Multidimensional positioning instrument to automate liquid handling and droplet manipulation

Abstract

A device for liquid handling and droplet manipulation includes a multidimensional positioning instrument having a manipulation head mounted thereon and containing one or more tools secured to the manipulation head. The one or more tools are selected from the group comprising: a camera, optical sensor, one or more permanent magnets, an electromagnet, pipettor, electrofusion head, a plunger holder, and a contact member. A stage is disposed adjacent to the multi-axis manipulator, wherein the stage is configured to receive one or more cartridges thereon. The multi-axis manipulator and / or stage are configured for relative motion along three orthogonal axes. A computing device is provided that includes software configured to control the relative motion of the multi-axis manipulator and stage to place the one or more tools over or in contact with selected regions of the one or more cartridges as part of series of pre-programmed operations or steps.

Description

2025-094-2MULTIDIMENSIONAL POSITIONING INSTRUMENT TO AUTOMATE LIQUID HANDLING AND DROPLET MANIPULATIONRelated Application

[0001] This Application claims priority to U.S. Provisional Patent Application No.63 / 748,263 filed on January 22, 2025, which is hereby incorporated by reference in its entirety. Priority is claimed pursuant to 35 U.S.C. § 119 and any other applicable statute.Technical Field

[0002] The technical field generally relates a multidimensional motion positioning instrument used to automate liquid handling and droplet manipulation. The instrument uses a customizable manipulation head and specialized software for precise fluid control in cartridges. The system automates fluid handling, including magnetically controlled droplet movement and electrofusion, for volumes ranging from microliters to hundreds of microliters. The adaptable design supports various laboratory applications, from imaging to sensing. Additionally, the cartridges are constructed with affordable, transparent materials (e.g., PET membranes, acrylic sheets) and enable precise droplet partitioning, merging, and bead separation, catering to diverse experimental needs.Statement Regarding Federally SponsoredResearch and Development

[0003] This invention was made with government support under HL 148182, and HL169695 awarded by the National Institutes of Health, and 1648451 awarded by the National Science Foundation. The government has certain rights in the invention.Background

[0004] Existing automated liquid handling devices are often bulky, limited in functionality, and require manual intervention for certain steps. Many also exhibit inefficient control, particularly during the magnetic manipulation of microfluidic ferrofluidic droplets, leading to less precise fluidic behaviors. International Patent Application Publication No. W02024050485 discloses a digital ferrofluidic device and method for multiplexed assays and viral testing. The device is able to automatically perform loop-mediated isothermal2025-094-2amplification and the detection of SARS-CoV-2 virus in clinical samples. To manipulate ferrofluidic droplets, individual addressable coils are located on a substrate beneath a microfluidic chip that contains ferrofluidic droplets. The individually addressable coils operate as an electromagnet when actuated and current is driven through the addressable coils. This substrate acts as a navigation floor for permanent magnets that act or operate as moveable ‘Terrobots'’ that are used to carry volumes or droplets of ferrofluid in the microfluidic chip. U.S. Patent Application Publication No. 2022 / 0379309 discloses a similar platform or system that is able to perform droplet sorting, dispensing, droplet generation, merging, mixing, filtering, and analysis that uses individually addressable coils in combination with moveable permanent magnets to earn,' volumes or droplets of ferrofluid within a microfluidic chip.Summary

[0005] This system and instrument address the challenges associated with automated liquid handling and the execution of reactions involving small fluid volumes in life science research, diagnostics, and laboratory testing. The invention includes several key components: a multidimensional positioning instrument, such as a 3D printer or robotic assembly system, with precise control over the localization of one or more manipulation heads; a microfluidic cartridge that houses reagents, samples, and other elements necessary for controlled reactions, operated by the manipulation head of the positioning system; and a software system designed to automate the 3D positioning system for executing reaction operations on the microfluidic cartridge either in parallel or sequentially. In certain embodiments, the microfluidic cartridge includes ferrofluid droplets, manipulated using stacked magnets on the manipulation head to achieve ferrobotic automation, containing, for example, biological samples such as cells, nucleic acids, proteins, sugars, lipids, and other biomarkers or combinations thereof. The invention also features feedback control mechanisms that allow for automated alignment of the manipulation head with the microfluidic cartridge.

[0006] The instrument addresses the limitations of existing automated liquid handling devices by offering a fully integrated, miniaturized solution that consolidates a wide range of automated functions into a single system. This innovation streamlines microfluidic liquid handling, significantly reducing human error by increasing precision in repetitive tasks, all while enhancing efficiency and throughput. In addition to its hardware advantages, the system is equipped with an intuitive GUI, making it accessible to users of various technical2025-094-2backgrounds. By automating complex laboratory' experiments, the system minimizes manual effort and reduces the risk of errors commonly associated with biological and biochemical assays. The instrument and system provide a seamless, high-precision alternative to traditional systems, meeting the growing demand for more advanced, user-friendly automation in life science research.

[0007] In one embodiment, a device for liquid handling and droplet manipulation includes a multidimensional positioning instrument comprising a multi-axis manipulator having a manipulation head mounted thereon and containing one or more tools secured to the manipulation head. A stage is disposed adjacent to the multi-axis manipulator, wherein the stage is configured to receive one or more cartridges and wherein the multi-axis manipulator and / or stage are configured for relative motion along three orthogonal axes (e.g.. x, y, and z). A computing device is provided that includes software configured to control the relative motion of the multi-axis manipulator and / or stage to place the one or more tools over or in contact with selected regions of the one or more cartridges as part of series of preprogrammed operations or steps.

[0008] A method of performing liquid handling and droplet manipulation using the device described above includes: loading reagents or a sample in the one or more cartridges; and executing the series of pre-programmed operations or steps to perform one or more operations including droplet generation, droplet transport, droplet merging, droplet mixing, droplet reaction, droplet incubation, or droplet splitting.Brief Description of the Drawings

[0009] FIG. 1A illustrates an exemplary enhanced multidimensional positioning instrument (three-axis) with functional embodiments controlled by a user interface for manipulating magnetic droplets in cartridges for general laboratory automation.

[0010] FIG. IB illustrates another exemplary enhanced multidimensional positioning instrument (three-axis) with functional embodiments controlled by a user interface for manipulating magnetic droplets in cartridges for general laboratory automation. FIG. IB further illustrates a computing device interfacing with the multidimensional positioning instrument (three-axis) along with a display that includes a Graphical User Interface (GUI).

[0011] FIG. 2A illustrates a tool (electrofusion head) used for contactless, high-voltage droplet merging.2025-094-2

[0012] FIG. 2B illustrates another embodiment of an electrofusion head that uses low-voltage electrode contacts that utilize direct contact with corresponding electrical contacts on the cartridge to transduce the electrical shock to droplets. Unlike the FIG. 2A embodiment, this electrofusion head contacts the top surface of the cartridge.

[0013] FIG. 2C illustrates the camera / sensor head of the system as well as an optical density sensor (right) that can be used in the system.

[0014] FIG. 2D is schematic illustration of the manipulation head using a contact member according to another embodiment, which can apply precise pressure on the top layer / flexible membrane of microfluidic cartridges to transport droplets.

[0015] FIG. 3A illustrates a laser-cut acrylic microfluidic cartridge and its fabrication method.

[0016] FIG. 3B illustrates a laser-cut superhydrophobic acrylic microfluidic cartridge and its fabrication methods.

[0017] FIG. 3C illustrates a 3D-printed microfluidic cartridge and its fabrication.

[0018] FIG. 4A demonstrates the z-axis-controlled liquid handling module on an exemplary 3D printer used as part of the multidimensional positioning instrument.

[0019] FIG. 4B illustrates a liquid handling module according to another embodiment that uses a syringe pump that is used to dispense or w ithdrawn fluids onto / from cartridges. This syringe pump-based liquid handling module provides small and large volume control.

[0020] FIG. 5A illustrates an Editor Panel of the intuitive GUI of the enhanced multidimensional positioning instrument. The Editor Panel shows a graphical representation of the active area of the multidimensional positioning instrument (e.g., a stage), along with shape outlines of the cartridges and motion paths for assay automation. It enables the user to run, edit, and create assay procedures in a user-friendly manner.

[0021] FIG. 5B illustrates the Camera Panel of the GUI of the enhanced multidimensional motion system. The camera panel allows the user to, inter alia, specify camera parameters, capture images, and navigate between the captured images.

[0022] FIG. 5C illustrates the G-Code Panel of the GUI of the enhanced multidimensional motion system. The G-Code Panel displays G-Code machine commands based on the user's inputs from the Editor Panel.

[0023] FIG. 5D illustrates the Syringe Setup Panel of the GUI of the enhanced multidimensional motion system. The Syringe Setup Panel allows the user to specify parameters for the syringe pump to introduce and transfer liquid samples.2025-094-2

[0024] FIG. 6A illustrates a set of fundamental magnetic droplet manipulations that may¬ be performed within a microfluidic cartridge according to one embodiment. Illustrated are droplet generation, droplet delivery, droplet dispensing, droplet merging, and droplet removal.

[0025] FIG. 6B illustrates the results of 1 pL droplet dispensing using ferrofluid of a variety of concentrations. A consistent volume of 1 pL is reported across a range of ferrofluid concentrations 2.5%-15.0%.

[0026] FIG. 6C are images of dispensed droplets of a variety' of volumes (0.5 pL to 5.0 pL) at 2.5% and 5.0 % ferrofluid.

[0027] FIG. 6D is a graph showing theoretical and experimental ferrodroplet volumes for volumes (0.5 pL to 5.0 pL) at 2.5% and 5.0 % ferrofluid.

[0028] FIG. 6E illustrates buffer droplet generation in cartridge having a nozzle designed for large volume droplet generation.

[0029] FIG. 6F illustrates a graph showing the buffer droplet volume generated (pL) as a function of pause time using 2.5% and 5.0% ferrofluids. As the pause time increases, the volume of the generated buffer droplets increases until it approaches saturation. A linear correlation is observed between the pause time and buffer droplet volume (R2> 0.99), indicating high predictability. Once the pause time exceeds 3 seconds, the droplet size surpasses the diameter of the stacked magnets, reducing the attraction effect and resulting in a non-linear correlation between pause time and buffer droplet volume.

[0030] FIG. 7A illustrates three methods for transporting droplets on a microfluidic cartridge. The first two (left) utilize a magnet while the last (right) uses pressure applied to a flexible membrane.

[0031] FIG. 7B schematically illustrates the manipulation head transporting the droplets using the methods of FIG. 7A.

[0032] FIG. 7C illustrates the characterization of magnetic droplet velocities for smallvolume droplets under different oil and particle conditions.

[0033] FIG. 8A illustrates the general implementation of automated serial dilution in a microfluidic cartridge according to one embodiment.

[0034] FIG. 8B illustrates the general implementation of biomolecule capture using magnetic beads in a microfluidic cartridge according to one embodiment.

[0035] FIG. 8C illustrates the general implementation of liquid handling and blood filtration in a microfluidic cartridge according to one embodiment.2025-094-2

[0036] FIG. 8D illustrates the general implementation of automated multiplexed reactions in a microfluidic cartridge according to one embodiment.

[0037] FIG. 8E illustrates both two-dimensional and three-dimensional implementations of thermal control in a microfluidic cartridge according to one embodiment.

[0038] FIG. 9 illustrates the workflow of automated serial dilutions (with a buffer) using a multidimensional positioning instrument with ferrofluid droplets according to one embodiment.

[0039] FIG. 10 illustrates a microfluidic cartridge design and workflow for an automated sandwich ELISA assay.

[0040] FIG. 11 illustrates the workflow for automated colorimetric PCR within a microfluidic cartridge and the colorimetric change caused by PCR amplification-induced pH drop.

[0041] FIG. 12 illustrates the general workflow of an automated agglutination assay on a microfluidic cartridge, using the example of an antibody-mediated reaction.

[0042] FIG. 13 illustrates an automatic nucleic acid library’ preparation process within microfluidic cartridges, enabling downstream next-generation sequencing.

[0043] FIG. 14 illustrates the workflow of automated antibacterial susceptibility testing (AST) within microfluidic cartridges.

[0044] FIG. 15 illustrates automated bacteria cell culture optical density measurement and passaging within microfluidic cartridges.

[0045] FIG. 16 illustrates the workflow of automated cell electroporation for gene delivery’ within microfluidic cartridges.

[0046] FIG. 17A illustrates the characterization of the transportation speed of droplets infused with ferrofluids (FF, 2.5% or 5.0%, uniformly distributed under a magnetic field) or magnetic beads (MBs) (clustered).

[0047] FIG. 17B shows the results that confirm robust droplet transportation was verified by moving a 2.5% ferrodroplet across a spectrophotometric sensor, occluding it, and then moving back to the origin, and repeating this transportation over 40,000 sec. The sensor detects the repetitive patterns as shown in the insets. The illustration shows the test setup on the print bed.

[0048] FIG. 17C shows the characterization of the dispenser for tiny volume aliquoting. The magnet moves straight across the dispenser to create 0.5-5 pL daughter droplets, or2025-094-2moves diagonally over, pauses, and moves out of the dispenser to create 6-9 pL daughter droplets.

[0049] FIG. 17D shows the volume accuracy of dispensed droplets with 2.5% or 5.0% FF is plotted, showing high correlation (2.5%, R2= 0.99; 5%, R2= 0.99).

[0050] FIG. 17E shows images of the pipeline to generate five 20 pL daughter droplets using the cartridge configuration of FIG. 6E: (i) Align the magnet to the nozzle and slowly move down to magnetically attract the parent ferrodroplet (100 pL). (ii) A part of the ferrodroplet flows out of the nozzle (width of 1.0 mm), and the pause time (At) controls the volume of a daughter droplet, (iii) Quickly move the magnet to the right to pinch off the daughter droplet, (iv) Move the daughter droplet to the storage chamber, (v) Lift the magnet and go back to the parent droplet position, (vi) Repeat four times until the parent droplet is fully consumed.

[0051] FIG. 17F shows plots of the volume accuracy, showing the generated daughter droplets of different target volumes between 10 and 25 pL (2.5%, R2= 0.99; 5.0%, R2= 0.99).

[0052] FIG. 17G shows the characterization of magnetic mixing of ferrodroplet contents. A 2 pL dye sample was electrofused with 8 pL buffer droplets and magnetically mixed by using different combinations of the number of rotation cycles and radii. Mixing images are shown from 10 to 30 cycles of rotations with a radius of 0 to 3.5 mm.

[0053] FIG. 17H shows the electro fused Mixing Index (dye-colored area ratio) is plotted for different numbers of rotation cycles and radii.

[0054] FIG. 171 shows an illustration of the MBs separator layout and major steps of bead capture. The stacked magnets align with the MBs-infused ferrodroplet (Step 1), then moves the droplet against a semicircular chamber (radius of ~0.5 mm) while the MBs are clustered and immobilized (Step 2). The magnet then extracts the ferrodroplet from the MBs, moving the droplet to the right (Step 3). The sequestered MBs are indicated in the bottom image by a red dashed circle.

[0055] FIG. 17J shows separation efficiency was quantified by comparing light transmission before and after separation. After separation, all the groups (10%, 50%, and 100% MBs solutions) achieved the same level of transparency compared to the 0% reference group. Obvious color changes can be observed before and after the separation, as shown in the insets for the 50% and 100% MBs solutions.2025-094-2

[0056] FIG. 18A illustrates a schematic workflow of a 5* serial dilution, including buffer droplet generation and dye sample dilution: (i) the buffer droplets were formed by collecting the ferrofluid (FF)-infused parent buffer drops from the buffer reservoirs (Step 1), dispensing them into individual daughter drops (Step 2), and then moving the daughter droplets up to the dilution chambers (Step 3). (ii) The dye samples, also infused with ferrofluid, were serially diluted by dispensing the sample and delivering it to the closest dilution chamber with a daughter buffer drop (Step 4). The dispensed sample and the daughter buffer drop are merged using contactless electrofusion, and the merged droplet is rotated magnetically to mix its contents (Step 5). Finally, this process was repeated several times across the sequential dilution chambers (Step 6).

[0057] FIGS. 18B-18D are graphs showing the normalized light intensity (l-R ratio)* vs dilution factor* for 2x (FIG. 18B), 5x (FIG. 18C), and 10x (FIG. 18D) serial dilutions with the Lo3DP using 2.5% FF droplets. The decreasing colorimetric signal with dilution matched well between the Lo3DP and the manual test tube methods.

[0058] FIG. 18E is a graph showing the normalized light intensity (l-R ratio * vs the dilution factor* for I0x serial dilution with the system using 10% FF droplets, showing signal saturation (arrowed on graph) that obscures the colorimetric readouts (Color signals were not significantly different, p = 0.5181, when diluting the sample 10 times). The curves of colorimetric signals matched well between the system and manual test tube methods. *Log2 scale for 2x and Logio scale for 5x / 10x serial dilutions.

[0059] FIG. 19A illustrates a schematic of the LAMP assay workflow for automated inparallel testing of primer sets. The initial positions of reagents such as LAMP primer sets (i)-(v), positive DNA and negative control (+ DNA & -), and LAMP master mix are shown. The workflow for verifying BRCA1 LAMP primer sets involves several steps following the loading of oil and all reagents: 1) Dispense and transport primer sets: the primer set (i) is moved to the dispenser adjacent to the LAMP master mix to split the parent droplet into two droplets, follow ed by sequential addition to the reaction mix droplets. This process is repeated for primer sets (ii)-(v). 2) Dispense and transport + DNA & - samples to merge with the LAMP master mix: move the negative control parent droplet to the dispensers to form five daughter ferrodroplets, which are then merged with five different LAMP master mix droplets to form reaction mix droplets. Then, this process is applied to the DNA + parent droplet. 3) Incubate and capture reaction images: perform in situ heating to 65°C, incubate for 30 minutes, and acquire endpoint colorimetric images of the LAMP reactions.2025-094-2

[0060] FIG. 19B illustrates a microfluidic cartridge temperature control and calibration setup: the illustration depicts the calibration process in which the stage (e.g., 3D printer printing bed) is heated from 20°C to 110°C while the thermal camera measures the temperature of both the print bed and microfluidic cartridge. A strong linear correlation is observed between the set temperature and cartridge temperature (R2= 1.00), with temperature stability’ at 65°C measured over ten trials (CV of 0.3%).

