads or experimental requirements. The limited scalability of current solutions is often due to their fundamental design architecture, which may not accommodate a wide range of throughput needs.

[0027] The future of both synthetic DNA manufacturing and NGS is geared towards more integrated and automated systems that can efficiently handle the entire design- build-test-learn cycle. There is a pressing need for solutions that can address the growing demand for high-throughput, complex assays while reducing the extensive and labor-intensive manual operations currently required.

[0028] Current automation approaches only partially address these needs, primarily by replacing human labor with robotized fluid handling solutions. However, these solutions often fail to address critical end-user needs such as miniaturization, reliability, flexibility, and programmability. While emerging automation approaches have begun to address fluid handling using microfluidics, they often ignore other critical functionalities, thus suffering from similar issues as incumbent solutions.

[0029] There is a pressing need to establish a common framework for all necessary basic functionalities to overcome the difficulties originating from compartmentalized instrumentation. An effective automation solution must be holistic, rather than a string of independent units, to truly address the needs of end-users. Currently, no automation solution offers such a holistic approach. There is a clear need to establish a modular approach that combines all required functionalities.

[0030] In view of the above, an objective of this disclosure is to provide a flexible and integrated platform capable of performing multiple steps of library preparation and other related processes in a single device. A particular objective is to combine IMEC VZW

[0031] PA 2024 / 160 PCT1

[0032] P626O5 / WO various key functionalities such as fluid manipulation, temperature control, and magnetic bead handling into a unified device. A further objective is to provide a solution that is broadly programmable and has the potential for high throughput of samples.

[0033] These and other objectives are achieved by the solutions described in the independent claims. Advantageous implementations are further described in the dependent claims.

[0034] A first aspect of this disclosure provides a fluid processing device comprising a plate-like structure for processing one or more droplets located on the plate-like structure. The plate-like structure comprises a plurality of functional pixels. Each functional pixel as a whole is adapted to selectively perform at least two of the following processing operations at different times on a droplet located on the functional pixel: manipulating the droplet; applying heat to the droplet; and applying a magnetic field to the droplet.

[0035] The plate-like structure of the fluid processing device may be a substrate or may comprise a substrate, wherein the functional pixels may be integrated into the substrate. The plate-like structure maybe part of a fluidic chamber for holding the one or more droplets.

[0036] Manipulating the droplet may comprise at least one of the following: moving the droplet into or out of a functional pixel (e.g., from or to another functional pixel); splitting the droplet (e.g., in multiple smaller droplets); and combining or mixing the droplet with one or more further droplets (e.g., into a larger droplet).

[0037] Notably, the terms “fluid” and “liquid” may be used interchangeably in this disclosure. The fluid / liquid may be water, or a water-chemistry-mix, or a suitable carrier liquid for samples, or may be a sample like blood or the like (body fluid). The fluid may be a sample or may include a sample, in particular, a droplet may comprise at least one sample. In this case, the fluid processing device may process the droplets for the purpose of implementing a sample library preparation IMEC VZW

[0038] PA 2024 / 160 PCT1

[0039] P626O5 / WO workflow. A sample may be an analyte sample (e.g., blood), or may also include other chemical fluids (e.g., reagents), which may be captured in the droplet(s).

[0040] Optionally, the area of the functional pixel maybe smaller than the cross-sectional area of a typical droplet. In an example implementation, the area of the functional pixel may be at least ten times smaller than the cross-sectional area of the typical droplet. For instance, the functional pixel may be less than 2mm x 2mm (if in a square shape, or in an equivalent area), in order to be small enough to process a single droplet, for example, with a volume of at least loonL. In some examples, the functional pixel maybe at least 10 micrometer (pm) x 10 pm (if in a square shape, or in an equivalent area), in order to be small enough to process a single droplet, for example, with a volume of ipL or larger. It is noted that the functional pixel maybe in a square shape, or in a rectangular shape, or in any shape that is suitable for manipulating the droplet. The shape of the functional pixel is not limited in this disclosure.

[0041] Optionally, when a particular pixel is adapted to perform one processing operation at one time instance, one or more its neighboring pixels that is not adapted to perform any processing operation may be adapted to sense the intensity of the processing operation and feedback the intensity to the device. For instance, when a pixel is adapted to perform heating, one or more its neighboring pixels (that are not performing any processing function) may be adapted to measure a relative temperature of the area of the pixel performing heating and feedback the temperature to the device. In this way, the device can be aware of whether there is any overheating issue and can adjust the temperature of the active pixel accordingly. This can be useful to prevent assay performance issues as biochemical reactions are sensitive to absolute temperature in the process. This can be also useful to address manufacturing process, or environmental variabilities related to circuit functionalities. The control of the device parameter such as current and voltage can be adjusted to overcome the unpredictable behavior of such variabilities. In the case of magnetic function of the pixel, this can be used to verify whether the intended function is executed properly. For example, one or more neighboring pixels adjacent to a pixel performing magnetic function can detect the presence of magnetic particles through sensing relative permeability of the IMEC VZW

[0042] PA 2024 / 160 PCT1

[0043] P626O5 / WO medium. In this disclosure, one or multiple pixels maybe used to perform sensing operations.

[0044] In an implementation of the fluid processing device, the plurality of functional pixels are arranged adjacently across a continuous part of the plate-like structure.

[0045] In a further implementation of the fluid processing device, each functional pixel comprises a conductive element configured to perform each of the at least two processing operations. Optionally, the conductive element maybe used to provide change in contact angle, and / or heating, and / or applying force to move the droplet, and / or applying magnetic force to the droplet or the objects in the droplets. The conductive element may be adapted to avoid direct electrical contact between the functional pixel and the droplet.

[0046] Optionally, the device may comprise a dielectric layer between the conductive element and the droplet. The dielectric layer may be used to prevent possible hydrolysis and other issues. A material of the dielectric layer may be, for example, polymer (e.g., parylene, PVC, PCC, PTFE), or non-organic (e.g., oxides, Silicon nitride (SiN), Silicon dioxide (Si02)).

[0047] In a further implementation of the fluid processing device, the device further comprises a plurality of metal coils embedded in the plate-like structure, wherein each functional pixel is associated with one or more metal coils, and each functional pixel as a whole is adapted to perform the at least two processing operations on the droplet located on the functional pixel by respectively applying different signals to the one or more metal coils.

[0048] In a further implementation of the fluid processing device, the one or more metal coils associated with a functional pixel are adapted to:

[0049] - manipulate the droplet located on the functional pixel in response to receiving a voltage with no current flow;

[0050] - apply heat to droplet located on the functional pixel in response to receiving an alternating current; or IMEC VZW

[0051] PA 2024 / 160 PCT1

[0052] P626O5 / WO

[0053] - apply the magnetic field to the droplet located on the functional pixel in response to receiving a direct current.

[0054] In a further implementation of the fluid processing device, each functional pixel comprises a plurality of stacked layers, wherein each layer is adapted to perform one or more of the at least two processing operations. A plurality of layers may be preferably disconnected from each other at the stack layers such that the processing operations are limited to specific functions they are designed for, respectively. A plurality of layers maybe connected to each other in the stack layer such that a function can be realized in the connected layers concurrently. This may have benefits in terms of enhancing the functionality, power delivery or sensing capabilities.

[0055] In a further implementation of the fluid processing device, each functional pixel comprises a first layer adapted to manipulate the droplet located on the functional pixel, and a second layer adapted to apply heat and the magnetic field to the droplet located on the functional pixel. Optionally, the first layer may be part of a microfluidic channel for droplet manipulation.

[0056] In a further implementation of the fluid processing device, each functional pixel comprises an electrowetting element in the first layer for manipulating the droplet, and an electrode in the second layer for applying heating and the magnetic field to the droplet.

[0057] Optionally, the electrowetting element may be an electrowetting on dielectric (EWOD) element, or an opto-electrical wetting element.

[0058] In a further implementation of the fluid processing device, each functional pixel comprises a first layer adapted to manipulate the droplet located on the functional pixel, a second layer adapted to apply heat to the droplet located on the functional pixel, and a third layer adapted to apply the magnetic field to the droplet located on the functional pixel. IMEC VZW

[0059] PA 2024 / 160 PCT1

[0060] P626O5 / WO

[0061] In a further implementation of the fluid processing device, each functional pixel comprises an electrowetting element in the first layer for manipulating the droplet, a thermal element in the second layer for applying heating to the droplet, and a magnetic element in the third layer for applying the magnetic field to the droplet.

[0062] It is noted that the wording of “first”, “second”, third” used in this disclosure are used merely to differentiate different layers, and are not intended to indicate any sequence for forming the layers. In this disclosure, the actual sequence for forming multiple layers is not limited. The first, the second, and the third layers may be stacked in any suitable order. The same applied to the two-layer design.

[0063] A specific order maybe optimized for various application scenarios. For instance, a layer closest to the droplet may be a layer for manipulating the droplet, such as an electrowetting layer. This configuration reduces the voltage required to induce wetting properties, thereby mitigating the risk of dielectric breakdown and simplifying circuit design to achieve the necessary voltage ranges and timing requirements.

[0064] As another example, a layer closest to the droplet may be a layer for applying the magnetic field to the droplet. This arrangement minimizes the electrical current needed to generate the magnetic field and its gradient, thereby enhancing power efficiency, reducing unwanted thermal dissipation, and improving circuit reliability.

[0065] In one example, a layer for applying heat to the droplet is not placed as the closest layer to the droplet. The thermal power may exhibit spatial dependency (e.g., due to resistive design), and short distances (such as being less than 50 microns to the droplet) can result in a non-uniform temperature distribution on and within the droplet. This non-uniformity may adversely affect biochemical reactions. Therefore, the thermal layer may be positioned at a sufficient distance from the droplet, which allows for a more uniform temperature distribution through heat diffusion, thereby ensuring optimal biochemical reactions. IMEC VZW

[0066] PA 2024 / 160 PCT1

[0067] P626O5 / WO

[0068] Optionally, a pixel size of each layer maybe the same or different. For instance, the size of a single thermal pixel maybe equal to N electrowetting pixel(s). The size of a single magnetic pixel may be equal to M electrowetting pixel(s). N and M are positive integers.

[0069] In a further implementation of the fluid processing device, one or more of the plurality of functional pixels are configured to provide light onto and / or detect light emitted by one or more droplets located on the one or more functional pixels.

[0070] In a further implementation of the fluid processing device, one or more of the plurality of functional pixels are configured to manipulate the droplet with dielectrophoretic (DEP) forces that relies on creating non-uniform electric fields across non-conductive materials. These materials can be the droplet itself or particles inside the droplet. This force can help concentrating, mixing, moving of objects or the droplet itself.

[0071] In a further implementation of the fluid processing device, the device further comprises one or more light emitting devices configured to emit the light of at least one predetermined wavelength, and / or one or more photodetectors configured to detect the light emitted and / or scattered by the one or more droplets located on the one or more functional pixels, wherein the one or more light emitting devices and the one or more photodetectors are arranged on or besides an outer surface of the one or more functional pixels.

[0072] Optionally, the light emitting device may be a light emitting diode. The photodetectors may be a CMOS sensor. The light emitting device may be configured to emit light of at least one wavelength, and the photodetector may detect light in at least that at least one wavelength and optionally other wavelengths.

[0073] In a further implementation of the fluid processing device, the device further comprises: IMEC VZW

[0074] PA 2024 / 160 PCT1

[0075] P626O5 / WO

[0076] - one or more input ports, each input port being configured to receive the one or more droplets and to provide the one or more droplets onto the plate-like structure; and / or

[0077] - one or more output ports, each output port being configured to eject the one or more droplets from the fluid processing device.

[0078] Optionally, the one or more input ports and / or the one or more output ports comprise respectively one or more piezoelectric micro-machined ultrasonic transducers (PMUTs). The PMUTs are adapted to actuate the one or more droplets, or a fluid containing the one or more droplets.

[0079] In a further implementation of the fluid processing device, each functional pixel is adapted to receive at least two different control signals at different times for respectively controlling each functional pixel to perform the at least two different processing operations.

[0080] In a further implementation of the fluid processing device, each functional pixel is connected with a circuitry. The circuitry comprises at least one electronic switching element (for each functional pixel) configured to selectively provide the at least two different control signals to each functional pixel at different times.

[0081] In a further implementation of the fluid processing device, the at least one electronic switching element is connectable to a common circuit line, wherein the common circuit line is adapted to produce the at least two different signals at different times.

[0082] In a further implementation of the fluid processing device, the at least one electronic switching element is configured to switch among a current waveform circuit line, a potential circuit line, and a ground circuit line at different times, so as to selectively provide the at least two different signals to each functional pixel at different times. IMEC VZW

[0083] PA 2024 / 160 PCT1

[0084] P626O5 / WO

[0085] In a further implementation of the fluid processing device, the at least two different signals for performing the at least two different processing operations differ in terms of their amplitude and / or frequency and / or waveform and / or duty cycle.

[0086] Optionally, the fluid processing device may be adapted to adjust the temperate of heating by adjusting the amplitude and / or duty cycle of the signal. The alternating current amplitude may be proportional to the power dissipation for simple harmonic signals. Alternatively, the signals may be shaped with pulse-width modulation. In such cases, the duty cycle may be adjusted to deliver desired level of power.

[0087] Optionally, the fluid processing device may be adapted to adjust the intensity of the magnetic force by adjusting the amplitude, frequency, and / or waveform of the signal.

[0088] The magnetic force is proportional to the magnetic field generated by a magnetic element. Therefore, the magnetic field strength can be modified through controlling a current signal amplitude, as the signal amplitude is proportional to the magnetic field. The signal frequency may also play a role as the impulse experienced by a magnetic particle in the vicinity of the magnetic field is proportional to time averaged force, polarity change of the magnetic field can determine the net force applied to particles in each time interval. Higher frequency signals thus results in rapid polarity change of the force and results in zero net magnetic force for a particle within the period of the signal. In periods shorter than the signal period signal, a net magnetic force is generated. The waveform may also determine the magnetic force through shape of the signal, the skewed, non- symmetric signals maybe used to create a net magnetic force in a time frame larger than the signal period.

[0089] Optionally, the fluid processing device may be adapted to adjust the force of manipulation by adjusting the amplitude of the signal. The surface tension forces acting on the droplet may be determined by the amplitude of the voltage applied through the droplet. The amplitude change thus has a proportional effect on the forces that create displacement or motion of the droplet. IMEC VZW

[0090] PA 2024 / 160 PCT1

[0091] P626O5 / WO

[0092] In a further implementation of the fluid processing device, the device further comprises a controller configured to address the plurality of functional pixels.

[0093] For instance, the controller may be configured to determine one or more target functional pixels (e.g., out of a pixel matrix comprising pixel rows and pixel columns) that are to be activated for performing different processing operations. Once the one or more target functional pixels are addressed (e.g., determined and / or selected), the circuitry mentioned above comprising at least one electronic switching element may be used to provide different signals to the one or more target functional pixels for performing different processing operations at different times.

[0094] A second aspect of this disclosure provides a fluid processing array comprising a plurality of fluid processing devices according to the first aspect or any implementation form thereof.

[0095] The fluid processing array may specifically be a sample processing array configured to perform a sample processing workflow. The fluid processing array may be used for (sample) library preparation, and could be included in a cartridge for library preparation.

[0096] In an implementation of the fluid processing array, the fluid processing array is a chip or a panel; or the fluid processing array comprises a plurality of chips, each chip comprising at least one of the fluid processing devices.

[0097] Any chip may be a bio chip, and may comprise multiple functional pixels, which may correspond to the multiple functional zones of a fluid processing device. A panel may be a planar arrangement of multiple such chips or fluid processing devices.

[0098] A third aspect of this disclosure provides fluid processing system comprising at least one fluid processing device according to the first aspect or any implementation form thereof. IMEC VZW

[0099] PA 2024 / 160 PCT1

[0100] P626O5 / WO

[0101] The fluid processing system may be a sample processing system configured to perform a sample processing workflow, for example, a sample library preparation workflow.

[0102] A fourth aspect of this disclosure provides a method for fluid processing, the method comprising: manipulating one or more droplets located on a plate-like structure comprising a plurality of functional pixels; and selectively applying at least two of heat, a magnetic field, and a manipulation force, in any order, to a droplet located on a functional pixel at different times, e.g., by applying different control signals.