[0061] FIG. 19C shows the validation of primer sets for BRCA1 breast cancer gene detection by colorimetric LAMP on the system. Successful amplification of the target gene induces a pH change, resulting in a clear color transition from pink to yellow. The positive droplet from primer set (i) remained pinkish, indicating insufficient amplification and a suboptimal primer set. In contrast, the positive droplets from the other primer sets (ii)-(v) displayed yellow coloration after incubation, indicating efficient amplification and effective primer sets, which matched well with gel electrophoresis, revealing distinct amplicon bands in sets (ii)-(v).

[0062] FIG. 20 illustrates the step-by-step droplet generation process (2.5% and 5% ferrofluid) and magnetic bead separation.

[0063] FIG. 21 A shows a graph (colorimetric properties of ferrodroplets) of the normalized dynamic range as a function of ferrofluid concentration for different wavelengths.

[0064] FIG. 21B shows a graph (fluorescent properties of ferrodroplets) of the fluorescent intensity as a function of ferrofluid concentration for different fluorescein concentrations.Detailed Description of Illustrated Embodiments

[0065] FIGS. 1A and IB illustrate a system 10 for liquid handling and droplet manipulation according to one embodiment. The system 10 enables enhanced multidimensional positioning of tools or instruments for laboratory automation and represents a significant advancement in streamlining and miniaturizing liquid handling tasks, especially for repetitive biological and biochemical assays. This innovative system 10 integrates adaptable platforms such as FDM 3D printers, CNC drilling machines, and custom robotic assembly systems, providing a versatile foundation for precise and efficient laboratory operations. At the core of this system 10 is a motion platform that incorporates a multidimensional positioning instrument 12 and stage 14 that supports multidimensional movements across up to eight axes (including rotational movement), including the X, Y, and Z axes, enabling precise positioning and operation of various functions. The2025-094-2multidimensional positioning instrument 12 includes a manipulation head 18 mounted thereon. The manipulation head 18 contains or is configured to contain on or more tools 20 that are secured to the manipulation head 18. The multidimensional positioning instrument 12 may include a frame 22 along with cross-member 24 on which the manipulation head 18 is mounted. The frame 22 may be fixed in position and the cross-member 24 may move vertically relative to the stationary frame 22 so as to provide vertical motion to the manipulation head 18 and tools 20 mounted thereon. For example, the cross-member 24 may be raised or lowered using a belt drive (not shown) that uses servos or motors to drive the belt and thus move the cross-member 24.

[0066] The manipulation head 18 is moveable laterally along the length of the crossmember 24. The manipulation head 18 may be driven using a similar belt drive (not show) that uses separate servos or motors to independently drive the manipulation head 18 laterally. The manipulation head 18 can be adapted for different functions depending on the type of tool 20 that is loaded thereon and actuated. In some embodiments the manipulation head 18 and the tool 20 to be used are positioned adjacent to a cartridge 30 to perform operations on fluids. Depending on the particular desired operation or function, the manipulation head 18 may incorporate various tools 20 such as magnets (electromagnets or permanent magnets), a camera or image sensor, optical sensor, pipettors, electrofusion head, syringe plunger holder, or contact member. The manipulation head 18 may hold single tools 20 or multiple such tools 20, e.g., any combination thereof, to facilitate operations on fluid samples. Some of the tools 20 may be permanently secured to the manipulation head 18 in some embodiments. In other embodiments, some or all of the tools 20 are removably secured to the manipulation head 18. The tools 20 may be secured to the manipulation head 18 using clips, fasteners, straps, custom receivers that are located on the manipulation head 18.

[0067] This manipulation head 18 enables comprehensive three-dimensional operations on fluids located within the one or more cartridges 30 disposed on the stage 14, offering benefits like enhanced freedom of control and improved z-axis functionality . Such features include the ability to vertically disengage from the cartridge 30 and re-engage at newly designated coordinates on the same cartridge 30 (or another cartridge 30) or to draw fluid samples into an upper level of a multi-level cartridge 30. The cartridge(s) 30 may include microfluidic cartridges having microfluidic features formed therein like channels, reservoirs, fluid holding regions. However, the cartridges 30 may contain larger volumes of fluid or lack microfluidic-sized features but still usable with the system 10.2025-094-2

[0068] These capabilities contrast with other microfluidic manipulation methods, such as electrowetting on dielectrics, and previously reported ferrobotic systems (see Emaminejad et al., Ferrobotic swarms enable accessible and adaptable automated viral testing. Nature 611, 570-77 (2022)), which, due to their reliance on a 2D planar array of electromagnets, are unable to achieve full 3D control. In one aspect, the manipulation head 18 is mounted on a z-axis manipulator such as the belt drive mechanism that moves the manipulation head 18 along the z-axis (e.g., up or down) while the cartridge 30 is held by a stage 14. The stage 14 may include a flat surface or the like (e g., 3D printer bed or floor) with areas or regions that accommodate the one or more cartridges 30. In one embodiment, the cartridge 30 is placed centrally on the stage 14. There is no need to secure the cartridge 30 to the stage 14 given the non- or low-contact nature of the interaction with the various tools20. Alternatively, there could be recesses, apertures, wells, or mounts in the stage 14 in which the cartridges 30 are loaded. In still another alternative, the stage 14 may be completely flat and the cartridges 30 secured to the stage 14 with fasteners, clips, or the like. The stage 14 may optionally incorporate one or more heaters 15 that are used to control the temperature of the conditions within the cartridges 30 (e.g., for assays). In some embodiments, a cooler 17 (e.g., Peltier cooler) may be integrated into the stage 14 to provide cooling functional ity or the ability to maintain the cartridge 30 and contents therein at low temperatures. Lateral movement in the x and y directions may be accomplished with the multidimensional positioning instrument 12 by moving the manipulation head 18 along the cross-member 24 that is able to impart movement in the x and y axis directions as seen in FIGS. 1 A and IB. This provides for motion in in three orthogonal axes while the stage 14 is stationary'. In another embodiment, the manipulation head 18 is mounted on a multidimensional positioning instrument 12 configured for linear motion in the z-direction and the stage 14 is a moveable stage for 2D motion (e.g., x and y motion provided by moving the stage 14 relative to the manipulation head 18 that moves in the z direction). It is also possible that combinations of the above are possible. For example, it is possible that the multidimensional positioning instrument 12 is able to move the manipulation head 18 in the x, y. and z directions while the stage 14 is also able to move in the x and y directions.

[0069] In one preferred embodiment, the tool 20 secured to the manipulation head 18 includes a plurality' of stacked magnets 32. The stacked magnets 32 includes permanent magnets mounted on the manipulation head 18, enabling high-precision movement and control of magnetic droplets 200 contained in the cartridge 30 over a range of volumes (0.5-2025-094-2100 pL) by directing them as needed for assays. An example of a permanent magnet that can be used for the stacked magnets 32 includes Nd2Fei4B ultra strong magnets with a diameter from 1-10 mm for directing ferrofluid droplets 200 of different volumes. Alternatively, the tool may include one or more electromagnets. An example includes electromagnets with a diameter of 8 mm and a height of 20 mm can be used to direct magnetic droplets 200 (including both microparticle-infused droplets and ferrofluid droplets). Larger particle diameters (~1 pm) are preferred for operation on droplets 200 with a weaker electro-magnet driven magnetic field.

[0070] Other magnetic tools 20 may be secured to the manipulation head 18 which are used to magnetically interact with the ferrofluid droplets 200 located on or in the cartridge 30. This may be a single magnet or even electromagnet. The motion of the of the manipulation head 18 is controllable such that the position, velocity, and acceleration of the manipulation head 18 (and tools contained thereon) can be tuned with high precision and accuracy. For example, when the tool 20 is a magnetic tool, the ferrofluid droplets 200 may be driven within the cartridge 30 in controlled directions and velocities as needed for the particular assay or application. The ferrofluid droplets 200 may be moved with a controlled velocity that, in some embodiments, may be a substantially uniform or constant velocity in the x, y planes of the cartridge 30. Alternatively, the ferrofluid droplets 200 may be moved with a controlled velocity that, in some embodiments, may be differ depending on the type or nature of the ferrofluid droplet 200 or the particular operations conducted in the cartridge 30. As noted below, this can be controlled using the scripts or instructions that control the relative motion of the manipulation head 18 and / or the moveable stage.

[0071] Additional tools 20 may also be mounted on the manipulation head 18. For example, a camera 34 or image sensor may optionally be mounted on the manipulation head 18. The camera 34 or image sensor may provide real-time visual feedback, assisting in the accurate control and monitoring of the system's operations and acting as an aid to the graphical user interface (GUI)'s feedback control system as described herein. The camera 34 or image sensor also enables real-time data collection as needed for the assay. In the working embodiment discussed herein, a camera holder (not shown) may be disposed on the manipulation head 18 that can accommodate diverse Raspberry Pi / USB cameras (such as Raspberry Pi Camera Module V2 and Arducam Hawkeye Ultra High-Resolution Autofocus Camera Module for Raspberry Pi). Note that these are just exemplary cameras 34 and other makes and models may be used. In some embodiments, the camera 34 is replaced by a2025-094-2single-pixel optical sensor (such as Sparkfun AS72651, AS72652, AS72653, and 7265x) for performing optical readouts on reactions in the cartridge 30.

[0072] Another example of a tool 20 that can be mounted on the manipulation head 18 includes an optional pipettor 36 that includes a pipette adapter 38 that holds a pipette tip 40. The pipettor 36 allows the system 2 to introduce samples and / or reagents into the cartridge 30 through inlet ports or reservoirs or remove fluids through outlet ports or reservoirs. This may include reaction products, product fractions, waste, and the like. In some embodiments, the manipulation head 18 with the stacked magnets 32 (or electromagnets) is used to remove magnetic fluids through an outlet port or reservoir in the cartridge 30. The pipettor 36 can be actuated through a connected actuator 42, such as a pressure source, pump, displacement mechanism, or syringe. The pipettor 36 is connected to the actuator 42 through tubing 44. The syringe or displacement mechanism may contain displacement oils / reagents for functioning as an electronically controlled pipettor 36 with volume control. It can perform regular liquid pipetting or inject fluids (e.g., magnetic fluids) into the cartridge 30, where they can be manipulated by stacked magnets 32 on the manipulation head 18. This reduces human errors associated with liquid handling tasks and improves accuracy, efficiency, and throughput.

[0073] The cartridges 30 enable intricate and precise fluid handling which, in some embodiments, may be on a microliter scale (0.5-100 pL). The different cartridge designs allow for the control of magnetic droplets 200 in a predefined way. enabling the execution of complex laboratory procedures with high precision and reproducibility. The magnetic droplets 200 may include a ferrofluid and / or larger magnetic beads. In a preferred embodiment, the ferrofluid is an aqueous biocompatible ferrofluid, like ferumoxytol (Feraheme), introduced at concentrations between (1-15% depending on the assay’s need). The magnetic droplets 200 can be used to perform a number of operations include splitting of fluid samples, merging of fluid samples (e.g., using electrofusion), mixing of fluid samples, aliquoting of fluid samples, and dilution of fluid samples. In other embodiments, the magnetic droplets 200 may include magnetic beads contained therein.

[0074] With reference to FIGS. 1 A and IB, the system 10 includes a display 46 that is mounted to or otherwise associated with the system 10. For example, the display 46 may be mounted to the frame 22. The display 46 includes a graphical user interface (GUI) 48 that facilitates intuitive control and programming of the system’s functions. The display 46 may include a touchscreen where the user can physically touch to interact with the GUI 48.2025-094-2Alternatively, the user may use a keyboard, mouse or the like to interface with the GUI 48 for a display 46 without touch functionality. The GUI 48 supports both the custom design of new workflows and the execution of existing biological assays. Communication between the GUI 48 and the system hardware occurs via a serial port, which sends G-Code that controls the manipulation of the multidimensional positing instrument 12 that provides the manipulation head 18 with 0.1 mm resolution, enabling diverse functionalities. The GUI 48 also controls the stage temperature via the one or more heaters 15, allowing optional programmable heating during assays.

[0075] An intuitive GUI 48 is essential for the user-friendly operation of the enhanced multidimensional positioning instrument 12, allowing users of varying technical backgrounds, including minimally trained individuals, to easily execute a wide range of laboratory assays. The key feature of this GUI 48 is the Editor Panel 50 (discussed below) with an interactive grid 58, which provides a graphical representation of the working surface of the stage 14 of the multidimensional positioning instrument 12, along with shape outlines of cartridges 30 and biological / biochemical assay procedures. The GUI 48 enables users to design new assay formats, program multi-step complex assays, and automatically execute diverse biological / biochemical assays (e.g., dilutions, ELISA, PCR, and LAMP) with minimal input. Additionally, users can save and edit existing assays.

[0076] The GUI 48 controls the multidimensional positioning instrument 12 by generating G-Code scripts or sending G-Code commands via serial or other communication to the CNC board the multidimensional positioning instrument 12. Through G-Code, the GUI 48 precisely manages the position, velocity, and acceleration of the manipulation head 18 or multiple manipulation heads 18 to meet the specific needs of each assay. The GUI 48 provides, in some embodiments. 0.01 mm resolution, demonstrating superior control precision and offering greater accuracy in sample manipulation within cartridges 30.Additionally, the GUI 48 may control the temperature of the multidimensional positioning instrument's stage 14, enabling precise heating and the creation of temperature gradients for certain biochemical assays. This precise control over all steps of biological / biochemical assays ensures accuracy and reproducibility from setup to execution.

[0077] Assay procedures and cartridges 30 are automatically aligned using the camera 34 or image sensor integrated into the manipulation head 18 to ensure precise positioning. The camera 34 captures high-resolution images of the cartridges 30, which contain spatial information about the cartridge geometry and alignment markers (e.g., colored circles on the2025-094-2comers). This enables the GUI 48 to determine the cartridge's relative location and rotation on the stage 14 of the multidimensional positioning instrument 12. It also superimposes assay protocols with the cartridge configuration by adjusting the offset of assay operation steps based on the alignment marker locations. Existing assays can be easily edited by modifying individual steps within the GUI 48 and saving them to the local PC (e.g., Raspberry' Pi or mini-Windows PC, or the like) or sending them to the CNC board via serial or other communication in G-Code format. Additionally, new assays can be designed by specifying the sequential steps of the entire assay protocol based on the cartridge outline 62.

[0078] In one embodiment, the GUI 48 includes four modular panels: (i) an Editor Panel 50 for manipulation-head navigation, (ii) a Camera Panel 52 for imaging and / or viewing the cartridge 30, (iii) a G-Code Panel 54 for command generation and visualization, and (iv) Syringe Setup Panel 56 for automated liquid handling. This structure provides coordinated control of droplet 200 movement, reagent dispensing, heating, assay procedure timing, and image acquisition. These four panels 50, 52, 54, 56 are navigated through the menu bar located at the top of the GUI 48. The GUI 48 may be implanted implemented in any number of computer programming languages. The working embodiment employed Python using the PySimpleGUI and Tkinter libraries.

[0079] G-Code is a common numerical control (NC) programming language used to control CNC (Computer Numerical Control) machines, 3D printers, and other automated manufacturing tools. It tells the machine how to move, where to move, and what operations to perform. The controls potion, sets position(s), controls speed, and manages unit operations. The Editor Panel 50 features a grid interface equipped with control buttons, allowing users to easily design and execute laboratory experiments or execute pre-programed scripts or instructions. The scripts or instructions may be carried out by software executed on a computing device 100 or microcontroller that is configured to control the relative motion of the multi-axis manipulator and / or the moveable stage. For example, this may be used to place one or more tools 20 over or in contact with selected regions of the one or more cartridges 30. This GUI 48 simplifies the programming process, making it accessible to users of varying technical expertise. The system 10 may also incorporate feedback control mechanisms that enable real-time monitoring and adjustments.

[0080] FIG. 5A illustrates the Editor panel 50, which facilitates communication between the user, the computing device 100 (e.g., local PC, Raspberry Pi or mini-Windows PC, Mac, Jetson Nano, etc.), and the multidimensional position instrument 12 by providing a visual2025-094-2representation of the stage 14 and allowing the user to specify the navigation path of the manipulation head 18 based on the design of the cartridge 30. Software running on the or executed by the computing device 100 operates the GUI 48 and also interfaces with the multidimensional position instrument 12 to control the operations of the multidimensional positioning instrument 12. Python was used in the working embodiment but it should be appreciated that other software languages may also be employed. The Editor Panel 50 contains a grid 58. which represents the physical layout of the stage 14 on which the one or more cartridges 30 are placed. The grid 58 can be shown or hidden by selecting or deselecting a Show Grid checkbox. The grid dimensions can be adjusted between 40x40 mm2and 200x200 mm2by zooming in or out using a left or right click on the grid 58 when the Toggle Zoom checkbox is selected. This feature allows the grid 50 to accommodate cartridges 30 of various dimensions and simplifies control over the manipulation head 18. The shape outlines of the cartridges 30 can be superimposed on the grid 58 using the Import Function 60, which uploads cartridge outline 62 in Drawing Interchange Format (DXF) (other formats may also be used). The user can specify the position of the cartridge outline 62 by clicking on the grid 58 and then lock the position by selecting the Lock DXFs checkbox. To enable high-throughput assaying and perform repetitive experiments, the program allows the user to import multiple cartridge designs. The positions of these cartridges 30 can also be adjusted by clicking and dragging the cartridge outline with the cursor. The interactive checkboxes menu 64, located beneath grid 58. provides control over various grid functions. This includes Show Grid (to show / hide the grid 58 on the virtual stage 14), Realtime Mode (to move the manipulation head 18 in real-time with each left click on the grid 58), Entire View (to zoom out to display the entire 200x200 mm2stage 14), Lock DXFs (to lock cartridge positions on the grid 58). Toggle Zoom (to enable zooming in or out with left or right clicks on the grid 58), and Toggle Capture (to enable adding capturing points for imaging with the camera or image sensor 34).