[0103] The method of the fourth aspect achieves the same advantages as the fluid processing device of the first aspect, and may be extended by respective implementations as described above for the fluid processing device of the first aspect.

[0104] A fifth aspect of this disclosure provides a computer program comprising instructions which, when the program is executed by the control unit (e.g., a processor therefor), causes the fluid processing system to perform the above actions, or to perform the method of the fourth aspect.

[0105] BRIEF DESCRIPTION OF THE DRAWINGS

[0106] The above described aspects and implementations are explained in the following description of embodiments with respect to the enclosed drawings:

[0107] FIG. 1 shows a schematic representation of a plate-like structure of a fluid processing device according to this disclosure.

[0108] FIG. 2A-2C show various configurations of a functional pixel of a fluid processing device according to this disclosure. IMEC VZW

[0109] PA 2024 / 160 PCT1

[0110] P626O5 / WO

[0111] FIG. 3A-3G show various examples of a functional pixel of a fluid processing device according to this disclosure.

[0112] FIG. 4A-4C show examples of a plurality of functional pixels according to this disclosure.

[0113] FIG. 5A-5B show an integration of optical detection capabilities into a fluid processing device according to this disclosure.

[0114] FIG. 6 shows an example of droplet manipulation.

[0115] FIG. 7 shows an example of applying heat and magnetic fields.

[0116] FIG. 8A-8C show different functions achievable by a single layer design of functional pixels of a fluid processing device according to this disclosure.

[0117] FIG. 9 shows an example of multiple functional pixels connected through a circuitry.

[0118] FIG. 10A-10B show examples of switching mechanisms for controlling pixels of a fluid processing device according to this disclosure.

[0119] FIG. 11 shows a further example of switching mechanisms for controlling pixels of a fluid processing device according to this disclosure.

[0120] FIG. 12 shows a method for processing a fluid according to this disclosure.

[0121] FIG. 13 shows an example of a plate-like structure of a fluid processing device according to this disclosure.

[0122] FIG. 14 shows an example of a fluid processing array according to this disclosure. IMEC VZW

[0123] PA 2024 / 160 PCT1

[0124] P626O5 / WO

[0125] FIG. 15 shows an example of a fluid processing system according to this disclosure.

[0126] DETAILED DESCRIPTION OF EMBODIMENTS

[0127] FIG. 1 illustrates a schematic representation of a plate-like structure 10 of a fluid processing device according to this disclosure. The fluid processing device is for processing one or more droplets 13 located on the plate-like structure 10. The plate-like structure 10 comprises a plurality of functional pixels 11a, 11b, 11c. It is noted that the size of the droplet and the size of the functional pixel depicted in FIG. 1 are for illustration purposes only. In general, a droplet may cover one or more functional pixels.

[0128] Each functional pixel 11a, 11b, 11c as a whole is adapted to selectively perform at least two of the following processing operations (or functions) at different times on a droplet 13 (or at least part of the droplet 13) located on the functional pixel. The processing operations comprises: manipulating the droplet (referred to as fluid control / manipulation); applying heat to the droplet (e.g., thermal incubation); and applying a magnetic field to the droplet (referred to as magnetic manipulation).

[0129] The plurality of pixels are arranged adjacently across a continuous part of the platelike structure 10. This arrangement allows for seamless and flexible manipulation of droplets across the entire surface of the device.

[0130] According to this disclosure, the fluidic functions (e.g. moving, mixing, splitting etc.) can be complemented in the same space with heating and / or magnetic force exposure to eliminate the need to transport the liquids, reagents etc. to different instruments via automated or manual processing. This disclosure discloses that the multiple functionalities may be combined as stacked continuous pixelated layers on a substrate. It also discloses that the certain functionalities can be IMEC VZW

[0131] PA 2024 / 160 PCT1

[0132] P626O5 / WO implemented with the same pixel element without a need to split into multiple layers.

[0133] An idea of this disclosure is to combine multiple functionalities into a single device sharing the same backbone of electronics control circuitry. The fundamental architectural structure relies on pixelated concept in which each of elements has 2D repeating pattern of functional pixels in an array format. This implies that the substrate of the device may contain functional pixels of magnetic, heating and EWOD elements and their connection pins to the electronic control circuitry that can be integrated to the substrate or provided via an external element linked to the substrates via necessary interconnects.

[0134] As an example, the substrate may be composed of a single layer. Each functional pixel of the single layer may be adapted to function as a magnetic or thermal or EWOD element depending on whether the pixel is powered via AC or DC, closed circuit or open circuit. A DC close circuit drive may result in a magnetic field resulting in directional magnetic forces on magnetic particles within the proximity of the pixel. As the strength of the magnetic field and its gradient diminishes as a function of distance from the element, it is preferred to minimize the gap between the element and the droplet in which the magnetic particles are located (<imm). AC close circuit drive rapidly changing alternating current (>5Hz) may cause the pixel (e.g., the conductive comprise therein) to behave as a heater as the net force on magnetic particles within the response time of a particle will be zero due to the polarity change. Furthermore, the same pixel can operate as an EWOD electrode when the circuit operates as a voltage source with no current (open circuit drive). Applied voltage on the electrode thus causes contact angle change on the affected droplet leading to droplet manipulation functions such as moving, mixing, splitting etc.

[0135] It is also possible that the same pixel can serve as a temperature or magnetic field sensor by measuring temperature dependent relative resistivity or relative permeability changes in the vicinity of the element with respect to a reference value. A resistance and inductance measurement through the electronic circuit thus can provide a correlation to the physical quantities like temperature or IMEC VZW

[0136] PA 2024 / 160 PCT1

[0137] P626O5 / WO presence of magnetic particles. Either the same actuation element or neighboring pixels can be utilized as sensors to provide a feedback mechanism to controller circuit to improve the reliability. This provides simplicity since all functionalities can be realized through single element design and require only control circuit adjustments.

[0138] This multi-functional capability of each pixel enables complex fluid processing operations to be performed within a single, compact device, significantly reducing the need for multiple separate instruments and minimizing the risk of sample loss or contamination during transfer between different processing stages. Further, by enabling each single functional pixel to selectively perform different processing operations, the size of the device can be reduced and thus the system bulkiness can be reduced.

[0139] Each functional pixel (11a, 11b, 11c) may comprise a conductive element configured to perform each of the at least two processing operations. For instance, the fluid processing device further comprises a plurality of metal coils (not shown in FIG. 1) embedded in the plate-like structure 10. Each functional pixel 11a, 11b, 11c is associated with one or more of these metal coils. The functional pixels 11a, 11b, 11c as a whole are adapted to perform the at least two processing operations on the droplet 13 located on the functional pixel by respectively applying different signals to the one or more metal coils.

[0140] These metal coils associated with a functional pixel 11a, 11b, 11c are adapted to:

[0141] - manipulate the droplet 13 located on the functional pixel in response to receiving a voltage with no current flow, e.g., utilizing the principle of electrowetting on dielectric (EWOD);

[0142] - apply heat to the droplet 13 located on the functional pixel in response to receiving an alternating current; or

[0143] - apply the magnetic field to the droplet 13 located on the functional pixel in response to receiving a direct current, generating a localized magnetic field.

[0144] This versatile design allows for precise control over droplet movement, temperature, and magnetic field application, all within a same single pixel. Such IMEC VZW

[0145] PA 2024 / 160 PCT1

[0146] P626O5 / WO integration enables complex biochemical procedures to be executed with high precision and minimal user intervention.

[0147] Optionally, the plate-like structure 10 may include a hydrophobic coating on its surface to enhance droplet manipulation capabilities. This coating helps to reduce droplet adhesion and improves the efficiency of droplet movement across the pixels.

[0148] In some embodiments, the fluid processing device may also include one or more input ports (not shown) for introducing droplets onto the plate-like structure 10, and one or more output ports (not shown) for retrieving processed droplets. These ports can be strategically placed around the perimeter of the plate-like structure 10 to facilitate easy integration with external fluid handling systems.