[0081] To create the path for the manipulation head 18 movement, the Realtime Mode, Toggle Zoom, and Toggle Capture checkboxes must be deselected, while the Lock DXFs checkbox must be selected. The path is created by left-clicking on the grid 58 or manual input via a manual point addition panel 66 to add individual waypoints 68; subsequently, added waypoints 68 are automatically linked with each other via links 70, creating a single movement path. Since the manipulation head 18 has several functional tools 20 (e.g.. stacked magnets 32, camera 34, and pipettor 36), the user can select the tool 20 to be used at each2025-094-2point. This selection is made by specifying a radio button (e.g., stacked magnets 32, camera 34, and pipettor 36) at the bottom of the GUI 48. Different offsets will automatically be applied to the path points based on the selected embodiment. Right-clicking on the grid 58 adds a skip point to the path; the manipulation head 18 will be raised before moving to the target location. Since the distance between the manipulation head 18 and cartridge 30 increases, the stacked magnets 32 will move to the target location without affecting the position of the magnetic droplets 200 within the cartridge 30. These operations enable high throughput in the system 10 by locating or transferring individual magnetic droplets 200 within the cartridge 30 without attracting other droplets 200. Additionally, multiple cartridges 30 can be operated within the same protocol, or several biological samples can be processed in parallel within the same cartridge 30. The user can also add time delays between subsequent movements by clicking the Add Delay button 72 and specifying a delay time in milliseconds for incubation or image capture purposes.

[0082] Instead of directly left or right-clicking on the grid 58, a new waypoint 68 can be added to the path manually by specifying the X. Y, and Z coordinates (Z Move or Z Skip) of the new waypoint 68, along with the manipulation head 18 movement speed, and clicking the Add Move button 74 or Add Skip button 76 to add a regular or skip point, respectively. Compared to clicking on the grid 58, the manual addition of waypoints 68 allows the user to specify coordinates with 0.01 mm resolution, providing more precise sample transfer and manipulation within the cartridge 30. This manual point addition panel 66 is located beneath the checkbox menu 64.

[0083] Once the path is created, it can be directly sent to the multidimensional position instrument in G-Code format using the Send via Serial button 78 or saved as a local G-Code file using the Save As button 80. The Send via Serial and Save as buttons 78. 80 are located in the menu 82 located above the grid 58. In addition to these, the menu 82 also contains the following buttons: New (to create a new path and discard current settings), Open (to open a saved path in G-Code format), Autohome (to home the axes of the 3D printer), and Import DXF button 60 (to import the cartridge outline 62 in DXF format).

[0084] Existing assays stored in G-Code format can be loaded into the GUI 48 by clicking the Open button within menu 24 and executed using the Run Assay button. To run an existing assay, the corresponding cartridge outline 62 of the particular cartridge 30 must be preloaded and superimposed on the grid 58. Information about the assay format, including the lot number of the cartridge 30, is stored within the G-Code assay file. The assay will only be2025-094-2performed if the cartridge type matches the assay format; otherwise, the GUI 48 will display an error in the Debug Terminal 90, prompting the user to load the correct assay protocol or cartridge 30. Prior to execution, the assay operation steps are precisely aligned with the cartridge configuration by adjusting the offset value based on the cartridge alignment marker locations on the stage 14. The alignment marker locations are determined from an image of the cartridge 30 captured by the camera 34, along with image analysis results of the camera's relative position within grid 58.

[0085] When the Toggle Capture checkbox is selected, left-clicking on the grid 58 adds capture points specifically for the camera 34 to capture. These points will be processed after the user clicks the Take Image button 86 within the Camera panel 52, as shown in FIG. 5B. This functionality allows the user to re-read assay results once the assay and automatic imaging are completed, especially in cases where the initial assay readout does not meet the user's needs, such as when the capture points are incorrect.

[0086] The Code Table 88 displays the current manipulation head path. Each entry in the table includes the movement t pe (Move, Skip, or Pause), the X, Y, and Z coordinates (in mm) of the corresponding point within the path (indicating where the manipulation head has moved to at this step), the pause time (in ms), and the movement speed (in mm / min). The Code Table 88 also allows users to select and manually modify a point (e.g., adjust its coordinates, movement speed, or pause time) as well as delete waypoints 68. The Debug Terminal 90 displays the movements of the manipulation head 18 based on user inputs. It also logs each action the user has performed on the grid 58 and reports any errors or warnings during system operations.

[0087] FIG. 5B shows the Camera Panel 52, where the user can specify and adjust key camera parameters such as exposure time (in seconds), camera-to-sample distance (Z height, in mm), and resolution by entering the values into the text boxes and clicking the Change Exposure 92, Change Z Height 94, and Change Resolution 96 buttons, respectively. The user can also specify the number of images to capture per location and the interval between subsequent captures (in seconds) to enable both endpoint (N=l) and time-lapse (N>1) image capture and analysis. These values can be entered in the input fields 98 located beneath the Change Resolution 96 button. The Auto Focus button 102 allows the user to obtain clear, focused images. The Take Image button 86 initiates image capture based on the user-specified parameters and the capture points defined on the grid 58 in the Editor Panel 50, as shown in FIG. 5A when Toggle Capture mode is selected. Captured images are displayed on2025-094-2a window 106 of the Camera panel 52, and the user can navigate between images using the Previous and Next buttons 108 located beneath the displayed images on the camera panel 52. All captured images are saved in a user-defined folder.

[0088] The Change Z Height button 94 in the Camera Panel 52 and the Z move value in the manual point addition panel of the Editor Panel 50 represent the same value; updating one will automatically update the other. FIG. 5C illustrates the G-Code Panel 54, which displays G-Code commands generated based on user inputs from the Editor Panel 50. The subsequent G-Code functions and their corresponding comments are displayed line-by-line within the preview window 110.

[0089] FIG. 5D illustrates the Syringe Setup Panel 56, which enables automated control of liquid handling, including the introduction of samples and buffers into the cartridges 30. The user can specify syringe pump parameters, such as the syringe type (e.g., 0.5 mL, 1 mL, 3 mL syringe) 1, operation volume (in pL), and flow rate (in pL / min, up to 1000 pL / min) 2. If the flow rate exceeds 400 pL / min, the program automatically selects the 1 mL syringe model. The user can also switch the syringe operation between push and pull modes via button / toggle 112, corresponding to fluid injection or withdrawal. The specified syringe parameters can be sent directly to the syringe pump in G-Code format by clicking the Submit via Serial button, or they can be saved as a local G-Code file using the Save as button.Syringe parameters are display ed in the preview window 114 as G-Code functions, along with comments. The syringe operation mode can be updated by clicking the Clear button and entering new parameters. The Submit via Serial, Save as, and Clear buttons are located in menu 116 beneath the syringe selection pulldown option 118.

[0090] A primary purpose of this system 10 is to automate laboratory experiments, thereby reducing the manual effort and potential for error in repetitive tasks required for biological or biochemical assays. Additionally, the system 10 operates on smaller fluid volumes compared to traditional automated liquid handling systems that use robotic pipettors, and it supports a broader range of operations, as further disclosed. These operations leverage the capabilities of the cartridge 30 and other functions of the multi-dimensional positioning instrument 12, such as heating, optical excitation, imaging, etc.

[0091] This invention introduces the concept and implementation of using an advanced and adaptable positioning instrument for general laboratory7automation, equipped with a multifunctional manipulation head 18 that consists of diverse tools 20, such as stacked magnets 32, a camera 34, an electrofusion head 120, and a mechanical head 122. as shown in2025-094-2FIGS. 1A, IB, 2A-2D. Each embodiment, or combinations of different embodiments, can be tailored to specific assays and other diverse biological and biochemical applications. This versatile manipulation head 18, integrated into various multidimensional tools 20, significantly enhances the precision and efficiency of automatic laboratory processes.

[0092] It should be appreciated that the multidimensional positioning instruments 12 may be based on or share components, drive trains, and like with commercial 3D printers (e.g., Creality, Prusa, and Easy Thread), a CNC drill. Of course, the multidimensional positioning instruments 12 do not need to be made from components of existing commercial instruments as the multidimensional positioning instruments 12 can be bespoke or custom made suitable for integration with the cartridges 30. These images highlight the adaptability of the multifunctional manipulation head 18 to several positioning instrument types. The concept of creating the manipulation head 18 for lab automation can apply to any multidimensional positioning instrument 12that uses G-Code or stepper motor pulse signals. It should be appreciated that while aspects of 3D printers were modified to create working embodiments of the system 10. the system 10 does not require modification of off-the-shelf equipment. Rather, it shows that existing drive mechanisms and supply chains that provide these components can be utilized to manufacture the multidimensional positioning instruments 12 described herein.

[0093] FIGS. 2B-2C illustrate the operation of droplet 200 merging using a contactless, high-voltage electrofusion head 120 (FIG. 2 A) or a pair of low-voltage electrode contacts 124 (FIG. 2B). The contactless electrofusion head 120 integrates a high-voltage generator 126 (100-2000 V) with stacked magnets 32 that are connected via a wire. Once the positioning instrument positions the stacked magnets 32 above the cartridge 30 enclosing the droplets 200, a high-voltage electrical shock is generated and transduced through the magnets 32. This shock traverses the air (within 2 mm) and the membrane / acrylic plate of the cartridge 30 (within 5 mm) to merge the droplets 200, in under one second. The contactless method eliminates the need for direct contact with the fluid sample and avoids complex on-chip electrode microfabrication steps, thus reducing costs and facilitating flexible assay design. This process enables contamination-free droplet merging in any channel or chamber within the cartridge 30 without incorporating electrodes into the cartridges 30.

[0094] In other embodiments, a tool 20 in the form of a pair of low-voltage electrode contacts 124 on the electrofusion head 120 utilizes direct contact to transduce electrical shocks and provides a method to merge droplets 200 in applications where high-voltage2025-094-2shocks may be disruptive. This feature is preferred for delicate electrical manipulations involving droplets 200 containing sensitive components, such as living cells and reactive biochemicals. Designed to deliver precise electrical shocks ranging from 0.1-2V, the low-voltage electrofusion method minimizes interference with biological processes. The cartridge 30 includes corresponding on-chip electrode contacts 128 that are connected to an electrically conducting path that leads to electrodes disposed within the cartridge 30 and designed to contact the droplets 200 directly. Once the manipulation head 18 moves the pair of low-voltage electrode contacts 124 makes contact with the on-chip electrode contacts 128, a low voltage is applied to merge the droplets 200 effectively.

[0095] FIG. 2C depicts a micro or mini camera 34, such as an Arducam autofocus camera module or a USB camera that can be mounted onto the manipulation head 18 using a custom holder 130, as shown on the left of FIG. 2C. Also, this custom holder 130 can be designed to accommodate various lenses, bandpass fdters, light diffusers, light sources, and other optical components, enabling a range of imaging modalities such as colorimetry, fluorescence, and chemiluminescence. Alternatively, the holder 130 can be used to mount an optical density sensor 132 and its light source, as illustrated on the right of FIG. 2C. High-illumination LEDs are mounted onto the sensor, providing bright and uniform illumination around the sensor's pinhole, which enables highly accurate optical density measurements across different channels. The SparkFun 6-channel optical sensor (AS7262 Visible Spectral Sensor, SparkFun Electronics, CO, USA), which contains six color channels (450 nm Violet, 500 nm Blue. 550 nm Green, 570 nm Yellow', 600 nm Orange, and 650 nm Red), serves as an example. Other light sensors with bandpass fdters in the ultraviolet and infrared ranges can also be used with the custom holder 130 on the manipulation head 18. The data obtained from the camera 34 or sensors (like optical density sensor 132) can be fed into the GUI 48 for data analysis and feedback control or to return a result for an assay to the user through the GUI 48 or encoded on a computer-readable medium. These concepts provide a versatile, high-precision imaging system with advanced positioning capabilities, unlocking new' possibilities for automating bioimaging and biophotonic applications.

[0096] FIG. 2D shows the manipulation head 18 including a contact member 138, which applies precise pressure, causing 0.1-0.4 mm deformation on the top layer / membrane 136 of cartridges 30 to transport droplets 200. Note that these droplets 200 do not have be magnetic given that pressure is used to transport these droplets 200. In scenarios w here the top of the cartridges 30 are sealed by the top layer / membrane 136, the mechanical head can apply2025-094-2localized pressure to manipulate the fluid within the cartridge 30. The top layer / membrane 136, which may be a 50-100 pm PET transparent sheet, reshapes into a sloped ceiling under pressure to guide transportation of droplets 200 within the cartridge 30 (illustrated in the direction of arrow A in FIG. 2D). This action forces the droplet 200 to move in the opposite direction when one side is compressed. Utilizing the mechanical head 30 and top layer / membrane 136 facilitates the transportation of aqueous droplets 200 without magnetic micro / nano-particles in the cartridge 30, enhancing optical transparency for a broad spectrum of biophotonic applications and assays.

[0097] The cartridges 30 described herein can be fabricated using various methods suitable for both prototyping and mass manufacturing. These methods can be broadly categorized into two main types: low-cost, fast prototyping methods (e.g., laser cutting and 3D printing) and low-cost mass-fabrication methods (e.g., plastic injection molding, hot-embossing). The materials for the cartridges 30 are preferably thermoplastics, which are both low-cost and functionalizable, although other materials such as glass, PDMS, elastomers, or other polymers that are preferably transparent in visible wavelengths may also be used.

[0098] For laser cutting methods used to create cartridges, the microfluidic network of channels, reservoirs, chambers, and other functional components are designed using 2D vector graphics software, such as Autodesk AutoCAD. The design is then transferred to an acrylic sheet, which is sandwiched with ultra-strong double-sided tape and together, are cut with a laser cutter. Transparent membranes made with inlets and outlets are subsequently used to sandwich the acrylic sheet containing the microfluidic network and other features formed therein. This fabrication method offers several advantages: the acry lic sheet, an industry' -standard material for laser cutting, provides excellent transparency, stiffness, and rapid prototyping speed. The sub-millimeter precision of laser cutting delivers ideal resolution for microfluidic-based cartridges 30. Additionally, the ultra-strong double-sided tape ensures robust adhesion between the different layers of the microfluidic cartridges, preventing leaks of oils and other aqueous phases. The PET transparent membranes are very- thin (50-100 pm), flexible, and offer optimal optical properties for visual inspection and quantitative measurement.

[0099] To provide a non-stick and contamination-free surface environment within the cartridges 30, a surface treatment using NeverWet superhydrophobic spray or trichlorosilane is applied to the inner walls of the acry lic sheet and the inner surfaces of the PET membranes (FIG. 3B). This superhydrophobic treatment imparts three key advantages to the microfluidic2025-094-2cartridges 30: first, preventing aqueous phase droplets 200 from adhering to the inner surfaces of the microfluidic network, eliminating the need for surfactants; second, facilitating easy droplet 200 merging, as the absence of surfactants allows in some cases for droplets 200 to be merged upon contact, without the need for electrocoalescence; and finally, minimizing cross-contamination in sensitive molecular experiments because the contents of the aqueous droplets do not leak or adhere to the superhydrophobic surface.

[0100] For the 3D printing methods used to create the cartridges 30, two pieces of a microfluidic cartridge are designed using 3D modeling software such as Autodesk Fusion 360 or SolidWorks and then printed using transparent SLA Fast 405 nm UV-Curing resin (e.g., Supper PP and ANYCUBIC Upgraded Standard 3D Printer Resin) on an LCD or DLP 3D printer. One piece contains the inlets and outlets, while the other piece includes a microfluidic network of channels, reservoirs, chambers, and other functional components. The two pieces can be bonded together using a clamp (or simply by superimposing a transparent weight, such as an acry lic square block) under 405 nm UV light treatment. This rapid prototy ping method has two key advantages: it is ideal for fabricating microfluidic cartridges 30 with micrometerscale features (such as <20 pm microfluidic channels) since current LCD 3D printers offer very high resolution. For example, the ELEGOO Mars Ultra printer can achieve a resolution of 18 pm in the X, Y, and Z axes. Second, it can create 3D microfluidic cartridges 30 or multi-layer microfluidic cartridges 30, which are difficult to achieve with laser-cutting methods. For instance, some 3D hook or weir structures, where a channel locally decreases in height, can be used to trap magnetic microparticles. A superhydrophobic treatment can be applied to the inner surface of the 3D-printed microfluidic cartridge 30 by injecting 10% trichlorosilane in Novec 7500 oil into the inlet of the microfluidic cartridge 30 and then washing it out with the Novec 7500 oil. Other methods for manufacturing the cartridges 30 include photolithography / PDMS casting and plastic injection molding. These are conventional standard methods for precision microfabrication or mass production that are known to those skilled in the art.

[0101] FIG. 3A illustrates the layout of the multiple layers of a laser-cut acrylic microfluidic cartridge 30, its fabrication method, and a photograph of the completed microfluidic cartridge 30. The fabrication process can be divided into the following steps: 1) sandwich the acrylic sheet with ultra-strong double-sided tape. 2) Perform laser cutting on the assembled acrylic sheet to form the microfluidic network and on the transparent membranes to create the inlets and outlets. 3) Clean the dust from the transparent membranes using2025-094-2ethanol or deionized water, and use a Kimwipe to remove any residue. 4) Remove the cover from the ultra-strong double-sided tape and bond the acrylic sheet with the transparent membranes. Press the surface to remove any bubbles, ensuring strong, leak-proof bonding between the different layers of the microfluidic cartridge.

[0102] FIG. 3B illustrates the layout of the multiple layers of a laser-cut superhydrophobic acrylic microfluidic cartridge 30, its fabrication method, and a photograph of the completed superhydrophobic microfluidic cartridge. The fabrication process can be divided into the following steps: Steps 1 to 3 are the same as the fabrication method mentioned in FIG. 3A. Step 4): remove the bottom side cover from the ultra-strong double-sided tape and bond the acry lic sheet with the transparent membranes. Step 5): use clear tape on the inner face of the membrane to cover the edges of the top transparent membranes containing the inlets and outlets. Step 6): apply the NeverWet superhydrophobic spray to the inner side of the top layer / transparent membrane 136 and the acrylic sheet (with the microfluidic network and bottom transparent membrane). Step 7): wait for about one hour until the superhydrophobic polymer fully grafts onto the surface and dries. Step 8): remove the top side cover from the ultra-strong double-sided tape and peel off the clear tape from the top transparent membranes containing the inlets and outlets. Step 9): bond the coated top layer / transparent membrane 136 with the coated acry lic sheet containing the microfluidic network. Press the surface to remove any bubbles, ensuring a strong, leak-proof bond between the different layers of the microfluidic cartridge.

[0103] FIG. 3C illustrates the layout of the two pieces of an alternative microfluidic cartridge 30: a 3D-printed microfluidic cartridge 30, its fabrication method, and a photograph of the completed 3D-printed microfluidic cartridge 30. The fabrication process can be divided into the following steps: 1) Print the two pieces using an additive 3D printer (e.g., LCD 3D printer); 2) Use a blade to cut the printed pieces from the print bed of the 3D printer, wash them with ethanol until no shiny, uncured photoresin remains, and then dry7them using an air blower; 3) Align the two pieces, and then place them into a compressor clamp for bonding; 4) Treat the 3D-printed microfluidic cartridge with 405 nm UV light for 15 minutes to ensure the photoresin at the interface of the two pieces is fully polymerized, providing a strong, leakproof bond between the pieces of the 3D-printed microfluidic cartridge 30. Other applicationspecific cartridges 30 may employ similar manufacturing methods, for example, a blood filtration microfluidic cartridge 30 (see e.g., FIG. 8C). The specialized fabrication process uses the same steps listed above (1-4) of the aforementioned process. However, the cartridge2025-094-2design includes a gap for a piece of blood cell filter membrane 140 (e.g., paper or the nitrocellulose membrane), a channel running 142 through the entire height of the cartridge (with the opening designed to fit snug to a pipettor tip 40), and micro-sized beams 144 to support the filter membrane 140. The filter membrane 140 is precisely placed in its designed reservoir during step 3, before the curing process.

[0104] FIG. 4A illustrates one embodiment on a liquid handling module 146 that enables precise liquid manipulation (e.g., injection, withdrawal, transfer, and mixing). It consists of a syringe holder 148 fixed to the stage 14 and a plunger holder 150 mounted on the manipulation head 18. Syringes 152 of various volumes (e.g., 0.5, 1, 3 rnL) can be securely fitted into the syringe holder 148. The plunger holder 150 has a complementary indent that mechanically engages with the plunger 154 of the syringe 152. By controlling the movement of the Z-axis of the manipulation head 18, both the volume (in pL) and the flow rate (in pL / min) can be adjusted. Specifically, changing the Z-axis height of the manipulation head 18 defines the distance the syringe plunger 154 moves, thereby altering the volume of liquid injected or withdrawn (1-3000 pL). Additionally, adjusting the Z-axis feed rate controls the movement speed of the syringe plunger 154, determining the flow rate (400-1000 pL / min).

[0105] In another embodiment of a liquid handling module 146, tubing connects a syringe needle (or outlet port of syringe 152) to a pipette tip adapter 38 located on the manipulation head 18, as illustrated in FIG. 4B. The pipette tip 40 can be attached to the adapter 38 by precisely positioning the manipulation head 18 and inserting the adapter 38 into the pipette tip 40, securing it to the manipulation head 18. Once attached, the enhanced multidimensional positioning instrument 12 functions as a mini liquid handling robot capable of performing standardized laboratory' tasks (e.g., introduction of samples or reagents into the microfluidic cartridge inlets, withdrawal of fluids from the microfluidic cartridge outlets, reagent mixing, cell culture media exchange, and ELISA washing). The pipette tip adapter 38 can also be replaced with a hard tube or glass capillary dimensioned to apply capillary pressure, which can be inserted into a cartridge 30 to function as a capillary liquid pump, offering additional capacity for microfluidic bioassays.

[0106] In this embodiment, a lead screw 157 is mechanically secured to the plunger 154 of a syringe 152. Rotation of the lead screw in response to actuation of a motor or servo advances or retracts the lead screw and thus the plunger 154. The flow rate range will depend on the lead value (8 mm) of the lead screw in the liquid handling module 146, such as a minimum of 400 pL / min for the C reality Ender 3D printer. To significantly lower the2025-094-2minimum achievable flow rate in our enhanced multidimensional positioning instrument, the liquid handling module 146 uses a 3D-printed miniaturized syringe pump 156 as illustrated in FIG. 4B. This design repurposes the filament extruder of the 3D printer by connecting its motor to an ultra-fine lead screw 157 (lead value 0.3 mm) using a coupler and employs two linear shafts held by ball bearings to maintain the stability of the plunger pushing plate 155 which contacts the plunger 154 of the syringe 152, achieving a flow rate range of 0.1-1000 pL / min. These extremely low flow rates enable the formation of nanoliter droplets 200 or the microfabrication of particles, such as single-cell / molecule encapsulated droplets (e.g., Nanovials, and PicoShell particles). In some embodiments, the liquid handling module 146 can accommodate a plurality of syringes 152 increasing assay throughput.

[0107] For example, the design can be expanded to a multi-channel configuration, such as a four-channel liquid handling module 146. By leveraging advances in the CNC industry, a CNC board capable of interpreting G-Code can control up to eight individual axes, allowing four of them to move the syringe plungers 154, while additional control axes are used for one or more manipulation heads 18. This setup enables the manipulation of multiple fluids with extreme precision, making it ideal for complex workflows such as multiplexed assays or parallel reactions. Enhanced control over the pump's position, velocity, and acceleration ensures smooth and consistent flow, preventing turbulence that could disrupt sensitive biological assays. By offering both large-volume / flow rate capacity and small-volume / flow rate precision, this system 10 provides unmatched versatility in handling a wide range of tasks, ensuring reproducibility and accuracy across diverse experimental protocols.

[0108] FIG. 6A illustrates several basic magnetic droplet manipulations carried out within a cartridge 30 in one embodiment of an automated bioassay system 10 driven by a multidimensional positioning instrument 12. A laser-cut, laminated acrylic microfluidic cartridge 30 is demonstrated here, featuring various functional elements such as inlets 158 for loading reagents, an outlet 1 0 for waste removal or sample collection, a hook-like structure or corrugation 162 (also referred to as a dispenser) for dispensing small droplets 200, a buffer reservoir 164 for storing 50-100 pL of reagents, and a nozzle 166a, 166b for generating larger volume droplets (10-30 pL). It also includes multiple reaction chambers 168 containing reaction mixtures generated from buffer droplet reservoirs. Reaction mixtures may initially be in a dried or lyophilized form or in a solution form. Ferrofluid droplets serve as "sample cargo," immersed in a surrounding oil phase (e.g., dodecane with or without surfactant like 1-3% Span 80, Novec 7500 oil with or without surfactant, such as 0.1-2% Picosurf), while2025-094-2stacked magnets 32 positioned by the manipulation head 18 provide the magnetic drag force, functioning as "cargo carriers." The cargo earners are controlled by an enhanced multidimensional positioning instrument 12 with a GUI 48, using the sample cargo, which contains different biological molecules and reagents, to perform bioassays automatically. For example, the basic movement of the stacked magnets 32 by the manipulation head 18 can be used to apply a magnetic drag force to transport ferrofluid droplets 200 (e.g., 2.5-5% ferrofluid) and mix their contents after droplet merging. This can be achieved by moving the stacked magnets 32 in a circular motion (e.g., with a radius of 3.5 mm and a speed of 1 cm / s) positioned 0.2 mm above the top layer or membrane 136 over the ferrofluid droplets 200, inducing a circular motion and stirring of the ferrofluid droplets 200. The magnetic drag force applied to the ferrofluid droplets 200 is used to perform various operations on the microfluidic cartridge 30. For instance, dragging a droplet 200 over the hook-like structure or corrugation 162 splits the droplet 200 and dispenses it into smaller droplets 200 (0.5-9 pL), and pulling a ferrofluid parent droplet 200 from the nozzle 166b and breaking it off as its neck generates large-volume buffer droplets 200 (10-30 pL). Additionally, a high-voltage electrical shock can temporarily break the protective shell if a surfactant stabilizes droplets, facilitating droplet merging. Alternatively, droplet merging can occur upon contact without surfactant using a superhydrophobic-coated microfluidic cartridge 30, as described herein.

[0109] FIGS. 6B-6D illustrates the performance, predictability, and accuracy of droplet generation in microfluidic cartridges 30 using various ferrofluid concentrations within sample droplets 200 surrounded by an oil phase. FIGS. 6B-6D shows two data graphs (FIGS. 6B and 6D) and photographs (FIG. 6C) demonstrate the consistency and predictability of droplet dispensing by driving a parent droplet 200 across a hook-like structure or corrugation 162. The volume of the dispensed daughter droplet 200 depends on the controlled dimensions of the hook-like structure 162 when the ferrofluid concentrations remain constant. By varying the speed of the dispensing process, the designs can counterbalance the increasing magnetic drag forces as ferrofluid concentrations increase. As shown in FIG. 6B, the dispensed droplet volumes remain at 1 pL (Coefficient of Variation (CV) = 3.88%) even as the concentrations of ferrofluid increase from 2.5% to 15%, demonstrating excellent consistency. The graph of FIG. 6D demonstrates the predictability of the designs, as one can predefine the dimensions of the hook-like structure 162 to control dispensing volume. Both the 2.5% and 5% ferrofluid experimental data closely match the theoretical values, ranging from 0.5 pL to 5.0 pL. These data also show the excellent functionality of low-concentration ferrofluid-infused droplets2025-094-2200, which offer greater optical transparency for colorimetric and fluorescent assays. The stacked magnets 32 used here lead to a stronger magnetic body force and the ability to actuate droplets with reduced ferrofluid concentrations of 2.5-5%.

[0110] For larger volumes, ranging from 10 to 30 pL, it is preferred to use larger stacked magnets 32 (outer diameter > 8 mm) for dispensing droplets using a hook-like structure or corrugation 162. However, using a large column of stacked magnets 32 decreases operational resolution and precision, which can lead to the unintentional attraction of multiple magnetic droplets 200 during operations. In a preferred embodiment, droplet dispensing in the 10 to 30 pL range is achieved using small-sized stacked magnets 32 (outer diameter < 6 mm) while maintaining precise droplet manipulation using a nozzle 166a, 166b, as illustrated in FIG. 6A. The shape and dimensions of the nozzle 166a. 166b can be adjusted according to different ferrofluid concentrations. For instance, a narrow straight nozzle 166a is ideal for generating 5% ferrofluid buffer droplets 200, as its small size reduces the flow rate, counteracting the stronger magnetic drag force and improving control and stability during buffer generation. Conversely, a larger triangular nozzle 166b is designed for generating 2.5% ferrofluid buffer droplets, as it reduces flow resistance and deformation of the parent droplet 200 required for dispensing and facilitates buffer droplet formation under lower magnetic drag forces. The buffer droplet generation process from these nozzles 166a, 166b involves localizing the stacked magnets 32 on the manipulation head 18 in the vicinity of the droplet 200 to attract the liquid flowing out from the nozzle 166a. 166b, pausing for a specific time period to allow the dispensed droplets 200 to reach the desired volume, and then rapidly moving the manipulation head 18 (1-2 cm / sec), pulling the droplet 200 to break the neck and moving it to designated positions, completing the buffer droplet generation.

[0111] To achieve programmable buffer droplet generation from such nozzles 166a, 166b using 2.5% and 5.0% ferrofluids, pause time characterization was performed, as shown FIG.6E. As the pause time increases, the volume of the generated buffer droplets 200 also increases until it approaches saturation. Specifically, a linear correlation is observed between the pause time and buffer droplet volume (R2> 0.99), indicating high predictability (FIG. 6F). However, as seen in FIG. 6F, once the pause time exceeds 3 seconds, the droplet size surpasses the diameter of the stacked magnets 32, reducing the attraction effect and resulting in a non-linear correlation between pause time and buffer droplet volume.

[0112] FIGS. 7A-7B illustrate three different methods for droplet transportation within a cartridge 30: (1) magnetic microparticle (bead)-infused droplet transportation driven by2025-094-2stacked magnets 32; (2) ferrofluid-infused droplet transportation driven by stacked magnets 32, and (3) pressure-based droplet transportation driven by a mechanical contact member 138 on the manipulation head 18 that interacts with the top layer / flexible membrane 136.

[0113] The first method, magnetic microparticle (bead)-infused droplet transportation driven by stacked magnets 32, involves magnetic microparticles / beads suspended within the droplet 200, forming tiny beads cluster when attracted by stacked magnets 32. One exemplary magnetic bead type is the Beckman Coulter AMPure XP, Lot # A63880, with a diameter of 1.0 pm ± 8%. These beads consist of three layers: a polystyrene core, a magnetite middle layer, and a carboxylate-modified polymer coating. The movement of the bead cluster drives the rest of the droplet 200 through the internal tension betw een w ater molecules. Since the cluster contains a high concentration of magnet-responsive particles, it can be separated from the droplet if pulled too rapidly. Thus, the droplet transportation velocity is relatively slow (< 30 mm / s). However, a notable advantage of this method is the ability' to separate the magnetic microparticles / beads from the droplet follow ing manipulation, enabling optical imaging of a pure sample droplet without optical absorption by magnetic microparticles, which is preferred for bioassays requiring optical readout with low signal above background levels.

[0114] The second method, ferrofluid-infused droplet transportation driven by stacked magnets 32, uses colloidal suspensions of magnetic nanoparticles (diameters < 100 nm), such as ferumoxytol (AMAG Pharmaceuticals, MA, USA). The nanoparticles remain evenly distributed within the droplet 200 and do not easily form clusters even under a strong magnetic field, enabling the strongest transportation capabilities among the different methods. Specifically, a transportation velocity of over 60 mm / s is easily achievable with minimal dosages of ferrofluids (e.g.. 2.5% and 5%). This characteristic also provides a solid foundation for various functionalities, such as droplet dispensing, mixing, and buffer droplet generation.

[0115] The third method is a pressure-based droplet transportation method, where fluid sample droplets 200 (with or without magnetic nanoparticles or microparticles) are transported by applying pressure to the top surface of the cartridge 30 using a contact member 138. This pressure is applied to atop layer / flexible membrane 136, chosen for its elasticity, allowing it to deform (~0.1-0.4 mm) and create a slight slope on the ceiling of the inner chamber of the microfluidic cartridge 30. This deformation effectively forces the droplet 200 to move along the fluidic channels without a magnetic drag force. This technique2025-094-2is particularly beneficial for assays that require completely uncontaminated samples, as ferrofluid or magnetic beads are not necessary for droplet transportation.

[0116] The characterization of droplet transportation velocities under various oil and particle conditions is presented in FIG. 7C. The data compares the performance of droplets 200 with different compositions: 5% ferrofluid droplets 200 moving in Novec oil (labeled 5% Novec), 5% ferrofluid droplets 200 in dodecane oil (labeled 5% DDC), 2.5% ferrofluid droplets 200 in Novec oil (labeled 2.5% Novec), 2.5% ferrofluid droplets 200 in dodecane oil (labeled 2.5% DDC), and 0.4% magnetic bead-infused droplets 200 moving in Novec oil (FIG. 17A), across a volume range of 0.5 pL to 10 pL. The graph shows that droplets 200 with 5% DDC achieve the highest speeds, consistently exceeding 120 mm / s across various volumes. Droplets 200 with 5% ferrofluid in Novec also maintain high speeds. In contrast, droplets 200 with 2.5% DDC and 2.5% ferrofluid in Novec exhibit moderate speeds, respectively. Droplets 200 containing magnetic microparticles / beads demonstrate the lowest speeds (FIG. 17 A).

[0117] FIG. 8 A illustrates the general implementation of automated serial dilution in a cartridge 30 (e.g., microfluidic cartridge 30). This process involves three general steps: 1) dispensing a droplet volume characteristic of the dilution factor (e g., for a 10X serial dilution, dispensing a 1 pL droplet from a 10 pL parent droplet), 2) merging this small droplet with a subsequent buffer droplet (e.g., for a 10X serial dilution, merging a 1 pL dispensed droplet with a 9 pL buffer droplet), and 3) thoroughly mixing the new droplet 200 (e.g., moving stacked magnets 32 in a circular motion with a radius of 3.5 mm and a speed of 1 cm / s for 30cylces). In this example, varying concentrations of the sample and serial droplets are indicated by color / saturation (black to lighter). A stack of magnets 32 on the manipulation head 18 is used to drive the droplet transportation, the droplet sample is moved to the hook-like structure or corrugation 162 for dispensing a small volume, and the excess is returned to its original reservoir. The dispensed droplet is then moved to the adjacent reservoir, occupied by a buffer solution of a specific volume characteristic to the dilution factor. Using an electrofusion head 120 or low-voltage electrode contacts 124, these two droplets 200 are merged and, with the circular movement of stacked magnets 32, thoroughly mixed. This process can continue indefinitely until the desired number of dilutions is completed. The dilution factors (usually 2-20X) and the number of times dilution can be performed are limited only by the precision of the manufacturing methods, which determine2025-094-2the minimal operable droplet volume and the size of the working plate of the multidimensional positioning instrument 12.

[0118] FIG. 8B illustrates the general implementation of biomolecule capture using specialized magnetic beads in a microfluidic cartridge 30, showing protein capture as an example. After the sample (S) is added to the primary reservoir, the liquid handler deposits magnetic beads (MB) coated with capture molecules into the sample. These capture molecules can be capture antibodies or fragments thereof (e.g., immunoglobulin G (IgG), nanobodies, scFvs, etc ), aptamers, oligonucleotides, biotin / streptavidin, other affinity reagents, or biomolecules with specific functional groups (e.g., -NH2, -COOH, -Phenyl, -azide, alkynes) that bond to target proteins / peptides through bioconjugation (e.g., covalent bonding or noncovalent interactions). This mixture then incubates under optimal binding conditions for the capture molecules to bind to the analyte of interest (AOI) in the sample. After incubation, stacked magnets 32 draw the beads (MB) bound to the AOI out from the sample droplet 200 by using a manipulation head 18 velocity above the threshold for droplet motion. The separated beads (MB) are then transported into the main reservoir and then into a subsequent droplet for further processing, washing, and / or quantification. For example, in one preferred workflow, the beads with AOIs are washed in a second droplet, pulled out of the second droplet, and introduced into a third droplet that contains a detection reagent that labels the AOI. This may include a second affinity reagent that binds the AOI, which includes a fluorescent marker, enzyme, oligonucleotide barcode, or other tag. The second affinity reagent may include a protein-reactive or nucleic acid-specific dye. Finally, the beads (MB) may be directly analyzed or removed again from the third droplet for analysis in another region or droplet using a camera 34, photodetector, or electrochemical measurement. In some embodiments, incubation for a period of time can be used to accumulate signals prior to detection. This implementation can be generalized to capture other biomolecules (e.g., nucleic acids, lipids, carbohydrates) by coating the magnetic beads (MB) with specific capture molecules know n in the art to have affinity7to bind these targets.