[0149] In this disclosure, the plate-like structure 10 may comprise a plurality of layers (other than the functional pixel layer). The functional pixels na / b / c may be embedded on a same substrate. The functional pixels na / b / c maybe implemented using a single layer design or multi-layer design.

[0150] The plate-like structure 10 may be fabricated using various technologies, such as printed circuit board (PCB) technology or semiconductor manufacturing processes, depending on the desired pixel density and performance characteristics. By integrating multiple functionalities into a single device, this fluid processing device offers significant advantages in terms of miniaturization, automation, and flexibility for various applications, including but not limited to DNA sequencing, protein analysis, and drug discovery. The ability to control each pixel in a programmable manner allows for the execution of complex, multi-step protocols without the need for manual intervention, thereby reducing human error and increasing reproducibility in laboratory processes.

[0151] FIG. 2A-2C illustrate various configurations of a functional pixel of the fluid processing device according to the present disclosure. IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO

[0152] FIG. 2A depicts a single-layer design for a functional pixel 211. In this configuration, a single layer (or substrate) is adapted to perform all three functions: droplet manipulation, heating, and magnetic manipulation. This design represents the most integrated approach, where a single conductive element, such as a metal coil, is utilized to achieve all three functionalities. The conductive element can be selectively controlled to:

[0153] - manipulate the droplet through electrowetting when a high voltage is applied with no current flow (open circuit);

[0154] - generate heat when an alternating current (AC) is passed through it; and

[0155] - create a magnetic field when a direct current (DC) is applied.

[0156] This single-layer design offers advantages in terms of simplicity of manufacture and potentially reduced thickness of the overall device. It also provides the ability to use the same element as a temperature or magnetic field sensor by measuring temperature-dependent relative resistivity or relative permeability changes in the vicinity of the element with respect to a reference value.

[0157] FIG. 2B illustrates a two-layer (or two substrates) stacked design for a functional pixel, combining two of the functions into a single layer. In this configuration, the pixel comprises two layers: a first layer 221 and a second layer 222. For instance, the first layer 221 may be dedicated to droplet manipulation, e.g., through electrowetting (EWOD); the second layer 222 may be designed to provide both heating and magnetic field application.

[0158] In this example, the element for magnetic induction is also used for heating. Accordingly, AC / DC current flowing through the element is adapted to cause heat or magnetic field as explained previously. When the circuit is open (e.g., via an implementation of circuit switch) a high voltage can be applied to the element without a current flowing through. This element can then be used for electrowetting actuation.

[0159] This design allows for some optimization of each layer for its specific function(s), potentially improving performance compared to the single-layer design. The thermal-magnetic combination in layer 222 may efficiently utilize the same IMEC VZW

[0160] PA 2024 / 160 PCT1

[0161] P626O5 / WO conductive element, as both functions rely on current flow (AC for heating, DC for magnetic field).

[0162] FIG. 2C illustrates a three-layer (or three substrates) stacked design for a functional pixel, where each layer is dedicated to a single layer, and all layers are vertically stacked (when viewed from top of the plate-like structure 10).

[0163] This configuration might be used in applications where the heating function needs to be closely integrated with droplet manipulation, while magnetic field application can be handled separately. It allows for optimization of the magnetic layer independently, which can be beneficial for applications requiring strong or precisely controlled magnetic fields.

[0164] Both stacked designs (FIG. 2B and 2C) offer a balance between integration and functional optimization. They allow for some separation of functions, which can be beneficial for reducing interference between different operations and potentially improving the overall performance of each function. These multi-layer architectures may help overcome potential performance trade-offs inherent in the single-layer design.

[0165] The choice between these alternative designs shown in FIG. 2A-2C may depend on various factors, including:

[0166] - specific requirements of the intended application;

[0167] - manufacturing considerations and available fabrication technologies;

[0168] - the desired balance between device thickness constraints, functional performance, and cost;

[0169] - the need for sensing capabilities integrated into the actuation elements.

[0170] Each design offers unique advantages. The single-layer design (FIG. 2A) may maximize integration, potentially reduces manufacturing complexity, and offers built-in sensing capabilities. The stacked designs (FIG. 2B and 2C) can offer more flexibility in optimizing each function and may provide better performance in scenarios where one function (e.g., magnetic field application) requires significantly different structural characteristics than the others. IMEC VZW

[0171] PA 2024 / 160 PCT1

[0172] P626O5 / WO

[0173] Splitting the functionalities into different layers can allow each functional pixel to be optimized for its own function. For example, the coils are good magnetic elements whereas meander structures are good thermal elements due to their geometric properties. On the other hand, EWOD electrodes are typically square and the borders between the electrodes can be further designed to optimized electrowetting actuation. Second, splitting the functionalities into multiple layers may also allow adjust the unit cell size for each function. For example, the EWOD unit cell size (pixel) can be smaller than thermal or magnetic unit cell sizes. In this way, thermal, magnetic and EWOD unit cells can be designed to have different dimensions. This can bring advantages for performance as elements with larger footprint may improve uniformity and speed of actuation.

[0174] It is worth noting that in all designs, the droplet manipulation layer (e.g., the EWOD layer) maybe prioritized to be closest to the droplets to minimize actuation voltage requirements. The EWOD layer can be designed as either a single plate (no additional ground layer on the opposing side of the droplet) or in a two-sided form (with an additional ground layer).

[0175] These alternative designs may allow for adaptation to various application needs and manufacturing capabilities while maintaining the core functionality of multipurpose, programmable fluid handling. The ability to adjust unit cell sizes for different functionalities in multi-layer designs can further enhance performance, allowing for optimized uniformity and speed of actuation at the expense of increased design and manufacturing complexity.

[0176] It is noted that the illustration of the stacked layers shown in FIG. 2 (and in the following figures) is schematic only, and does not show a real implementation of the stacked layers. For instance, the stacked layers maybe disconnected from each other (e.g., stacked with a distance from each other), such that processing operation functions are limited to specific layers they are designed for, respectively. It is also possible that a plurality of layers may be connected to each other such that a processing function can be realized in the connected layers concurrently. For instance, in FIG. 2C, layers 231, 232, and 233 may be IMEC VZW

[0177] PA 2024 / 160 PCT1

[0178] P626O5 / WO disconnected from each other such that each processing function is limited to each layer. However, for the heating layer 233 itself, there may be multiple layers that each comprises a heating element, so as to enhance the heating function of the heating layer 233.

[0179] FIG. 3A-3G illustrate various examples of the functional pixel designs, showing different arrangements of the thermal, magnetic, and droplet manipulation (using EWOD as an example) functionalities. These figures demonstrate the flexibility of this disclosure in accommodating various design requirements and manufacturing processes.

[0180] FIG. 3A shows a single-layer design of each pixel based on the configuration shown in FIG. 2A. FIG. 3B-3D show two-layer designs of each pixel based on the configuration shown in FIG. 2B. FIG. 3E-FIG. 3G show three-layer designs of each pixel based on the configuration shown in FIG. 2C.

[0181] It is noted FIG. 3B and FIG. 3C show a double-sided design where the droplet may be placed (e.g., sandwiched) between two layers described according to this disclosure. In this scenario, the functional pixels of the two layers maybe aligned.

[0182] FIG. 4A-4C shows examples of a plurality of functional pixels according to this disclosure.

[0183] FIG. 4A illustrates a 3D view of an array of functional pixels based on the singlelayer design (corresponding to FIG. 2A). In this configuration, multiple functional pixels na / b / c may be arranged in a grid pattern. Each pixel na / b / c is achieved using a single layer 211 that incorporates all three functionalities - droplet manipulation, thermal, and magnetic functions. This arrangement shows the compact nature of the single-layer design when implemented across multiple pixels. The top surface of these pixels (or the affected area of these pixels) may form a continuous plane where droplets can be manipulated, heated, and subjected to magnetic fields. This design offers a slim profile and potentially simpler manufacturing process, as all functionalities are integrated into one layer across the entire plate-like structure. IMEC VZW

[0184] PA 2024 / 160 PCT1

[0185] P626O5 / WO

[0186] FIG. 4B-4C depicts a 3D view of an array of functional pixels based on the multilayer design (corresponding to FIG. 2B-2C, respectively).