[0119] FIG. 8C illustrates the general implementation of liquid handling and blood filtration in a cartridge 30 using the manipulation heads 18 described herein. This design can filter and extract serum from a blood sample, enabled by a custom pipette tip 40 and adaptor 38 to generate sufficient pressure. Attached to the manipulation head 18, a custom pipettor 36 is fitted to an adaptor 38 that holds a pipette tip 40 and snugly fits into the blood filtration cartridge 30. The cartridge 30 features a top layer with an inlet 31 and an indent to2025-094-2accommodate a Vivid GX blood plasma separation membrane or filter membrane 140 and a bottom layer with a with a reservoir or cavity with the filter membrane 140 being supported by beams 144. Blood is dispensed from the pipette-tip-adaptor 38 apparatus once attached to the cartridge 30 and then expelled at a controlled rate to ensure filtration without hemolysis. The blood serum passes through the filter membrane 140, supported by pillars or beams 144 that prevent membrane 140 collapse or tearing, and the filtered serum then flows through the channel 142 and into a bottom cavity into another microfluidic cartridge 30 (or the same cartridge 30) for further processing, such as magnetic droplet manipulation (dosing high concentration ferrofluid or magnetic microparticles / beads into the serum sample droplet for several potential downstream automated laboratory experiments as discussed herein).

[0120] FIG. 8D illustrates the general implementation of automated multiplexed reactions in a microfluidic cartridge 30 driven by a manipulation head 18. In this example, three different solutions containing different detection reagents are each dispensed into individual reaction chambers 168. A sample is then introduced into the bottom sample reservoir 172. Stacked magnets 32 on the manipulation head 18 drag the sample through two dispensers which are hook-hke structure or corrugation 162, dividing the original droplet 200 into three equal partitions 200a, 200b, 200c. Each partition droplet 200a, 200b, 200c is subsequently dragged to a corresponding reaction chamber 168, where it is merged and mixed with the reagents to initiate a reaction such as hybridization reactions, binding / affmity reactions as discussed herein, nucleic acid amplification reactions, enzymatic reactions, or the like. This design holds potential for various applications and assays, capable of facilitating a high number of potential reactions (up to approximately 20 or more) within a single cartridge. Multiple reactions can be all of the same ty pes with different targets, e.g., nucleic acid amplification tests (NAATs) with different primers in each sub-reaction, or different reaction ty pes in each sub-reaction, like NAATs, immunoassays, enzymatic assays, etc.

[0121] FIG. 8E illustrates both 2D and 3D implementations of thermal control in a microfluidic cartridge 30 on a heated stage 14 of a multidimensional position instrument 12. These two designs, one flat and one contoured, enable thermocycling, which is essential for various biological assays such as PCR and applications requiring precise temperature control. In the 2D implementation, different temperature zones are controlled by covering sections of the stage 14 with materials that have varying thermal insulation properties. When a cartridge 30 is placed on the stage 14, it is heated accordingly using heater(s) 15. Zones on the cartridge 30 experience different levels of heat, enabling effective temperature control. For2025-094-2instance, metal transfers heat more efficiently than an acrylic sheet and an acrylic sheet more than air. Placing metal, acrylic, or paper or leaving an area exposed to air can create several different temperature zones ranging from 25°C to 100°C. In the 3D implementation, temperature zones are created by the varying height or thickness of the cartridge 30 components placed above the heated stage 14. As the distance from the heated stage 14 increases, the temperature decreases. This gradient is visualized on the adjacent temperaturegradient bar. By moving samples containing nucleic acid molecules, primers, polymerases, and other reagents among the different temperature zones using the manipulation head 18, thermal cycling or high-temperature processes such as PCR, LAMP, melting curve analysis, and nucleic acid extractions can be facilitated, accommodating diverse applications.

[0122] An example of implementing precise temperature control on a 3D microfluidic cartridge 30 for molecular diagnostics is through melting curve analysis. This post-PCR technique, commonly used to detect genetic variations in nucleic acid sequences, can identify mutant gene targets from clinical samples using a single fluorescent channel, simplifying system design and reducing costs. In this method, the PCR Reaction Buffer and clinical sample template are loaded into designated wells on the microfluidic cartridge 30. The manipulation head 18 moves the magnetic droplet 200, merging and mixing the PCR Reaction Buffer with the sample. The droplet 200 is then transferred to a 95°C zone for 5 minutes of pre-denaturation. The sample undergoes a 10-second denaturation step at 95°C. followed by a 45-second annealing step at 60°C. This denaturation and annealing cycle is repeated 35-40 times to complete PCR amplification.

[0123] After amplification, high-resolution melting (HRM) analysis (e.g., using MeltDoctor™ HRM Reagent Kit, slot #4425557, ThermoFisher) is performed by gradually moving the droplet 200 through a temperature gradient on the slope of the 3D microfluidic cartridge 30, from 50°C to 95°C, at a controlled rate of 0.2°C per second. Fluorescence imaging is conducted every second using a single-channel light sensor, which reduces system complexity and cost. The fluorescence intensity data are transmitted in real-time to the system's GUI 48 for analysis. The analysis software generates a melting curve by plotting fluorescence intensity against temperature, enabling the identification of melting peaks. By analyzing these peaks, specific genetic mutations, such as cancer-related markers, can be identified from the clinical sample. This miniaturized, cost-effective system 10, using a single fluorescent channel, offers a streamlined, multiplexed solution for clinical diagnostics while maintaining high precision in detecting genetic variations.2025-094-2

[0124] FIG. 9 illustrates the workflow of automated serial dilutions using a multidimensional positioning instrument 12 with a manipulation head 18 containing stacked magnets 32 to transport 2.5% ferrofluid droplets 200 in a microfluidic cartridge 30. The process begins with inserting a 10 pL sample droplet in sample reservoir 172 and one or two 16-27 pL parent buffer droplets into respective buffer reservoirs 164 via the multidimensional positioning instrument 12. Both solutions are infused with ferrofluid, allowing for magnetic manipulation by stacked magnets 32 by the moving manipulation head 18. This setup enables partitioning the parent buffer droplets into equal-sized droplets, each characteristic of the specified dilution factor (for 2X, 5X, and 10X dilutions, these are 5, 8, and 9 pL buffer droplets, respectively). The dilution process starts by dispensing the sample droplet (for 2X, 5X, and 10X dilutions, these are 5, 2. and 1 pL, respectively), then moving the dispensed (smaller) droplet 200 into the adjacent merging / mixing chamber 174, where it contacts the existing buffer droplet (for 2X, 5X, and 10X dilutions, these are 5, 8, and 9 pL, respectively). An electrofusion head 120 or low-voltage electrode contacts 124 induces the merging of the droplets 200, followed by thorough mixing achieved through a stirring motion driven by the motion of the manipulation head 18 comprising the stacked magnets 32. This dispensing, moving, merging, and mixing cycle is repeated across subsequent merging / mixing chambers 174, progressively diluting the sample until the desired dilution factor is achieved (for example, 16, 625, and 10,000 times). The rightmost buffer droplet 200 serves as a control, allowing for colorimetric comparison and assessment of dilution accuracy, for instance, when using dye to visualize dilution precision.

[0125] FIGS. 18B-18E presents comparative dilution results (2X, 5X, and 10X) from droplet operations using the system 10 versus traditional test tube operations, using a dye sample for colorimetric comparison. In this example, the dye concentration in the droplets serves as an indicator for determining the precision of dilutions. Droplets with fewer dilutions display a lower grayscale value (darker color) than those with higher dilutions. As shown in the images, the droplet diluted 10,000 times appears much brighter than the droplet containing the original stock concentration of analyte / dye. The plots display 2X, 5X, and 10X serial dilutions, comparing data from the automated system 10 and manual methods. The close alignment between the two datasets, particularly with a ferrofluid concentration of 2.5%, demonstrates the enhanced multidimensional positioning instrument’s abi 1 ity to consistently and accurately perform serial dilutions at small volumes. This is evidenced by the overlap in log2(l -R) or log io( 1 -R) values between the automated and manual methods,2025-094-2where Ji represents the ratio of the grayscale value of diluted sample droplets to that of the control droplet. These results support the efficacy of the multidimensional positioning instrument 12 with ferrofluid droplets as a reliable tool for repetitive, routine laboratory tasks like serial dilutions, especially in applications requiring high precision at microliter scales.

[0126] FIG. 10 presents a microfluidic cartridge design and workflow for an automated sandwich ELISA assay driven by a manipulation head 18. This application begins with the fabrication of a microfluidic cartridge 30 that includes a superhydrophobic coating 185 and an affinity region 186. The affinity region 186 comprises affinity capture reagents that bind to analytes and may comprise microbeads, hydrogels, or other thermoplastic materials conjugated to the affinity capture reagents. Affinity capture reagents preferably comprise antibodies with affinity’ to a target analyte that is specific to the target analyte but may comprise other reagents with affinity to the analyte, such as aptamers, proteins, peptides, nucleic acids, streptavidin, biotin, streptavidin / biotin combinations, or fragments thereof.

[0127] The microfluidic cartridge 30 is initially filled with Novec 7500 oil to enable droplet manipulation. Samples, detection agents, such as detection antibodies that form a complex with the analyte, and parent washing buffer droplets each mixed with 5% ferrofluid are loaded into their respective wells. Detection agents may also comprise aptamers, antibody fragments, proteins, etc. Detection agents are preferably labeled with enzymes, fluorophores, oligonucleotide barcodes, or other secondary binding sites for detection. Visualization substrates such as TMB for enzyme-labeled detection agents and stop solutions are combined with magnetic beads to eliminate any interference with colorimetric detection from the ferrofluid. The parent washing buffer droplet 200p is then divided into three equal partitions by the liquid handler, pipetting into the inlet 158 and moving to designated chambers 173, or from a buffer droplet generator that accurately produces droplets through a nozzle near the parent droplet reservoir and distributed across three separate chambers 173 or wells.

[0128] The assay progresses by sequentially transporting droplets to the affinity' region. An example process can be demonstrated using an NT-proBNP sandwich ELISA assay, involving the following steps: 1) Move the sample droplet containing NT-proBNP analyte from chamber 188 to the affinity region 186. The sample droplet may be from plasma or plasma extracted following a blood filtration operation, as shown herein, introduced into the inlet of the cartridge. Following this step, incubate for 30 minutes to bind the analyte to the affinity region. 2) Move the detection agent containing droplets (containing detection antibody to NT-proBNP bound to HRP) from chamber 190 to the affinity region, merge with2025-094-2the sample droplet, and incubate for 30 minutes. 3) Sequentially move the three washing buffer droplets to the affinity’ region. 4) Move the TMB substrate droplet from chamber 192 to the affinity' region and incubate for 10 minutes. 5) Move the stop solution from chamber 194 to the affinity region and incubate for 1 minute. 6) Drag out the magnetic beads from the solution and measure the OD450 of the cleared droplet using a Sparkfun optical sensor and excitation with high-illumination LEDs. This ELISA cartridge 30 design can be tailored to different types of ELISA assays for diverse target analytes, offering numerous possibilities for diagnostics and analyte quantifications.

[0129] FIG. 11 illustrates the workflow for automated colorimetric PCR within a microfluidic cartridge 30 using a manipulation head 18 and the colorimetric change caused by PCR amplification-induced pH drop. The sample containing a DNA molecule analyte of interest is introduced into the cartridge 30 through an input chamber 196 (left side) for sample loading; the sample droplet 200 is then merged with the PCR reaction mix droplet using the manipulation head 18 and electrofusion head 120. The PCR reaction mix includes forward and backward primers, colorimetric PCR master mix, and DNA monomers. The mixed droplet 200 is then mixed using circular motions of the stacked magnets 32 and moved by the manipulation head 18 through a denaturation zone at 95 °C to melt double-stranded DNA, an annealing zone at 60°C for primer binding to the DNA template, and an elongation zone at 72°C for new DNA strand synthesis by the Taq polymerase. Repeated motion through these spatially distributed temperature zones using the manipulation head yields cycles of the PCR reaction. The times of each of these reactions are also controlled to achieve complete melting, annealing, and elongation. Example times for each step include 1 minute of denaturation, 30 seconds of annealing, and 30 seconds of elongation. Droplets 200 may include ferrofluids and magnetic particles or can be controlled using pressure applied by a contact member 138 secured to the manipulation head 18 which contacts the top layer / flexible membrane 136 above the droplet 200. Additional cartridge features include a magnetic beads storage area 199 to pull a magnetic bead containing droplets 200 for fixation and imaging and a colorimetric result visualization area (right side of cartridge 30) in proximity to the readout of the colorimetric reaction. The reaction may also include other readout modalities, like fluorescence readout using double-stranded DNA intercalating dyes (EvaGreen, acridine orange) or sequence-specific readout using molecular probes or other hybridization-based labeling of the amplified DNA. In addition to PCR, other NAATs can be2025-094-2performed using similar workflows, such as LAMP, rolling circle amplification, isothermal PCR. etc.

[0130] The system 10 employs a thermal control strategy by heating the stage 14 to 95-98°C with one or more heaters 15 and using materials with varying thermal conductivities such as metal, acrylic, paper, foam, and air to create three distinct spatially-distributed temperature zones critical for different PCR phases. The cartridge 30 is filled with oil to prevent evaporation and maintain a controlled environment essential for the thermal cycling of the reaction mix and target DNA. For sample preparation, DNA solution (10 ng / pL) is combined with PCR primer mix, PCR master mix, a pH indicator, such as Phenol red (B21710.30, Thermo Fisher Scientific, USA), and magnetic beads, such as Beckman Coulter AMPure XP. Lot # A63880, with a diameter of 1.0 pm ± 8%. to a total volume of 20 pL — adjustable based on the application (e.g., 10 pL, 25 pL, 50 pL). This mixture is either created on the microfluidic cartridge 30 through merging and mixing of separate droplets 200 as described herein or is loaded into the input well of the cartridge 30 after mixing. The pH indicator in the PCR master mix enables visualization of the chromatic shift in response to pH changes, which occur due to proton release dunng the hydrolysis to form the pyrophosphate ion, a byproduct of DNA amplification. In one embodiment, a pH stabilizer or buffer is not included in the PCR mix, as it buffers the pH decrease and subsequent chromatic shift necessary for visual results interpretation.

[0131] The magnetic beads facilitate the physical movement of the sample, controlled by external stacked magnets 32 on the manipulation head 18, for collecting, transporting, and amplifying the sample within the confines of the cartridge 30. The PCR cycle including denaturation, annealing, and elongation is repeated over multiple cycles by moving the droplet with the manipulation head (e.g., 35 times) to ensure sufficient DNA amplification. Post-amplification, the magnetic beads are preferably moved to the storage area using the manipulation head for separation from the droplet 200, enabling a clearer measurement of the chromatic shift for improved result interpretation. A result visualization area (right side of cartridge 30) is configured to be transparent for excitation and detection of chromatic changes from the PCR amplification process, transitioning from pink to yellow (as pH decreases), indicative of successful amplification and significant presence of the target DNA (>l-5 ng / uL) in the input sample. Similar w orkflow s can also be applied for RT-PCR, starting with an RNA target analyte, where an additional reverse transcription step is achieved by mixing2025-094-2reverse transcriptase reaction mix with the RNA target to create cDNA for the downstream PCR workflow.

[0132] FIG. 12 illustrates the general workflow of an automated agglutination assay for the detection of an analyte on a microfluidic cartridge 30 using magnetic droplets 200, exemplified through an antibody-mediated reaction. Initially, a serum sample following isolation via a blood filter like that disclosed herein or a direct whole blood sample is added to the primary reservoir 202, bypassing the blood filtration step. Magnetic beads (MBs) are subsequently introduced into the reservoir to facilitate the manipulation and transportation of the sample. Alternatively, a solution of MBs may be pre-loaded on the cartridge 30 and merged with the serum or blood sample when introduced into the cartridge. The automated liquid handling module 146 or user then dispenses a positive control (+) and a negative control (-) sample into their designated chambers, setting the baseline for visual comparisons to the assay's final results with the sample containing the analyte.

[0133] In this specific embodiment, antigen-coated particles are added to the sample, potentially containing an antibody analyte specific to the antigen on the particles, and the positive control sample includes a concentration of analyte that binds to antigen-coated particles to induce agglutination. The antigen-coated particles have an affinity to an antibody analyte and, following incubation, can form multiple bridges between particles that lead to large clusters of agglutinated particles. The volume of these droplets typically ranges from 25-50 pL, optimizing the interaction surface area and reagent efficiency while ensuring adequate mixing and reaction kinetics. For alternate applications, these antigen-coated particles can be replaced with antibody-coated or other alTinity-agent-coated particles to detect specific antigen analytes present in the sample, broadening the assay's utility to encompass antigen-mediated agglutination.

[0134] In one exemplary agglutination assay using an agglutination test kit (BactiStaph™ Latex, slot # R21143), latex beads coated with proteins like fibrinogen and IgG are used as detection particles. The analyte in the sample is Staphylococcus aureus, which expresses protein A and clumping factor on its surface. These bacterial proteins bind to the coated beads, causing the beads to aggregate, leading to visible agglutination. When the sample containing ferrofluid (typically 25-50 pL) is mixed with the bead solution in the microfluidic cartridge 30, mixing motion is achieved through the circular motion of the stacked magnets 32 (as mentioned in the detailed description of FIG. 6A), followed by a short incubation of 1 to 3 minutes. If A. aureus is present, the beads form bridges via bacterial proteins, causing2025-094-2agglutination that can be observed visually or measured quantitatively through light scattering or imaging-based analysis.

[0135] The assay also includes positive and negative controls. The positive control contains a known concentration of S. aureus, ensuring proper agglutination, while the negative control lacks an analyte to confirm that agglutination is specific to S. aureus

[0136] A visual comparison to the control droplets is then conducted to determine the occurrence of agglutination and the correct function of the assay, thereby confirming the presence or absence of the target antibody (or antigen) in the serum or whole blood sample. This method allows precise detection of bacterial presence through either qualitative visual clumping or quantitative measurements of light scatter or imaging, offering a reliable tool for bacterial identification in clinical samples.

[0137] This microfluidic cartridge 30 design for automated agglutination assays can be adapted to detect a w ide variety of clumping substances, broadening its application across multiple fields. By using either antigen-coated or antibody-coated particles, the system 10 can be configured to detect specific antibodies, antigens, proteins, and nucleic acids that hybridize to multiple probes or other biomolecules involved in agglutination reactions. Such versatility makes this assay suitable for various medical diagnostics, such as identifying infectious diseases or autoimmune disorders where rapid and specific antigen-antibody interactions are critical. In food safety, this assay can be utilized to test for allergens or bacterial pathogens, offering a quick and reliable method to ensure product safety.Furthermore, in research settings, the system’s adaptability allows for studying various biomolecular interactions and developing new’ biochemical assays.

[0138] FIG. 13 demonstrates an automated library preparation process within a microfluidic cartridge 30 for next-generation sequencing (NGS). NGS library’ preparation begins with already extracted and purified DNA or cDNA mixed with magnetic beads, which is then fragmented into smaller pieces through enzymatic digestion or mechanical shearing, depending on the protocol (e.g., sonication or tagmentation). In starting from RNA for this process, the DNA or cDNA is inserted into the microfluidic cartridge 30 via inlet 204 after reverse transcription, if necessary, where it undergoes fragmentation. The fragmented DNA is then ligated to sequencing adapters containing platform-specific motifs, barcodes for multiplexing, and primers for downstream amplification. The incorporation of these adapters enables the fragmented DNA to bind to the sequencing platform, allowing the identification and amplification of specific sequences, e.g. through polony formation. The manipulation2025-094-2head 18 facilitates the precise mixing of reagents for fragmentation and adapter ligation, in sequence using chambers 206. ensuring optimal efficiency in these critical steps.

[0139] Following adapter ligation, the DNA fragments are released from the magnetic beads and amplified using polymerase chain reaction (PCR), typically with 6-8 cycles, to ensure sufficient yield without over-amplifying the library, which could introduce bias. PCR amplification on the cartridge 30 is achieved as described herein in other sections. After amplification, a purification step removes excess primers, adapters, and other unwanted reaction components, often through magnetic bead-based cleanup (e.g., AMPure beads). Clean up steps may be performed through merging with wash droplets followed by pull down of DNA attached to magnetic beads and replacement with new buffer droplets. In one embodiment the manipulation head 18 moves in the x-y plane the magnetic beads to a holding zone 208 with a permanent magnet, lifts off in an orthogonal direction (e.g., z direction) and brings in new ferrofluid-containing droplets for washing and subsequent reactions, and release of DNA from the magnetic beads.

[0140] This method significantly reduces the labor and time traditionally required for library preparation, where manual processes and expert intervention can extend up to 8-10 hours for a few samples (e.g., 8-16). Incorporating this technique into a microfluidic cartridge 30 enables a highly efficient, automated workflow' for next-generation sequencing (NGS) library preparation. This streamlined process enhances the throughput of sample processing and reduces variability in data quality, making it particularly useful for high-throughput genomic studies. Moreover, the system's ability to automate and miniaturize complex sample preparation procedures facilitates in-depth analysis of RNA and DNA, thereby providing comprehensive insights into gene expression, mutational profiles, and cellular heterogeneity.

[0141] FIG. 14 illustrates the w orkflow of automated antimicrobial susceptibility testing (AST) within microfluidic cartridges 30, focusing on high-throughput analysis to assess the effectiveness of antimicrobials against bacterial or fungal growth. This process uses growth media like Luria-Bertani (LB) or Mueller-Hinton Broth (MHB) tailored for bacterial culture or other growth media that can support the culture of a broad spectrum of bacteria and / or fungi.

[0142] The procedure begins outside the microfluidic cartridge 30, where a liquid handling module 146 prepares a stock solution of sample bacteria mixed w ith ferrofluid in the growth medium. The sample bacteria may be obtained from a clinical sample, such as blood, a blood culture, urine, stool, or other body fluid. The sample bacteria may also be obtained2025-094-2from surfaces or environmental samples (e.g., water, soil, etc.). This stock solution droplets containing ferrofluid is merged and facilitates controlled transportation of the droplets containing the bacteria within the microfluidic cartridge 30 using a magnetic manipulation head 18. However, other stock solutions containing no ferrofluid or magnetic beads may also be used as described herein. A parent droplet of this stock solution (20-30 pL) is then added to the microfluidic cartridge 30 and precisely dispensed into smaller, equal-volume fractions ranging from 1-10 pL using the hook-like structures or corrugations 162. These fractions are efficiently distributed into individual chambers or wells 176 on the microfluidic cartridge 30 using the translational motion of the manipulation head 18.

[0143] Following the distribution of the bacterial stock solution, each bacterial culture droplet undergoes individual incubation within its compartment, enabling isolated growth and facilitating parallel processing essential for AST. The AST is initiated by introducing antibiotics / antimicrobials at vary ing concentrations into each droplet, achieved through an eight times automated serial dilution (can be 2X, 5X, and 10X) process as described herein (e.g., FIG. 9). A stock solution of the antibiotic is introduced into the cartridge through an inlet port or may also be stored in solution or dried on the cartridge where it is rehydrated by the buffer. The stock solution of antibiotic is mixed with an appropriate buffer or growth medium (5-9 pL) and serially diluted to produce separate droplets containing a gradient of antibiotic concentrations in chambers 208.

[0144] Due to the superhydrophobic coating of the microfluidic cartridge 30 or through the use of electrofusion as described herein using the high voltage pulse generator or low -voltage electrode contacts 124, the serially diluted antibiotic droplets 200 are directed to separate designated chambers or w ells 176 and merged with the bacterial culture droplets 200. This merging is followed by thorough mixing through the circular motion of the stacked magnets 32 with the manipulation head 18 and incubation under controlled conditions (e.g., 37 degrees Celsius) to ensure optimal bacterial growth. Bacterial growth is measured through excitation with light (e.g., using an LED) and measurement of scattered and / or absorbed light using a photodiode or other light sensor or camera (e.g., optical density (OD) measurement). Bacterial growth may alternatively be measured using a fluorescent or colorimetric metabolic dye. Time-dependent measurements can be used to determine the grow th curves, and a minimum inhibitory concentration (MIC) and other metrics may be derived. Control wells without bacteria or with bacteria but no antibiotics can act as a baseline negative and positive control, respectively. The precise environmental control within the microfluidic cartridge 302025-094-2(e.g., through control of the temperature of the stage 14) allows for an accurate assessment of bacterial susceptibility to various antibiotics, which is measured as an impaired growth rate compared to positive control conditions. Susceptibility of the sample bacteria to multiple antibiotics can be achieved by including additional stock antibiotics in the cartridge or through different inlets. Separate serial dilutions with buffer / media may be performed and merged with additional bacterial stock solution drops to expand to additional antibiotic measurements. In some workflows, multiple antibiotic types can be dosed together into a single bacterial stock solution drop in order to investigate the synergistic effects of antibiotics. The automated setup minimizes manual intervention, enhancing AST’s reproducibility and scalability.

[0145] FIG. 15 illustrates the automated process of bacterial cell culture measurement and passaging within microfluidic cartridges 30 driven by a manipulation head 18. The system 10 features a microfluidic cartridge 30 coated with a superhydrophobic layer strategically positioned on stage 14 that sits between a light source 180 and a sensor 182. This setup begins by introducing a sample of growth medium containing ferrofluid and a sample of bacteria into the cartridge 30 using a liquid handling module 146 or through manual introduction. The sample of bacteria is merged with a droplet 200 of growth medium using the manipulation head 18. The sensor 182 detects light transmitted through the growth droplet 200 from the light source to continuously measure the ODeoonm, which reflects the optical density and growth progression of bacteria in the medium. The multidimensional positioning instrument's stage 14 is capable of heating to specific temperatures, such as 37°C, which is optimal for different bacterial cell growth. Continuous monitoring of the ODeoonm allows the system 10 to track cell concentration and growth phases accurately, determining a threshold time when the culture reaches the necessary density for passaging.

[0146] When the culture achieves the target optical density, typically between ODeoonm 0.8 to 1.0, the system 10 utilizes stacked magnets 32 on a moving manipulation head 18 to control the transportation of the droplet 200 containing the cultured cells. The droplet is mechanically divided through a hook-like structure or corrugations 162, dispensing a smaller volume droplet 200 (1-5 pL) that is subsequently moved and merged with a new droplet of growth medium (10-25 pL) (right side of cartridge 30). This precise manipulation of cell culture volumes ensures consistent and accurate cell culture management, facilitating ongoing experiments with minimal manual intervention. Sources of bacteria at different densities can be obtained for additional experiments on the microfluidic cartridge or droplets2025-094-2containing bacteria, which can be moved to an outlet region for retrieval and further use. Samples of bacteria may also be moved to a lysis droplet to lyse and release bioproducts for further analysis (optical, electrochemical, fluorescence, etc., using appropriate transducers as know n in the art). Applications include the production of biological products, amplification of plasmids, directed evolution or proteins or cells, or related biotechnology applications. Although the embodiment for the culture of bacterial cells is described, related embodiments for the growth of other cell types are also disclosed, including yeast, mammalian (including suspension-adapted cells or adherent cells that are bound to cell carriers), or insect cells. Different growth media may be included for each respective cell type, and the types for growth will depend on the cell type.

[0147] This automated process can be repeated indefinitely until the area of the microliuidic cartridge 30 or the working plate of the instrument is reached. By integrating optical density measurements with magnetic droplet manipulation, this system 10 offers a highly efficient tool for cell culture assays, especially in scenarios demanding high precision and reproducibility. This technology is particularly beneficial for applications that require stringent control over cell growth conditions, supporting advanced research and development in microbiology and cellular biology.

[0148] FIG. 16 illustrates the w orkflow' of cell electroporation for gene delivery' within microfluidic cartridges 30. This setup features a microfluidic cartridge 30 with a superhydrophobic coating positioned on a thermally controlled stage 14, cooled to 0°C (e.g.. with cooler 17), to provide a stable, low-temperature platform for maintaining the integrity of electrocompetent cells and ensuring preferred conditions for electroporation.

[0149] In this embodiment, the procedure for electroporation includes: First, introducing a droplet 200 containing electrocompetent cells into the inlet well of the cartridge 30 using a liquid handling module 146 or manual pipetting. Magnetic beads or ferrofluids are added to the droplet 200 to facilitate precise control over the transportation of the droplet 200 w ithin the cartridge 30 using a magnetic manipulation head 18. Second, introducing a droplet 200 containing the desired plasmids into an inlet and transporting it to the droplet 200 containing electrocompetent cells, which merges upon contact with the cell-containing droplet 200. Third, positioning the merged droplet 200 accurately between a pair of electrodes 184 (Power + and Ground G). Fourth, delivering precise electrical pulses to initiate electroporation by an electrofusion head 120. This electrical stimulation temporarily permeabilizes the cell membranes, enabling the uptake of plasmid DNA into the cells to complete genetic2025-094-2transformation. Fifth, the magnetic beads are separated out in the main chamber of the cartridge 30, isolating the transformed cells for further analysis or culturing on the cartridge 30 or placing the droplet 200 near an outlet port for recovery from the cartridge 30 and downstream use. The merged droplets 200 in the electroporation assay typically range from 1-10 pL to optimize the volume for effective cell-plasmid interaction.

[0150] This automated electroporation for gene delivery assay on a microfluidic cartridge 30 can be modified for a wide range of gene delivery methods, including chemical transfection techniques like Calcium Phosphate Transfection and DEAE-Dextran, as well as the incorporation of viral vectors such as lentiviruses or retroviruses. The plasmid-containing droplet contains differing reagents or viral vectors for gene delivery . Example applications include the introduction of corrective genes in gene therapy applications or the introduction of genes for expression on the surface of cells for downstream cellular immunization.Additionally, the precision and control provided by the microfluidic cartridge 30 make it an ideal tool for high-throughput studies necessary for exploring complex genetic interactions or protein expression. The gene introduction module can be combined with cell culture modules and other modules described herein to achieve more complex integrated assay types.

[0151] Specific Functional Embodiments of the Manipulation Head

[0152] The manipulation head 18, mounted on the system's multidimensional positioning instrument 12, is designed to support multiple interchangeable components or tools 20, making it highly adaptable to various laboratory tasks such as liquid handling, imaging, mechanical manipulation, magnetic droplet control, and electrofusion-based droplet merging.

[0153] One key embodiment is the liquid handling modulel46, which, in one embodiment, utilizes a 3D-printed syringe holder 148 and plunger pressor 150. This system 10 allows precise liquid manipulation, including injection, withdrawal, and mixing of fluid volumes from 1 to 3000 pL. It can also function as a mini liquid handling robot with a pipette adapter 38, offering the ability7to introduce or withdraw samples from microfluidic cartridges 30. Flow rates range from 0.1 to 1000 pL / min. accommodating both single and multi-channel configurations for tasks such as microparticle fabrication and cell culture media exchange.

[0154] For imaging, the manipulation head 18 can be equipped with a camera 34 or image / optical sensor capable of real-time feedback and advanced imaging functions such as colorimetry, fluorescence, and optical density measurement. The manipulation head 18 includes a camera holder is designed to support various lenses, filters, and light sources, ensuring flexibility for diverse experimental setups.2025-094-2

[0155] In mechanical manipulation, the manipulation head 18 includes a rod-like extension (e.g., contact member 138 that applies precise pressure to the top layer / membrane 136 of the cartridge 30, guiding droplet transport through controlled membrane deformation. This non-magnetic method is ideal for droplet 200 movement in applications where optical transparency is crucial.

[0156] Magnetic control is achieved, in a preferred embodiment, through stacked permanent magnets 32 (preferably more than four stacked magnets, and up to twenty or more stacked magnets) or electromagnets integrated into the manipulation head 18, allowing precise manipulation of ferrofluid droplets 200 (0.5-100 pL). These magnets 32 provide highly accurate droplet control, including movement, splitting, and merging within the microfluidic cartridge 30.

[0157] Lastly, an electrofusion head 120 enables droplet merging using high- or low-voltage electrical shocks. High voltage shocks may be applied without physically touching the cartridge while low voltage may be applied using a low-voltage electrode contacts 124 that contacts electrode contacts 128 on the cartridge 30. This functionality supports complex reactions within the cartridge 30, with high-voltage setups used to merge droplets 200 without contact to the fluid or low-voltage electrode contacts 124 that contact fluid used for merging droplets 200 while minimizing the interference to sensitive biological experiments.

[0158] Cartridges: Variations, Magnetization of Droplets, & Basic Functions

[0159] Manufacturing of Microfluidic Cartridges:

[0160] The microfluidic cartridges 30 are designed to accommodate magnetic droplet control, droplet merging, and visual monitoring. The fabrication process utilizes various methods depending on the needs ranging from rapid prototyping (e.g., laser cutting, 3D printing) to mass manufacturing (e.g., plastic injection molding, hot embossing).Thermoplastics are commonly used due to their affordability and functionalization potential, but other materials, such as glass or PDMS, can be employed for more specialized applications.

[0161] In laser cutting, the microfluidic network is designed using CAD software and transferred to acrylic sheets, sandwiched with double-sided tape, and cut using a laser cutter. These layers are then bonded with transparent membranes that contain inlets and outlets, creating an optically transparent and robust microfluidic cartridge. PET membranes ensure compatibility for visualization, while superhydrophobic treatments prevent droplet adhesion, eliminating the need for surfactants. A similar approach is used for 3D-printed cartridges,2025-094-2where designs are created in modeling software, printed using UV-curing resins, and treated with UV light to secure the bonding. The 3D printing method offers superior resolution, achieving features as small as 20 pm, enabling the creation of multi-layered microfluidic cartridges or intricate structures to trap magnetic particles.

[0162] Magnetization of Droplets:

[0163] Two primary methods are employed for droplet magnetization to control fluid droplets in the microfluidic cartridge 30. The first involves ferrofluid-infused droplets, which consist of magnetic nanoparticles (<100 nm in diameter) that remain evenly dispersed within the droplet under magnetic fields. These droplets 200 enable high-speed (> 60 mm / s) manipulation and provide a strong foundation for various operations, such as dispensing, merging, and mixing. The second method utilizes magnetic microparticles suspended in the droplet. When exposed to magnetic fields, these particles cluster together, creating a magnetic "core" that drives droplet movement. While this method offers slower droplet transportation (<30 mm / s), it allows for the separation of the magnetic beads from the droplet, enabling clear optical imaging of the sample, which is crucial for assays requiring minimal interference from magnetic materials.

[0164] Basic Functions and Applications:

[0165] The laser-cut laminated microfluidic cartridges 30 are equipped with various functional elements, such as inlets for reagent introduction, outlets for waste collection, and structures for precise droplet dispensing. The manipulation head 18 applies magnetic forces to move ferrofluid droplets 200, allowing operations like droplet splitting and buffer droplet generation. Small volumes (0.5-9 pL) can be dispensed with high precision by pulling a droplet 200 across a hook-like structure or corrugation 162. Larger droplets 200 (10-30 pL) are dispensed from a nozzle 166b by modulating the nozzle width and pause time. The dispensing process is highly predictable, with a direct relationship between pause time and droplet volume.

[0166] The flexibility of the manipulation head 18 and cartridge design supports several laboratory functions:

[0167] Serial Dilution:

[0168] Automated serial dilution is achieved by dispensing a droplet 200 indicative of the desired dilution factor, merging it with a buffer droplet, and mixing. Stacked magnets 32 transport and control these droplets 200, enabling precise dilutions from 2X to 20X, depending on cartridge precision and instrument size.2025-094-2

[0169] Biomolecule Capture with Magnetic Beads:

[0170] For capturing biomolecules (e.g., proteins, peptides), magnetic beads coated with specific capture molecules are introduced into a sample droplet 200. After incubation, the beads bind to the target molecules and are transported out of the sample using stacked magnets 32. The beads can then be processed further, washed, or quantified in subsequent droplets.

[0171] Liquid Handling and Blood Filtration:

[0172] The cartridges 30 facilitate liquid handling tasks like media exchange and reagent addition, as well as specialized applications like blood filtration. A pipette tip adapter 38 creates the necessary pressure to separate blood serum through a plasma separation membrane. Once filtered, the serum can undergo further processing using magnetic droplet manipulation.

[0173] Multiplexed Reactions:

[0174] For applications requiring multiple parallel reactions, the cartridge 30 is designed with multiple reservoirs and reaction chambers, each holding different reagents. The sample is divided into smaller droplets 200, merged with corresponding reagents, and processed. This approach supports up to 20 simultaneous reactions or more, making it ideal for multiplexed assays such as biomarker detection and quantification.

[0175] Thermal Cycling for Biological Assays:

[0176] The cartridges 30 can also support thermal cycling, which is critical for processes like PCR. Temperature zones are created by varying the insulation properties of materials on the stage 14 or adjusting the height of cartridge components relative to a heated stage (e.g., FIG. 8E). These zones enable temperature gradients from 25°C to 100°C, allowing precise control for nucleic acid amplification, melting curve analysis, and related assays.