[0187] In all three figures FIG. 4A-4C, the individual functional pixels 11a, 11b, 11c (as labeled in FIG. 1) are arranged to form a larger, cohesive operating surface. The plate-like structure 10 provides a continuous area where droplets 13 can be manipulated, heated, and / or exposed to magnetic fields as needed.

[0188] FIG. 5A and 5B illustrate an integration of optical detection capabilities into the fluid processing device.

[0189] In addition to droplet manipulation, heating and magnetic functionalities, the device also integrates optical detection capabilities, as detailed in FIG. 5A and 5B.

[0190] One or more of the plurality of functional pixels 11a, 11b, 11c may be configured to provide light onto and / or detect light emitted by one or more droplets located on the one or more functional pixels 11a, 11b, 11c. For this purposes, the droplet processing device may comprise one or more light emitting devices 54 configured to emit the light of at least one predetermined wavelength, and / or one or more photodetectors 55 configured to detect the light emitted by the one or more droplets located on the one or more functional pixels 11a, 11b, 11c, wherein the one or more light emitting devices 54 and the one or more photodetectors 55 are arranged on or besides an outer surface of the one or more functional pixels 11a, 11b, 11c. The photodetectors can comprise semiconductor elements, such as photodiodes and optical filters, which may be in the form of a single or multiple layers, such as on or in the same substrate. The optical filters may be spectrally transparent within a certain bandwidth in a visible range (3oonm - looonm), whereas they may be adapted to block the light in the other spectrum. The light source can be a light emitting diode (LED) with a selected emission bandwidth, beam angle and illumination power. The light source may also contain additional elements such as integrated polarizers, filters etc. to enable selectivity of the light matter interaction. IMEC VZW

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[0193] FIG. 5A shows a configuration of the fluid processing device with light emitting devices 54 and photodetectors 55 arranged in different layers (51 and 52) of the plate-like structure 10. In this arrangement, the top layer 51 is made partially optically transparent to allow light to pass through. This design allows for direct optical access to the droplets from above, enabling both illumination and detection of light-based reactions or markers within the droplets.

[0194] FIG. 5B shows an alternative configuration of the fluid processing device with light emitting devices 54 and photodetectors 55 arranged in a same layer (51 and 52) of the plate-like structure 10. In this case, the bottom layer of the device is made partially optically transparent. This configuration may be advantageous in scenarios where the top surface needs to remain unobstructed, or when integrating the device with other equipment that requires top-side access.

[0195] It is noted that in some configurations, a plurality of pixels may be covered by a single light emitting device 54 and / or a single photodetector 55. It is not necessarily that each function pixel is dedicated to a single light emitting device 54 or a single photodetector 55.

[0196] In general, the photodetector(s) 55 maybe aligned with the functional pixel(s) in a relationship of m:n, wherein m and n are positive integers. That is, m photodetector (s) 55 maybe aligned with n functional pixel(s).

[0197] The integration of these optical elements expands the capabilities of the fluid processing device in several ways:

[0198] - Reaction monitoring: The system can detect specific reactions occurring within the droplets, such as fluorescence or colorimetric changes, enabling real-time monitoring of assay progress.

[0199] - Luminescence detection: For assays involving luminescent markers, such as in some PCR applications or bead-based assays, the photodetectors can capture and quantify the light emitted.

[0200] - Droplet feedback: The optical system provides a non-invasive method for monitoring droplet presence, position, and potentially composition, offering critical feedback for precise droplet operations. IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO

[0201] - Assay versatility: The addition of optical detection capabilities significantly broadens the range of assays that can be performed on the device, including many common biological and chemical assays that rely on optical readouts.

[0202] - Quality control: The optical system can be used to verify the presence and quality of reagents or samples in real-time during processing.

[0203] This optical detection means can be implemented either as an array of photodetector pixels in close proximity to the functional pixels, or as a camerabased system observing selected regions or the whole area of the device. The choice between these implementations would depend on factors such as required spatial resolution, sensitivity, and cost considerations.

[0204] By incorporating these optical capabilities, the fluid processing device becomes a more comprehensive lab-on-chip solution, capable of not only manipulating and processing droplets but also analyzing their contents and providing real-time feedback for process control and results readout.

[0205] FIG. 6 illustrates an example of droplet manipulation. FIG. 6 depicts a droplet positioned on a hydrophobic dielectric layer comprising electrodes. There are two states of the droplet: one without applied voltage and one with applied voltage.

[0206] When no voltage is applied, the droplet maintains its initial contact angle with the surface, demonstrating hydrophobic behavior. When a voltage is applied to the electrode, an electric field is created between the electrode and the conductive liquid droplet. This electric field changes the surface energy at the solid-liquid interface, resulting in a decrease of the contact angle. The droplet appears to "wet" the surface more, spreading out over the activated electrode.

[0207] This change in contact angle is the fundamental mechanism that allows for droplet manipulation in EWOD-based devices.

[0208] This principle maybe applied to move droplets across a surface (e.g., the plate-like structure). Initially, the droplet is positioned over one or more electrodes. By selectively activating adjacent electrodes and deactivating the current electrode, IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO the droplet is induced to move towards the activated electrode. This process can be repeated to move the droplet across the surface in a controlled manner.

[0209] This principle can be extended to perform various droplet operations, such as moving individual droplets, merging multiple droplets, and splitting a single droplet into multiple droplets, etc. These basic functionalities enable critical liquid handling operations such as: metering reagent volumes, mixing multiple reagents, diluting samples, and applying buffer exchanges.

[0210] It is noted that though FIG. 6 demonstrates a EWOD-based manipulation, other principles maybe used to achieve similar functionality, such as:

[0211] - opto-electrical wetting, where light pulses create a wetting effect on a hydrophobic and electro-optic substrate; and

[0212] - acoustic actuation of droplets.

[0213] It is noted that the acoustic actuation may be integrated with electrowetting for enhanced functionality.

[0214] The device in this disclosure comprises a 2D array of pixels, which can be as small as a few pixels or as large as thousands, arranged in various geometric layouts to suit different application needs.

[0215] This droplet manipulation (e.g., EWOD-based) allows for precise control and movement of droplets across the surface of the device. When combined with the heating and magnetic field capabilities described earlier, it enables a wide range of complex fluid processing operations to be performed on a single, integrated platform.

[0216] FIG. 7 shows an example of heat and magnetic field generation.

[0217] Manipulating magnetic particles within droplets is a key functionality for many biological assays. The top-right side of FIG. 7 demonstrates the magnetic flux density generated by a conductive element (e.g., a metal coil), which is capable of creating localized electromagnetic fields. IMEC VZW

[0218] PA 2024 / 160 PCT1

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[0220] This metal coil is designed as an alternative to traditional commercial magnetic plates used in well-plate format assays. Instead of a large, uniform magnetic field, this design creates a spatially non-uniform magnetic field and gradient that can exert precise electromagnetic forces on magnetic particles (such as ferromagnetic or superparamagnetic beads) encapsulated in a droplet in its vicinity.

[0221] FIG. 7 shows a representation of the magnetic flux density, measured in Gauss (G), generated when a direct current (DC) is applied to a coil within the pixel. The magnetic field strength is highest near the coil and decreases with distance, creating a gradient that is crucial for particle manipulation.

[0222] This design allows for:

[0223] - Localized control: The ability to selectively activate individual pixels in a 2D array, enabling precise spatial control over magnetic forces.

[0224] - Versatile manipulation: Attractive or repulsive forces can be used to move, immobilize, or separate magnetic particles within droplets.

[0225] - Integration with other functions: Similar to EWOD electrode pixels, these magnetic pixels can be part of a multifunctional array for comprehensive droplet manipulation.

[0226] The effectiveness of the magnetic manipulation is optimized by maximizing the product of field strength and spatial gradient. This maybe achieved through design considerations:

[0227] - adjusting the distance between the coil and the droplet;

[0228] - adjusting the number of conductor windings in the coil;

[0229] - adjusting the current through the conductor; and

[0230] - considering the magnetic permeability of the space within the winding.

[0231] While FIG. 7 shows a coil implementation, it's worth noting that other conductor geometries, such as simple linear conductors, could also be used to generate the required electromagnetic fields. The conductors can be made of pure metals or metal alloys, offering flexibility in manufacturing and performance optimization. IMEC VZW

[0232] PA 2024 / 160 PCT1

[0233] P626O5 / WO

[0234] This pixel design represents a significant advancement in integrating magnetic particle manipulation into a compact, multifunctional microfluidic device, enabling complex assay protocols that require precise control over magnetic beads for purposes such as purification, analyte capture, selection, and buffer exchange. FIG. 7 also demonstrates a localized heating, which is crucial for many biological assays. The bottom-right side of FIG. 7 illustrates the surface temperature distribution generated by a same conductive element (that are used for generating the magnetic field), designed to create localized resistive heating.

[0235] This solution offers an alternative to conventional commercial heating plates that use resistance-based or heat pump methods in well-plate format assays. Instead of uniform heating across a large area, this disclosure allows for precise, localized temperature control.

[0236] FIG. 7 shows a representation of the surface temperature, measured in degrees Celsius (°C), generated when an alternating current (AC) is applied to the conductive element within the pixel. The temperature is highest directly above the heating element and decreases towards the edges of the pixel, allowing for efficient heat transfer to a droplet in its vicinity.

[0237] There are several effects of this solution:

[0238] - Localized heating: The ability to selectively activate individual pixels in the plate-like structure, enabling precise spatial control over temperature.

[0239] - Efficient heat transfer: Heat generated from the resistive element is transferred to droplets via conduction and convection.

[0240] - Optimized geometry: The design parameters of the thermal element are optimized to achieve uniform and efficient heat transfer between the element and the droplets.

[0241] Notably, this same pixel element may also serve as a temperature sensor. Temperature changes alter the native resistivity of the conductive wire, allowing the effective resistivity to be measured by sense circuitry to estimate the temperature of the droplet in its vicinity. This can be implemented in the following ways: IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO

[0242] The same pixel can act as both heater and sensor during operation, and / or adjacent pixel can be used as proximal temperature sensors for neighboring heating pixels.

[0243] While the figure shows a coil geometry, it's important to note that various other geometries can be used to create pixelated elements for resistive heating, such as meander wires, rings, or linear elements. The choice of geometry can be optimized based on the specific requirements of the application and the manufacturing process.

[0244] Furthermore, a range of materials can be used for these thermal elements, including metals, semiconductors, polymers, or composites, offering flexibility in design and performance optimization.

[0245] This pixelated heating design represents a significant advancement in integrating precise temperature control into a compact, multifunctional microfluidic device. It enables complex assay protocols that require accurate and localized temperature regulation for activating and sustaining biochemical reactions, enhancing the capabilities of the overall fluid processing device.

[0246] FIG. 8A-8C demonstrates how a single conductive element, likely a metal coil, can be used to perform three different functions within a single pixel of the fluid processing device. This figure shows the versatility of the single-layer design, where one pixel can be selectively controlled to provide thermal, magnetic, or fluidic manipulation.

[0247] FIG. 8A shows an example of thermal actuation mode of a target pixel. The target pixel with a conductive element is connected to an AC (alternating current) power source.

[0248] The "AC current ON" label indicates that an alternating current is flowing through the conductive element, while no current is flowing for elements of other pixels labeled with “AC current OFF”. This causes resistive heating in the area of the IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO target pixel with “AC current ON”, which in turn heats the droplet (and particles comprised in the droplet) above it.

[0249] FIG. 8B shows an example of magnetic actuation mode of a target pixel. The conductive element of the target pixel is connected to a DC (direct current) power source. The "DC current ON" label shows that a direct current is flowing through the conductive element, while no current is flowing for elements of other pixels labeled with “DC current OFF”. This generates a magnetic field within the area of the target pixel, which can interact with magnetic particles of the droplet. The “DC current” can be interpreted as a uni-directional current flow. The amplitude and the waveform of the signal can vary such as being constant, harmonic (e.g. sinusoidal) or chirped, toothsaw etc. The mean amplitude of the signal can be correlated to the magnetic force therefore the strength of the force can be adjusting through adjusting the signal amplitude. The “DC current” may be also interpreted as a pre-dominantly uni-directional current. In other words, the current can be allowed to flow in reverse directions as long as it is predominantly in time pointing to one direction.

[0250] FIG. 8C shows an example of fluidic actuation mode, exemplary using the principle of electrowetting on dielectric (EWOD).

[0251] The conductive element of a pixel is connected to a to a DC power source, labelled as “DC current ON”. This is to retain the magnetic particles. If magnetic manipulation is not desired, this pixel can be provided with no current or voltage. Alternatively, the pixel is switched to “open circuit state” that will cut of current flow through the device even though voltage can be still applied. This mode of operation can be useful for fluidic operations or sensing functionality.

[0252] Conductive elements of the neighboring pixels are provided with high voltages, labelled as “High Voltage” in FIG. 8C. This creates an electric field that can change the wetting properties of the droplet, allowing for droplet manipulation.

[0253] In FIG. 8A-8C, a droplet is shown above the conductive element, representing the fluid that is being manipulated by the pixel. It is also possible that a part of a IMEC VZW

[0254] PA 2024 / 160 PCT1

[0255] P626O5 / WO droplet or multiple droplets may be manipulated by multiple pixels. In the latter case, the droplets can be subject to multiple actuations simultaneously. Alternatively, the multiple pixels can be employed to perform the same function on the same droplet. This brings the advantage of manipulating large droplets and small droplets with the same device design architecture.

[0256] FIG. 9 shows an example of multiple functional pixels connected to controller 91 comprised in the fluid processing device 90. The controller 91 is adapted to individually address functional pixels. The controller 91 maybe a microcontroller.

[0257] FIG. 9 shows that the fluid processing device 90 may have a matrix (columns and rows) arrangement of the functional pixels. The device may comprise row addressing lines 92 and column addressing lines 93, which are respectively interfaced with the controller 91. Further, switching elements 95 are provided, one switching element 95 for each of the functional pixels loa / b / c. The controller 91 may use the addressing lines 92, 93 and the switching elements 95 to individually address each functional pixel na / b / c. The switching element 95 may be referred to as inter-pixel switching element 95, which is used to address (e.g., determine and select) which pixel for performing at least two of the processing operations at different times.

[0258] As an example, the controller 91 can be configured to control, through the switching element 95, one or more of the functional pixels loa / b / c to be activated, so as to perform at least two of droplet manipulation, heating, and magnetic function to one or more droplets 13 at different times.

[0259] The controller 91 may comprise a processor or processing circuitry (not shown) configured to perform, conduct or initiate the various operations of the controller 91 described above. The processing circuitry may comprise hardware and / or the processing circuitry may be controlled by software. The hardware may comprise analog circuitry or digital circuitry, or both analog and digital circuitry. The digital circuitry may comprise components such as application-specific integrated circuits (ASICs), field-programmable arrays (FPGAs), digital signal processors (DSPs), or multi-purpose processors. The controller 91 may further comprise memory IMEC VZW

[0260] PA 2024 / 160 PCT1

[0261] P626O5 / WO circuitry, which stores one or more instruction(s) that can be executed by the processor or by the processing circuitry, in particular under control of the software. For instance, the memory circuitry may comprise a non-transitory storage medium storing executable software code which, when executed by the processor or the processing circuitry, causes the various operations of the controller 91 to be performed. In one embodiment, the processing circuitry comprises one or more processors and a non-transitory memory connected to the one or more processors. The non-transitory memory may carry executable program code which, when executed by the one or more processors, causes the controller 91 to perform, conduct or initiate the operations or methods described herein.

[0262] FIG. loAand 10B illustrate switching mechanisms that enable the multi-functional capabilities of functional pixels in a fluid processing device.

[0263] In this disclosure, each functional pixel may be adapted to receive at least two different control signals at different times for respectively performing at least two different processing operations.

[0264] FIG. 10A shows a pixel comprises a single layer with conductive formations, for instance representing the metal coil or electrode structure discussed previously. The pixel is connected with a circuitry comprising a switching element 103a. The circuitry is capable of provide at least two distinct waveforms, which can vary in amplitude, frequency, waveform shape, and / or duty cycle, to the pixel through the switching element 103a. The switching element 103a may also be referred to as intra-pixel switching element, which is used to control which processing function to perform at the corresponding pixel.