[0177] In summary, the microfluidic cartridges 30, combined with the manipulation head 18 and magnetization techniques, offer a versatile platform for automating complex laboratory workflows. Whether performing serial dilutions, capturing biomolecules, or conducting multiplexed reactions, this system 10 enhances precision, throughput, and reproducibility across a wide range of biological applications.

[0178] HOW TO USE

[0179] The system 10 uses a multidimensional positioning instrument 12 and microfluidic cartridges 30 to automate liquid handling and droplet manipulation. Below is a step-by-step guide to operating the system, covering both hardware and software aspects.2025-094-2

[0180] Step 1: Setting Up the Hardware

[0181] Powering the System 10: Connect the multidimensional positioning instrument 12 to a power source (e.g., wall power source or battery) and built-in Raspberry Pi or other computing device 100. Ensure the stage 14 is clean and leveled.

[0182] Attaching the Microfluidic Cartridge: Securely position the cartridge 30 on the stage 14. Depending on the assay, the cartridge 30 may contain multiple inlets for reagent loading and outlets for waste or sample collection.

[0183] Selecting and Installing the Manipulation Head: Choose the appropriate manipulation head 18 based on the required operation such as magnets 32 for droplet control, pipettors 36 for liquid handling, or cameras 34 for imaging. Attach the manipulation head 18 to the multidimensional positioning instrument 12 if not already mounted thereon.

[0184] Syringe Setup (if applicable): Insert a syringe 152 into the syringe holder 148 for liquid handling operations. Connect the plunger 154 to the Z-axis plunger holder 150 or the repurposed extruder syringe pump. Ensure that the correct syringe volume (e g., 0.5 mL, 1 mL, or 3 mL) is correct for the experiment.

[0185] Step 2: Configuring the Software

[0186] Launching the GUI: Start the GUI 48 on the connected computing device 100. The software offers control through four primary panels: Editor Panel 50, Camera Panel 52, G-Code Panel 54. and Syringe Setup Panel 56.

[0187] Importing Cartridge Design in Editor Panel: In the Editor Panel 60, import the design of the microfluidic cartridge 30 (DXF format). Place the cartridge outline 62 on the grid 58 and adjust its position to match the actual cartridge 30 on the stage 14.

[0188] Camera Alignment and Calibration: Use the camera 34 (if installed) to align the system 10 with the microfluidic cartridge 30. The camera 34 captures an image of the cartridge 30 and automatically aligns the software's virtual grid 58 with the physical setup, using visual or fiducial markers for precise operation.

[0189] Step 3: Defining the Path for Manipulation

[0190] Creating Movement Paths: Add path waypoints 68 on the grid 58 to define the movement of the manipulation head 18. This can be done by clicking on the grid 58 or manually inputting X, Y, and Z coordinates. Use this method to specify where droplets will be transported, split, or merged.2025-094-2

[0191] Tool Selection: At each step, select the appropriate tool 20 for the operation, such as magnets 32 for droplet movement, pipettor 36 for fluid dispensing, or the camera 32 for imaging. The system 10 will automatically adjust the path based on the tool chosen.

[0192] G-Code Generation and Review: Once the path is complete, the system 10 generates G-Code commands. Review these commands in the G-Code Panel 54 to ensure accuracy before proceeding.

[0193] Step 4: Running an Assay

[0194] Loading an Existing Protocol (if applicable): To run a pre-configured protocol, load the G-Code file corresponding to the assay by selecting "Open" in the menu. Ensure the correct microfluidic cartridge 30 is loaded.

[0195] Starting the Assay: Click "Run Assay" to initiate the experiment. The system 10 will automate all steps according to the pre-programmed commands, including liquid handling, droplet manipulation, or reagent mixing.

[0196] Monitoring the Experiment: Use the Camera Panel 52 to monitor the assay in real time. Adjust camera parameters such as zoom and exposure to capture images of the droplets 200 or reagents in action.

[0197] Step 5: Liquid Handling Operations

[0198] Automating Liquid Handling: Use the Syringe Setup Panel 56 to control the volume, time, and flow rate of the syringe 152 for liquid handling tasks. The system 10 supports both injection and withdrawal modes for handling samples or reagents within the cartridge 30.

[0199] Droplet Manipulation: For operations involving magnetic droplets (e.g., ferrofluid), the system 10 will automatically use the manipulation head’s stacked magnets 32 to transport or merge droplets. Ensure the manipulation path is defined correctly to avoid accidental attractions of multiple droplets.

[0200] Step 6: Data Collection and Image Capture

[0201] Capturing Images: Use the Camera Panel 52 to specify capture points. Once the "Take Image" button 86 is clicked, images will be collected based on pre-set positions, allowing for analysis of droplet movement, reagent mixing, or reaction outcomes.

[0202] Saving Results: Save captured images and assay results to a local directory for future analysis. Additionally, save any new paths or protocols created during the experiment for re-use.

[0203] Step 7: Post-Experiment Adjustments2025-094-2

[0204] Modifying Assay Parameters: After the assay is complete, the system 10 allows for re-analysis or re-running of specific steps. Modify paths or capture points as needed without restarting the entire experiment.

[0205] Reviewing Data: Inspect captured images and G-Code logs to confirm that all steps were executed correctly. Adjust parameters for future runs if needed to improve accuracy or efficiency.

[0206] Experimental

[0207] System Infrastructure and Automated Microfluidic Operations

[0208] A fully integrated system 10 was developed that includes hardware and software infrastructure capable of performing programmable, droplet-based bioassays. The system 10 includes multidimensional positioning instrument 12 illustrated in FIG. IB. The stage 14 held the microfluidic cartridge 30 as well as racks for holding test tubes (for holding liquids) and pipette tips 40. A pipette tip ejector 198 was also located on the stage 14 which includes a notice for capturing a pipette tip 40 located on a pipette adaptor 38. Vertical motion of the manipulation head 18 ejects the pipette tip 40 into a waste receptable. The microfluidic cartridge 30 is filled with an immiscible oil environment surrounding ferromagnetic droplets 200 is engineered to perform digital microfluidic bioassays. The frame 22 and external components include a touchscreen display 46 for user interaction, a liquid handling module 146, and a manipulation head 18 that enables numerous functions, such as liquid introduction, droplet transportation and merging, and optical detection.

[0209] The external components are crucial for creating a modular infrastructure for miniaturized lab automation. Due to G-Code's universal compatibility, the system 10 is generalizable to broader 3D gantry systems like low-cost consumer-grade printers (~4"), standard 3D printers (~10"), and liquid handling robots (~20"), plus other CNC motion systems. Each of these devices is capable of being used as the multidimensional positioning instrument 12. A repurposed 3D-printer extruder-based liquid handling module 146 can be expanded to four channels and controlled by G-Code commands, enabling easy programming and integration of liquid-transfer tasks. There are three major components mounted on the manipulation head 18 in this embodiment. As seen in FIGS. IB, 2A, this includes a magnetic / electrofusion head 120 for droplet transportation and contactless merging, a pipettetip adaptor 38, and an optical module that can include a camera 34 or other optical sensing device. The user can enter coordinates or create motion paths on the touchscreen GUI 482025-094-2interface, and G-Code will be generated to control the precise localization of various functional components as described herein.

[0210] The workflow operational sequence from experimental design to data analysis can be divided into two major parts: design and execution. The design process includes the creation of bioassay procedures, design of the microfluidic cartridge 30 and rapid laser-cut fabrication, and spatial motion path programming through the GUI 48. Next, the execution process comprises liquid retrieval & introduction, droplet manipulation, and optical imaging and analysis. In detail, the pipettor 36 on the manipulation head 18 collects and dispenses oil and ferrofluid-infused aqueous reagents to form ferrofluid-based droplets 200 within the microfluidic cartridge 30. The system 10 subsequently executes a programmed series of droplet operations, such as transportation, dispensing, merging, and mixing. After that, the optical module or camera 34 automatically performs image capture or spectrophotometric measurement, followed by algorithmic data recognition and quantitative analysis.

[0211] Characterization and Quantitative Performance of Droplet Manipulation

[0212] Microfluidic automation is the key to miniaturizing the experimental footprint and reaction volumes. A series of quantitative experiments were conducted to evaluate the system’s capabilities in ferrofluidic droplet transportation, dispensing, mixing, and magnetic bead (MB) separation. These modular operations are fundamental to matching standard lab procedures and automating assays.

[0213] Droplet transportation speed is one contributing factor to the operating time of the device’s automation technology. Droplet transportation speeds were measured by setting a specific feed rate (F value) in G-Code commands. Maximum speeds that the droplet 200 could follow with a 4.5 cm displacement of the stacked magnets 32 w ere recorded and plotted in FIG. 17A. Droplets 200 with ferrofluid at concentrations of 2.5% and 5.0% maintained high mobilities, comparable to previously reported speeds where even higher ferrofluid concentrations (e.g., 7.5%, 10.0%, and 12.5%) were required. In contrast, MB-laden droplets 200 exhibited clustering and slower transportation, further revealing the superior homogeneity and magnetic responsiveness of ferrofluid-containing droplets 200 for digital microfluidic operations. Both ferrofluid-containing droplets 200 (with lower ferrofluid concentrations) and MB-laden droplets achieved high levels of optical transparency.

[0214] Robust, repetitive droplet operations are essential for practical applications and a broader adoption of the system 10. Repeated transportation of 2.5% ferrofluid-containing droplets 200 was demonstrated in FIG. 17B. Specifically, the stacked magnets 32 of the2025-094-2manipulation head 18 moved the droplet 200 across a spectrophotometric sensor, repeatedly occluding it and returning to the origin at a rate of 0.4 cycles per second for 40,000 seconds. The optical readout curve showed light intensity (a.u.) over these repeat cycles. The curves of two representative time periods, as shown in FIG. 17B (Inset 976-1, 000s and Inset38, 976-39, 000s), exhibited identical motion patterns, validating long-term transportation repeatability.

[0215] Dispensing reagents and samples into smaller fractions is fundamental for life science research. As shown in FIGS. 17C-17F, high-precision dispensing and droplet generation was achieved with a well-suited volume coverage, 0.5-25 pL, by utilizing two types of droplet manipulation structures, including hook-based dispensers 162 and nozzlebased droplet generators 166a. 166b. The system 10 moves the stacked magnets 32 to drag a parent droplet across hook-based structures 162 (FIG. 17C) in the microfluidic cartridge 30 and generate 0.5-9 pL daughter ferrofluid-containing droplets 200. FIG. 17D shows that the dispensed volumes matched well with the target volumes programmed through the hook geometry for two different ferrofluid concentrations (2.5% ferrofluid (FF): R2= 0.99; 5.0% FF: R2= 0.99). The deterministic, consistent dispensing capability across a wide range of FF concentrations (2.5% - 15%) has been validated.

[0216] The droplet splitting event is governed primarily by the volume of the hook structure 162 and the movement path of the guiding magnets 32. To validate this concentration-independent performance, a total of 108 droplets were dispensed across six FF concentrations (z.e., 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0%), with 18 droplets 200 imaged and analyzed under each condition. The dispensed droplets 200 remained tightly centered around a target volume of approximately 1.00 pL, yielding an overall coefficient of variation of 3.88% (FIG. 6B). Representative droplets 200 across all concentrations exhibited identical sizes, shown in the inset images, confirming that the uniformity of the dispensing mechanism was independent of magnetic responsiveness. This level of consistency supports its use in workflow s that require precise droplet dispensing.

[0217] Once the target droplet volume exceeds 9 pL, it becomes suboptimal to operate using the stacked magnets 32 with a 3 mm diameter. One workaround is to increase the diameter of the stacked magnets 32, w hich introduces a stronger magnetic field and a larger cross-sectional area. However, using larger stacked magnets 32 to manipulate a droplet 200 can result in a lower positioning accuracy and a higher risk of attracting non-targeted droplets 200. Thus, a nozzle-based droplet generator 166b was developed that can dispense a larger2025-094-2amount of liquid from 10 to 25 pL (FIG. 6B). By positioning the stacked magnets 32 over the nozzle neck 166b (FIG. 17E (i)). a part of the fluid from the parent droplet 200 flows out from the nozzle 166b (FIG. 17E (ii)). The period during which the stacked magnets 32 are overhanging is defined as pause time At (sec). By precisely controlling the At, specified amounts of liquid flow from the nozzle 166b and are pinched off (FIG. 17E (iii)). This droplet generation is repeated several times to create multiple copies for parallel reactions (FIG. 17E (vi)). The individual steps of droplet manipulation to create the many aliquots are detailed in the caption of FIG. 17E.

[0218] The correlation between At and the resulting volumes of nozzle-generated ferrofluid-containing droplets 200 was investigated. FIG. 6F showed that the relationships exhibited excellent linearity (R2= 0.99) for both 2.5% FF and 5.0% FF before At reached ~3 sec, where the volumes of fluid flowing out of the nozzle 166b were proportional to At. When the sizes of daughter droplets 200 reached the size of the stacked magnets 32, the volumes tended to be saturated, and relationships between At and volume transitioned to be non-linear. Based on the quantitative relationship acquired above, the nozzle-based droplet generators 166b achieved target volumes ranging from 10 to 25 pL (FIG. 17F). The operations with 2.5% and 5.0% FF both showed high linearity (R2= 0.99) across eight target volumes, providing a foundation for accurate downstream bioassays.

[0219] To achieve bioassay reproducibility, reagent mixing is a key step that ensures a uniform distribution of assay contents in a liquid environment. A "magnetic vortexing" method was used that involves rotation of the stacked magnets 32 over a heterogeneous ferrofluid-containing droplet 200 in a mixing chamber. Magnetic mixing efficiency was quantified by measuring Mixing Indices of mixed, dye-colored ferrodroplets (FIG. 17G). Controlled rotational motions of the stacked magnets 32 stirred the inner contents of an unmixed droplet 200 and enabled uniform color homogenization within 30 cycles with a rotation radius of 3.5 mm (FIGS. 17G-17H). These results demonstrate the system’s capability to efficiently homogenize droplet contents through programmable magnetic stirring, without physical agitation or contamination.

[0220] Magnetic beads (MBs) are commonly used in solid-phase bioassays to selectively enrich analytes, usually equipped with functional groups on their surfaces for specific binding of target molecules. Separating MBs is an important step for washing out non-target molecules or concentrating analytes of interest. The loading and subsequent removal of MBs in droplets 200 was validated in the system 10 (FIG. 171). By moving the stacked magnets 322025-094-2along predefined paths, MBs were successfully immobilized against a semicircular chamber wall while the surrounding ferrofluid was extracted, effectively isolating the beads. As shown in FIG. 17J, optical transmission analysis and microscopic images revealed that postseparation transparency levels in 10%, 50%, and 100% MB solutions were identical to those of the 0% reference control, confirming complete removal of magnetic particles.

[0221] Automated Serial Dilution

[0222] To evaluate the system’s capability for automating workflows consisting of a multi-step sequence of the demonstrated liquid handling tasks (e.g., droplet transportation, dispensing, and mixing), the multidimensional positioning instrument 12 was programmed to automate serial dilutions using ferrofluid-containing droplets (FIG. 18 A). FIG. 18A illustrates the process: a sequence of magnetic dispensing, transport, electrofusion, and mixing steps digitally controlled by the system 10 without human intervention. In detail, the two major steps are i) Buffer Droplet Dispensing: collecting the parent buffer droplets from reservoirs, dispensing them into daughter droplets with designated volumes (e.g., 8 pL for 5* serial dilution), and distributing daughter droplets to the dilution chambers located on the top row; ii) Dye Sample Dilution: dispensing the sample into a daughter droplet e.g., 2 pL for 5*) and transporting it to the proceeding dilution chamber for contactless electrofusion and mixing by magnetic stirring. This process iterates several times until the final dilution in the last chamber is completed.

[0223] Quantitative testing evaluated the accuracy of serial dilutions with the system 10. Several dilution experiments (i.e., 2x, 5x, and 10x) were conducted to verify the capabilities and robustness of this system 10. In detail, the system 10 was programmed to serially dilute a dye sample from a starting concentration (e.g., l / 10x stock for 5 ) to concentrations several orders of magnitude lower (e.g., dilution factors after each step of 5, 25, 125. and 625 for 5x), followed by image acquisition and colorimetric quantification.

[0224] As shown in the FIGS. 18B-18C, colorimetric changes from darker to lighter can be observed. For example, when the dilution factor reached 10,000 during the 10x serial dilution, the original light-brown color of the ferrofluid-containing droplet became observable. Standard dilutions were conducted with test tubes, pipettors, and a vortexer to measure comparative colorimetric changes to be plotted alongside measurements made with the system 10. Results showed that the normalized light intensity (1 -R ratio) of the tw o methods matched well (2*: R2= 0.99; 5< R2= 0.99; 10x; R2- 1.00) with minimal deviations across replicate trials.2025-094-2

[0225] An advantage of the system 10 is the high magnetic strength of its stacked magnets 32, allowing for digital microfluidic operations with minimal concentrations of infused ferrofluid (FF). By comparing the colorimetric signal curves in FIGS. 18D and 18E, an obvious signal saturation (p = 0.52 > 0.05, indicating no significant colorimetric changes after diluting the original stock sample 10 times) can be observed with 10% ferrodroplets utilized. The droplet images shown below the saturated signal curve remain uniformly dark, showing that excessive iron oxide nanoparticles can obscure optical signals. Further validation of this optical advantage is illustrated in FIGS. 21 A and 21B. Despite the aforementioned signal saturation, the light intensities of system 10 and Test Tube groups still matched well at 10x with 10% FF (R2= 0.99), showing robust performance across diverse fluid compositions.

[0226] The system 10 performs serial dilution by combining two precisely engineered dispenser structures: one for buffer droplets 200 and one for sample droplets 200. Each dispenser (hook structure 162) produces a fixed droplet volume defined by its cross-sectional geometry. By selecting appropriate combinations of sample and buffer volumes (e.g., 5 pL + 5 pL for 2x, 2 pL + 8 pL for 5x, or 1 pL + 9 pL for 10x), the system 10 can generate a wide and tunable range of dilution factors. Because the dispensers can be fabricated across a continuum of droplet volumes (e.g., 0.5-9.5 pL), the system 10 can theoretically produce dilution ratios ranging from 2 to 20* on a single microfluidic cartridge 30. Once the volumes are defined, the system 10 executes a programmed sequence of dispensing, merging, and magnetic stirring to produce each dilution step in an automated, repeatable manner. Below, we provide detailed procedures for 2* and 5x / 10x serial dilution, illustrating how these principles are implemented.