[0265] The state of the switching element 103a (open or closed) and the kind of signal it provides when closed determines the functionality of the pixel, allowing for independent operation of the pixel by controlling the switches and the applied waveform. For instance, if line 102a is closed and a DC current is applied through line 101a, then the pixel is adapted to generate a magnetic field. If line 102a is closed and an AC current is applied through line 101a, then the pixel is adapted to IMEC VZW

[0266] PA 2024 / 160 PCT1

[0267] P626O5 / WO generate heat. If line 102a is open, and a high voltage is applied to line 101a, then the pixel is adapted to perform droplet manipulation (as illustrated in FIG. 6).

[0268] FIG. 10B shows a pixel comprises multiple layers. Each layer may be connected individually through a switching element 103b. Similarly, the state of the switching element 103b (open or closed) and the kind of signal it provides when closed determines the functionality of the pixel.

[0269] FIG. 11 shows a further example of switching mechanisms for controlling pixels of a fluid processing device according to this disclosure. Based on FIG. 10A or FIG. 10B, the switching element maybe configured to switch among a current waveform circuit line 111, a potential circuit line 112, and a ground circuit line 113 at different times, so as to selectively provide the at least two different signals to each functional pixel iia / nb / nc at different times.

[0270] The switching mechanism shown in FIG. 11 is designed to selectively connect the pixel to these lines at different times, enabling the pixel to perform various functions based on the electrical input it receives. For instance, the pixel on the left is connected (represented by a filled dot) to the potential line 111 on one end with another end floating. This left pixel is adapted to apply electrowetting on the droplet located in its vicinity. The pixel on the right is connected to the wavefront line 112 on one end with another end connected with the ground. This right pixel is adapted to generate magnetic field.

[0271] It is noted that the features introduced in FIG. 2-11 are applicable to the functional pixel na / b / c introduced in FIG. 1.

[0272] FIG. 12 shows a method 100 according to this disclosure. The method 1200 may be used for fluid processing, and can be performed by the fluid processing device 10. The method comprises a step 1201 of manipulating one or more droplets 13 of a fluid or liquid, which are located a plate-like structure of the device. The method 1200 further comprises a step 1202 of selectively applying at least two of heat, a magnetic field, and a manipulation force, in any order, to the one or more droplets IMEC VZW

[0273] PA 2024 / 160 PCT1

[0274] P626O5 / WO at different times by applying different control signals at different times to each functional pixels.

[0275] FIG. 13 shows a plate-like structure of a fluid processing device according to this disclosure. The plate-like structure is formed by a grid-like pattern of functional pixels, each represented as a square unit. These pixels are arranged in a form of array, forming a plate-like structure 1301 (which is an example of the plate-like structure 10 in FIG. 1) as an active area of the fluid processing device. As an example, each pixel has a dimension of 200pm wide. This microscale design allows for precise control over small volumes of fluid, which is crucial for many microfluidic applications.

[0276] Each pixel in the array represents a multi-functional unit capable of performing various operations such as droplet manipulation (e.g., through electro wetting), heating, and magnetic field generation, as described in previous figures. The regular arrangement of these pixels ensures uniform coverage across the device's surface, allowing for consistent performance regardless of a droplet's position.

[0277] It is noted that the number of pixels and the size of each pixel in FIG. 13 are for illustration purposes only.

[0278] FIG. 14 shows an example of a fluid processing array 1410 comprising multiple fluid processing devices 1401 according to this disclosure. These devices 1401 are arranged in a grid pattern, forming a larger, more comprehensive fluid processing system, such as a cartridge. Each fluid processing devices 1401 in the cartridge has its own input / output ports and therefore is independently operable. The cartridge can be consumable device in the sense that it needs to be disposed after a certain period of usage. The driver, sensing electronics can be situated in a reader box and the actuation and sensing on cartridge can occur through electronic contacts between the cartridge and reader. The variable scalability can be achieved through multiplexing the readers within the same instrument architecture. Readers can be accessed in parallel with a single instrument that is the main unit that establishes the workflow programming and user interface. Such scalability provides users a flexibility of operating one sample in a cartridge or hundreds of samples in parallel IMEC VZW

[0279] PA 2024 / 160 PCT1

[0280] P626O5 / WO for the same or different workflows or in a batched format depending on the needs. This overcomes the existing difficulty of batching samples in large quantities for the same workflow due to lack of capability of executing parallel workflows.

[0281] FIG. 15 shows an example of a fluid processing system 1500. In this system 1500, multiple fluid processing arrays 1410 maybe programmed and controlled, forming a fully functional and automated laboratory instrument.

[0282] In FIG. 13 - FIG. 15, the form factor of pixels, the array size and format can be designed for given application requirements. Larger the array size of pixels, larger the programmability freedom it provides. As each functionality is presented in a distributed fashion on the substrate, it allows various workflows to be implemented without the limitation of number of functionalities. Furthermore, layered integrable structure provides the benefit of miniaturization as all critical functionalities are within the same substrate and controlled with the same functional pixels. Thus, the samples, reagents, chemicals etc. can be all present in the same platform without need for transfer between instruments.

[0283] It is noted that the functional pixels in this disclosure may be controlled individually with a circuitry to actuate different functions separately in a sequence or simultaneous fashion..

[0284] To further improve the device's magnetic manipulation capabilities, a permanent magnet system may be incorporated. This system enhances the magnetostatic forces generated by the functional pixels. The magnetic force, being proportional to the product of the magnetic field gradient and its magnitude, can be significantly increased by introducing a global magnetic field. This is achieved by positioning the device in proximity to an external magnetic element, such as a permanent magnet or an array of magnets. These magnets are arranged to create a strong magnetic field (preferably >iooG) that extends across the entire device, penetrating through the droplets. This global field complements the localized fields generated by individual pixels, enhancing overall magnetic particle manipulation capabilities. IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO

[0285] The performance of the fluid processing device can be further optimized by implementing a global thermal control system. This system maintains the device temperature within a range of 4°C to 6o°C, as required by specific assays or workflows. The thermal control system may comprise a thermal interface on at least one of the substrate surfaces not in contact with the droplets. This interface may be thermally coupled to a heat pump, facilitating efficient heat transfer to or from the device. Temperature sensors placed on or near the substrate provide feedback, allowing precise temperature regulation.

[0286] To enhance the reliability and reusability of the device, the surfaces of the platelike structure (the side contacting the fluid) can be coated with specialized layers. These coatings, typically composed of polymers or organic materials, serve multiple purposes, such as:

[0287] - Prevention of surface fouling;

[0288] - Reduction of electrostatic charging;

[0289] - Protection against dielectric breakdown of underlying layers. These treatments can be applied to both the top and bottom plates of the device, ensuring consistent performance over multiple use cycles.

[0290] The fluid processing device of this disclosure may be integrated with a fluidic distribution network that connects the input and output ports to external fluidic interfaces, such as pipettes. This network enables controlled distribution of reagents or samples to multiple locations on the device, facilitating parallel operations. The fluidic network maybe constructed from materials such as plastic or glass and can incorporate channels, chambers, and valves for precise fluid transfer.

[0291] The fluid processing device may be designed to be controlled by external or embedded controller circuitry. This allows for the execution of predefined operations stored in local or external memory. The controller can dynamically combine different zones of the device based on reagent volumes or workflow requirements. Additionally, the control system can interact with the device's sensor network, adapting operations in real-time to compensate for local failures by redirecting processes to functional zones. IMEC VZW

[0292] PA 2024 / 160 PCT1

[0293] P626O5 / WO

[0294] The fluid processing device can be manufactured using various technologies, such as:

[0295] - Printed Circuit Board (PCB) Technology: Offers reliability, costeffectiveness, and scalability. PCBs allow for higher current densities, beneficial for magnetic element functionality.

[0296] - Flat Panel Technology: Enables the production of high-density pixel arrays with smaller critical dimensions, allowing for smaller droplet volumes and potentially more precise control.

[0297] - Hybrid Approach: Combining PCB and Flat Panel technologies can leverage the strengths of both, though at the cost of more complex integration.

[0298] The choice of manufacturing technology depends on specific application requirements, such as desired droplet volumes, magnetic force strengths, and cost considerations. Regardless of the chosen technology, the core concept of combining multi-functional elements to achieve programmable and reliable workflows remains central to the invention.

[0299] These additional features and manufacturing considerations enhance the versatility, efficiency, and practical feasibility of the fluid processing device, making it adaptable to a wide range of microfluidic applications in research and diagnostic settings.

[0300] In conclusion, the fluid processing device described herein represents a significant advancement in the field of laboratory automation, particularly for complex biological assays such as genome and transcriptome sequencing applications. This innovative system addresses several critical challenges faced by researchers and laboratory professionals in conducting high-throughput, multi-step analytical procedures.

[0301] The device's core strength lies in its ability to automate the manipulation of reagents, chemicals, and biological materials, performing necessary assays and biochemical reactions on input samples within a single, integrated platform. IMEC VZW

[0302] PA 2024 / 160 PCT1

[0303] P626O5 / WO

[0304] By consolidating multiple functionalities into a single device, this invention eliminates the need for numerous dedicated instruments and consumables. This integration not only streamlines complex workflows but also reduces the potential for errors associated with manual handling and transfers between different instruments.

[0305] The compact design of the fluid processing device allows for more efficient use of laboratory space. This feature enables researchers to deploy multiple units within limited areas, facilitating increased parallelization of workflows and overall throughput.

[0306] The device's flexible and programmable nature significantly reduces sample waiting times and eliminates the need for large batch processing of identical samples. This programmability offers users greater control over their workflows, improving overall laboratory efficiency and reducing organizational complexities.

[0307] Unlike many existing solutions that are limited to specific throughput ranges, this device offers remarkable scalability. It is capable of efficiently handling both low- throughput (fewer than 16 samples) and high-throughput (over 1000 samples) applications, providing a versatile solution adaptable to varying research needs and scales of operation.

[0308] By addressing these longstanding challenges in laboratory automation, this invention not only improves current research capabilities but also opens new possibilities for high-throughput biological assays.

[0309] It is noted that in the claims as well as in the description of this disclosure, the word “comprising” does not exclude other elements or steps and the indefinite article “a” or “an” does not exclude a plurality. A single element may fulfill the functions of several entities or items recited in the claims. The mere fact that certain measures are recited in the mutual different dependent claims does not indicate that a combination of these measures cannot be used in an advantageous implementation.

Claims

IMEC VZWPA 2024 / 160 PCT1 P626O5 / WOClaims1. A fluid processing device comprising: a plate-like structure (10) for processing one or more droplets located on the plate-like structure (10), wherein the plate-like structure (10) comprises a plurality of functional pixels (11a, 11b, 11c), wherein each functional pixel (11a, 11b, 11c) as a whole is adapted to selectively perform at least two of the following processing operations at different times on a droplet located on the functional pixel: manipulating the droplet; applying heat to the droplet; and applying a magnetic field to the droplet.

2. The fluid processing device according to claim 1, wherein the plurality of functional pixels (11a, 11b, 11c) are arranged adjacently across a continuous part of the plate-like structure (10).

3. The fluid processing device according to claim 1 or 2, wherein each functional pixel (11a, 11b, 11c) comprises a conductive element configured to perform each of the at least two processing operations.

4. The fluid processing device according to any one of claims 1 to 3, further comprising a plurality of metal coils embedded in the plate-like structure (10), wherein each functional pixel (11a, 11b, 11c) is associated with one or more metal coils, and each functional pixel (11a, 11b, 11c) as a whole is adapted to perform the at least two processing operations on the droplet located on the functional pixel (11a, 11b, 11c) by respectively applying different signals to the one or more metal coils.

5. The fluid processing device according to claim 4, wherein the one or more metal coils associated with a functional pixel (11a, 11b, 11c) are adapted to: manipulate the droplet located on the functional pixel (11a, 11b, 11c) in response to receiving a voltage with no current flow;IMEC VZW PA 2024 / 160 PCT1 P626O5 / WO apply heat to droplet located on the functional pixel (11a, 11b, 11c) in response to receiving an alternating current; or apply the magnetic field to the droplet located on the functional pixel (11a, 11b, 11c) in response to receiving a direct current.

6. The fluid processing device according to any one of claims 1 to 3, wherein each functional pixel (11a, 11b, 11c) comprises a plurality of stacked layers (221, 222; 231, 232, 233), wherein each layer (221, 222, 231, 232, 233) is adapted to perform one or more of the at least two processing operations.

7. The fluid processing device according to claim 6, wherein each functional pixel (11a, 11b, 11c) comprises a first layer (221) adapted to manipulate the droplet located on the functional pixel, and a second layer (222) adapted to apply heat and the magnetic field to the droplet located on the functional pixel.

8. The fluid processing device according to claim 7, wherein each functional pixel (11a, 11b, 11c) comprises an electrowetting element in the first layer (221) for manipulating the droplet, and an electrode in the second layer (222) for applying heating and the magnetic field to the droplet.

9. The fluid processing device according to claim 6, wherein each functional pixel (11a, 11b, 11c) comprises a first layer (231) adapted to manipulate the droplet located on the functional pixel, a second layer (232) adapted to apply the magnetic field to the droplet located on the functional pixel, and a third layer (233) adapted to apply heat to the droplet located on the functional pixel.

10. The fluid processing device according to claim 9, wherein each functional pixel (11a, 11b, 11c) comprises an electrowetting element in the first layer (231) for manipulating the droplet, a thermal element in the second layer (232) for applying heating to the droplet, and a magnetic element in the third layer (233) for applying the magnetic field to the droplet.

11. The fluid processing device according to any one of claims 1 to 10, wherein one or more of the plurality of functional pixels (11a, 11b, 11c) are configured toIMEC VZWPA 2024 / 160 PCT1P626O5 / WO provide light onto and / or detect light emitted by one or more droplets located on the one or more functional pixels (11a, 11b, 11c).

12. The fluid processing device according to claim 11, further comprising one or more light emitting devices (54) configured to emit the light of at least one predetermined wavelength, and / or one or more photodetectors (55) configured to detect the light emitted by the one or more droplets located on the one or more functional pixels (11a, 11b, 11c), wherein the one or more light emitting devices (54) and the one or more photodetectors (55) are arranged on or besides an outer surface of the one or more functional pixels (11a, 11b, 11c).

13. The fluid processing device according to any one of claims 1 to 12, further comprising: one or more input ports, each input port being configured to receive the one or more droplets and to provide the one or more droplets onto the plate-like structure; and / or one or more output ports, each output port being configured to eject the one or more droplets from the fluid processing device.

14. The fluid processing device according to any one of claims 1 to 13, wherein each functional pixel (11a, 11b, 11c) is adapted to receive at least two different control signals at different times for respectively controlling each functional pixel (11a, 11b, 11c) to perform the at least two different processing operations.

15. The fluid processing device according to claim 14, wherein each functional pixel (11a, 11b, 11c) is connected with a circuitry (95), the circuitry comprising at least one electronic switching element configured to selectively provide the at least two different control signals to each functional pixel (11a, 11b, 11c) at different times.

16. The fluid processing device according to claim 15, wherein the at least one electronic switching element is connectable to a common circuit line (101a, 101b), wherein the common circuit line (101a, 101b) is adapted to produce the at least two different signals at different times.IMEC VZWPA 2024 / 160 PCT1P626O5 / WO17. The fluid processing device according to claim 15, wherein the at least one electronic switching element is configured to switch among a current waveform circuit line (111), a potential circuit line (112), and a ground circuit line (113) at different times, so as to selectively provide the at least two different signals to each functional pixel (11a, 11b, 11c) at different times.

18. The fluid processing device according to any one of claims 14 to 17, wherein the at least two different signals for performing the at least two different processing operations differ in terms of their amplitude and / or frequency and / or waveform and / or duty cycle.

19. The fluid processing device according to any one of claims 1 to 18, further comprising a controller (91) configured to address the plurality of functional pixels (11a, 11b, 11c).

20. A fluid processing array (1410) comprising a plurality of fluid processing devices (1401) according to one of the claims 1 to 19.

21. A fluid processing system (1500) comprising at least one fluid processing device according to one of the claims 1 to 19.

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