[0227] 2x Serial Dilution Procedure.

[0228] 1) Position the serial dilution microfluidic cartridge 30 at the center of the stage 14 (e.g., print bed).

[0229] 2) Start the program, load the experimental G-code, and click run.

[0230] 3) The system 10 pipettes a 25 pL buffer droplet into the bottom-left buffer reservoir (FIG. 4A). Then, the system 10 pipettes 10 pL of the sample into the top-left chamber. Align the stacked magnets 32 over the buffer reservoir.

[0231] 4) The stacked magnets draw the 25 pL buffer droplet out of its well.2025-094-2

[0232] 5) The buffer droplet is moved diagonally to the first dispenser 162, dragged into the dispenser, and then split. This results in a 5 pL buffer droplet nestled in the first dispenser.

[0233] 6) Step 5 is repeated three times for the 2nd, 3rd, and 4th dispensers (only two are illustrated in FIG. 4A), resulting in three 5 pL buffer droplets nestled in the dispensers. The leftover 5 pL buffer droplet is moved into the top-right chamber for background control.

[0234] 7) The four buffer droplets in the dispensers are sequentially dragged into mixing chambers on the top of the cartridge (e.g., 1st dispenser buffer droplet to the 1st mixing chamber (going left to right) and 2nd dispenser buffer droplet to the 2nd mixing chamber).

[0235] 8) The 10 pL sample droplet is dragged into the first chamber dispenser and split. The free 5 pL droplet is returned to the sample chamber, and the 5 pL droplet nestled in the chamber dispenser is moved to the 1st mixing chamber.

[0236] 9) The droplets in the mixing chamber are merged via electrofusion stimulus (high or low voltage or merged uninfluenced when using a superhydrophobic coated cartridge 30), and the stacked magnets 32 stir the unmixed droplet 30 cycles using a clockwise circular rotation.

[0237] 10) Steps 8 & 9 are repeated three times in the 2nd, 3rd, and 4th mixing chambers, splitting the respective fully mixed droplet and moving the dispensed droplet into the next mixing chamber.

[0238] 11) The final 10 pL droplet in the 4th mixing chamber is split following Step 8, but the 5 pL droplet nestled in the chamber dispenser is moved into the hook at the base of the right-most well instead of into the control droplet chamber.

[0239] 12) The automated serial dilution is complete, and the stacked magnets 32 move outside the cartridge 30.

[0240] 5x / 10x Serial Dilution Procedure.

[0241] 1) Position the serial dilution microfluidic cartridge 30 at the center of the stage 14 (e.g., print bed).

[0242] 2) Start the program, load the experimental G-code, and click run.

[0243] 3) The system 10 pipettes 24 / 27 pL buffer droplet into the left buffer reservoir and a 16 / 18 pL buffer droplet in the right buffer reservoir (FIG. 4A), located at the bottom-left comer of the cartridge (all volumes listed for 5x / 10x, respectively). Then, the system 10 pipettes 10 pL of the sample into the top-left chamber. Align the stacked magnets 32 over the right buffer reservoir.2025-094-2

[0244] 4) The stacked magnets 32 draw the right 16 / 18 pL buffer droplet out of its well.

[0245] 5) The right 16 / 18 pL buffer droplet is moved diagonally to the first dispenser 162, dragged into the dispenser, and then split. This results in an 8 / 9 pL buffer droplet nestled in the first dispenser 162.

[0246] 6) The leftover 8 / 9 pL buffer droplet is moved into the 4th mixing chamber. The droplet in the first dispenser 162 is dragged to the 3rd mixing chamber.

[0247] 8) The stacked magnets align over the left 24 / 27 pL buffer well and draw it out of its well.

[0248] 9) Step 5 is repeated twice for the 1st and 2nd dispensers 162, resulting in another two 8 / 9 pL buffer droplets nestled in the dispensers 162. The leftover 8 / 9 pL buffer droplet is moved into the top-right chamber for background control.

[0249] 10) The buffer droplet in the 1st dispenser is dragged to the 2nd mixing chamber, and the buffer droplet in the 2nd dispenser is dragged to the 1st mixing chamber.

[0250] 11) The 10 pL sample droplet is dragged into the first chamber dispenser and split. The free 8 / 9 pL droplet is returned to the sample chamber, and the 2 / 1 pL droplet nestled in the chamber dispenser is moved to the 1st mixing chamber.

[0251] 12) The droplets in the mixing chamber are merged via an electrofusion stimulus (high or low voltage or merged uninfluenced when using a superhydrophobic coated cartridge 30), and the stacked magnets 32 stir the unmixed droplet 30 cycles using a clockwise circular rotation.

[0252] 10) Steps 11 & 12 are repeated three times in the 2nd, 3rd, and 4th mixing chambers, splitting the respective fully mixed droplet and moving the dispensed droplet into the next mixing chamber.

[0253] 11) The final 10 pL droplet in the 4th mixing chamber is split following Step 11, but the 2 / 1 pL droplet nestled in the chamber dispenser is moved into the hook at the base of the right-most well instead of into the control droplet chamber.

[0254] 12) The automated serial dilution is complete, and the stacked magnets move outside the cartridge.

[0255] Automated Colorimetric LAMP

[0256] To assess the applicability of the system 10 for molecular diagnostics, a fully automated colorimetric loop-mediated isothermal amplification (LAMP) workflow was implanted by the system 10 for BRCA1 primer set validation in breast cancer diagnostics (FIGS. 19A-19C). The assay leveraged the system’s integrated fluid handling, droplet2025-094-2electrofusion, thermal regulation, and imaging capabilities to execute all steps of a LAMP reaction without manual operation. As illustrated in FIG. 19A, after loading the initial reagents (e.g., LAMP primer sets (i)-(v), positive DNA (+ DNA), negative control, and LAMP master mix), the workflow began with the dispensing of primer set droplets 200, followed by + DNA & negative control droplets 200. These reagents were merged into LAMP mix droplets 200 located in the top chambers. Then, the stage 14 of the system 10 was heated up to 65 °C for 30 minutes of isothermal amplification, followed by image capture and analysis.

[0257] Thermal regulation and calibration of the microfluidic cartridge 30 were conducted as shown in FIG. 19B. The stage 14 provided uniform and accurate heating from ~25 to 110 °C. After heat transfer from the stage 14 to the microfluidic cartridges 30, there was an excellent linear correlation between the programmed stage 14 temperature and the measured microfluidic cartridge temperature (Y = 0.91X + 1.173; R2= 1.00). Based on the equation, temperature control was achieved on a microfluidic cartridge 30 over a programmable range from ~25 to 100 °C. Temperature stability during isothermal incubation at 65 °C was exceptional, with a coefficient of variation (CV) of only 0.3 % over ten independent trials. This result confirmed that the 3D printer's heated bed which functioned as the stage 14 can be directly harnessed to maintain precise thermal conditions for bioassays.

[0258] As shown in FIG. 19A, an on-chip colorimetric LAMP assay as demonstrated that validated efficient primer sets for breast cancer detection with the BRCA1 target gene. After incubation, reactions with an efficient primer set produced a visible color change from pink to yellow due to a decrease in pH associated with efficient DNA amplification, while negative controls remained pink. Among the five primer sets tested, set (i) showed minimal color transition, indicating inefficient amplification, whereas sets (ii)-(v) yielded clear color changes to yellow, indicating successful amplification. The based colorimetric obtained by the system 10 results closely matched gel electrophoresis outcomes for bulk assays, which confirmed amplicon formation only in sets (ii)-(v). These findings demonstrated that the system 10 can reliably perform automated, in parallel nucleic acid amplification assays with integrated colonmetric detection.

[0259] Variations of droplet generation nozzles across ferrofluid concentrations

[0260] The system 10 employs two nozzle geometries, a 1-mm narrow nozzle and a 2-mm triangular wide nozzle (FIG. 20), to accommodate changes in magnetic responsiveness associated with varying FF concentrations. Droplets with higher FF infusion (e.g., 5.0% FF)2025-094-2exhibit a strong magnetic response, readily deforming and flowing through a nozzle structure. In contrast. lower-FF droplets (e g., 2.5% FF) respond weakly to magnetic actuation, resulting in reduced mobility and greater difficulty in overcoming geometric constraints like a narrow nozzle. To compensate for this weakened magnetic responsiveness, wider nozzles are employed to reduce the hydrodynamic barrier to droplet extrusion. Thus, 1-mm nozzles are paired with 5% FF droplets and 2-mm nozzles are paired with 2.5% FF droplets, ensuring reliable and repeatable droplet generation across ferrofluidic compositions.

[0261] In addition to nozzle width, two structural and operational adjustments enable robust droplet pinch-off with weaker magnetic responsiveness. First, triangular sharp edges are incorporated into the 2-mm nozzle design, providing a geometrically favorable break point that promotes clean droplet neck rupture. Second, as shown in FIG. 20. an additional "pull-up" step is introduced. After positioning the stacked magnets above the nozzle, the daughter droplet is pulled slightly upward by 1 mm before the lateral pinch-off This maneuver elongates the droplet neck to facilitate rupture. It simultaneously engages the stronger magnetic field at the edge of the stacked magnet column, thereby improving traction for low-FF droplets. Together, these structural and operational strategies enable efficient extrusion and pinching of both high-FF (5.0%) and low-FF (2.5%) droplets, thereby maintaining volume consistency within the 10-25 pL range.

[0262] Discussion

[0263] The system 10 achieves an exceptionally low hardware cost compared to standard liquid handlers and conventional microfluidic systems, often below $500. Existing low-cost custom liquid handling systems still have drawbacks in precision and throughput. Lab-on-a-Chip technologies have achieved remarkable miniaturization and precision of reaction volumes. However, their commercial implementation and widespread use have been limited by their reliance on bulky and costly instruments, such as precision pumps for fluid control and high-voltage electronic controllers for EWOD actuation. The system 10 balances miniaturization and functionality, providing digital microfluidic control within a compact, self-contained platform that integrates droplet manipulation, thermal regulation, and real-time imaging. Moreover, by leveraging magnetic droplet actuation, the system 10 enables programmable, non-contact liquid handling without mechanical wear or intricate circuitry, enhancing reliability and ease of use.

[0264] The versatility of the system 10 is exemplified by its ability to automate temperature-dependent nucleic acid amplification assays, such as colorimetric LAMP. These2025-094-2assays offer an attractive alternative to qPCR by operating at a constant temperature and producing visible colorimetric outputs that can be interpreted without specialized instrumentation. Despite their simplicity, LAMP workflows require precise liquid handling, controlled incubation at defined temperatures, and consistent optical readout; factors that traditionally limit scalability and reproducibility in resource-limited or decentralized settings. The system 10 addresses these challenges by integrating programmable reagent dispensing, on-chip thermal regulation, and image-based detection. This enables high-throughput screening and optimization of primer sets, accelerating assay development for a broad range of genetic targets.

[0265] The current system 10 implementation supports key droplet operations such as transportation, merging, heating, and optical analysis, integrating additional sensing modalities, such as fluorescence, impedance, or electrochemistry, enables multiplexed and kinetic assays in addition to colorimetry. From a software perspective, incorporating artificial intelligence (Al) and large language models (LLMs) could infuse adaptive intelligence into every step of lab automation: from microfluidic cartridge design and path planning to G-Code generation and real-time data interpretation. Acting as an "experimental co-pilot," an LLM-enhanced system 10 could autonomously generate protocols, detect anomalies, and interpret outcomes, lowering the barrier for non-expert users. These advances, building on the system architecture, can pave the way for accessible, democratized, and intelligent laboratory automation.

[0266] Methods

[0267] Materials for the Multidimensional Positioning Instrument

[0268] The system 10 was constructed using low-cost, widely accessible components, such as 3D-printer parts. The base motion system that formed the multidimensional positioning instrument 12 was derived from Ender-3 (Creality, Shenzhen, CN) and Prusa MK4S (Prusa Research, Prague, CZ) 3D printers. Custom connectors, holders, and other mechanical components were 3D-printed in-house using PLA filament (SUNLU, CN). The user interface GUI 48 was hosted on a Raspberry Pi 5 (8 GB), connected to a 10.1 " touchscreen (ROADOM, CN) for GUI display 46 and a 5" touchscreen (ELECROW, CN) for the liquid-handling module operations. For optical imaging, a 64 MP Autofocus Camera 34 (Arducam Technology, CN) was mounted on the manipulation head 18. Spectrophotometric measurements were performed using the AS7262 Visible Spectral Sensor (SparkFun Electronics, CO, USA). Magnetic droplet actuation used stacked Neodymium-Iron-Boron2025-094-2(NdFeB) magnets 32 (1 mm thick each, >10 mm in total, 3 mm in diameter) and ferumoxytol (Ferraheme, AMAG Pharmaceuticals, MA, USA) as the magnetizer in the droplet 200. All microfluidic cartridges 30 were filled with fluorinated oil (Novec 7500, 3M, MN, USA) containing 0.5 % biocompatible surfactant (Pico-Surf, Sphere Fluidics, NJ, USA). A standard piezoelectric voltage generator 216 from a commercial fire lighter (BIC Multi-Purpose Party- Lighter) provided the electrical pulses for contactless droplet merging.

[0269] Microfluidic Cartridge Fabrication

[0270] microfluidic cartridges 30 were fabricated by assembling multiple layers of transparent films (Apollo, LD Products, CA, USA), double-sided adhesive tapes (9474LE 300LSE, 3M, MN, USA), and an acrylic sheet (Acry lic United States, NY, USA). The multilayer stack consisted of a 1 mm acrylic sheet sandwiched between two 150 gm adhesive layers, with 100 gm transparent films on the top and bottom to enclose the microfluidic channels (FIGS. 3A and 3B). 2D microchannel patterns were designed in Autodesk AutoCAD and precisely cut on acrylic sheets using a laser cutter (Speedy 100, Trotec Laser, MI, USA). The transparent films were laser-cut to define the overall cartridge 30 outline and inlet / outlet ports, then sequentially cleaned with 100% ethanol and deionized water. The cleaning process was repeated twice. When no surfactant was used, superhydrophobic coating was required to prevent droplet adhesion. The coating procedure involved: (1) treating the uncovered microfluidic cartridge 30 (without the top transparent film) with NeverWet base coat and top coat (Rust-Oleum, USA), followed by 30 min of resting in the fume hood for superhydrophobic surface formation; (2) treating a separate transparent film with the same coating method; and (3) aligning and bonding the treated transparent film to the coated microfluidic cartridge 30 to enclose the channels.

[0271] Graphical User Interface (GUI)

[0272] The GUI 48 was developed to enable user-system communication and streamline microfluidic automation (FIGS. 1A, IB, 5A-5D). The GUI 48 supports both the custom design of new workflows and the execution of existing biological assays. Communication between the GUI 48 and the system hardware occurs via a serial port, which sends G-Code that controls the three-axis positioning of the manipulation head 18 with 0.1 mm resolution, enabling diverse functionalities. The GUI 48 also controls the stage 14 (print bed) temperature, allowing programmable heating during assays. The GUI 48 comprises four modular panels: (i) Editor Panel 50 (FIG. 5A) for manipulation-head navigation, (ii) Camera Panel 52 (FIG. 5B) for microfluidic cartridge imaging, (iii) G-Code Panel 54 (FIG. 5C) for2025-094-2command generation and visualization, and (iv) Syringe Setup Panel 56 (FIG. 5D) for automated liquid handling. This structure provides coordinated control of droplet movement, reagent dispensing, heating, assay procedure timing, and image acquisition. The GUI 48 was implemented in Python using the PySimpleGUI and Tkinter libraries.

[0273] Transportation Speed Measurement

[0274] To characterize the maximum speed at which ferrofluidic-containing droplets 200 could be reliably tracked, measurements were conducted using a microfluidic cartridge 30 with a 50 mm * 10 mm x 1 mm chamber. The cartridge 30 was positioned at the center of the stage 14, and the stacked magnets 32 were lowered until the bottom surface was -0.2-0.5 mm above the cartridge surface, without making physical contact. Ferrodroplets ranging from 0.1 pL to 100 pL in volume and containing 0.5% to 15% ferumoxytol were characterized (Table 1). For each test, a single ferrodroplet was magnetically guided along a 50-mm straight trajectory by displacing the stacked magnets via G-Code. The magnet velocity was controlled by adjusting the feed rate (F value) in the G-Code. A trial was considered successful if the droplet 200 remained intact and followed the stacked magnets for >45 mm. Starting from a low transportation speed, like 0.5 cm / s, the speed was increased in 0.5 cm / s increments. Testing continued until the droplet 200 either failed to follow the stacked magnets 32 or broke apart. The highest speed at which the droplet 200 completed a successful follow-up was recorded as its maximum trackable velocity. To compare ferrofluid droplets 200 with particulate suspensions, parallel measurements were performed on droplets 200 containing 50% magnetic beads (AMPure XP # A63880, Beckman Coulter, -1 pm diameter, 0.5-10 pL droplet volumes), following a similar procedure.

[0275] Since the system 10 uses stacked magnets 32 that generate strong magnetic fields, droplets 200 with 0.5%~2.5% FCs can move easily (Table 1). Even at low FF concentrations using 2.5% FF droplets 200 can maintain excellent mobility’ while minimizing decreases in the dynamic range of the colorimetric modality. Across both colorimetric and fluorescent characterization, 2.5% ferrofluid provided the best balance between optical clarity’ and magnetic mobility. Lower concentrations provided excellent transparency but reduced magnetic responsiveness, whereas higher concentrations (>5%) caused substantial signal attenuation, obscuring low-intensity colorimetric and fluorescent readouts. At 2.5% FF, droplets 200 remained highly mobile under magnetic actuation while preserving sufficient optical transparency for quantitative measurements. This concentration was therefore selected as the standard condition for all system assays2025-094-2Table 1

[0276] Long-term Transportation and Validation

[0277] To evaluate the long-term stability of ferrodroplet transportation on the system 10, a microfluidic cartridge 30 was fabricated with a 40 mm x 10 mm x 1 mm chamber. A 10 pL. 2.5% ferrodroplet was loaded into the oil-filled channel. The cartridge 30 was positioned directly above a mini-spectrophotometer placed on stage 14, using a custom chip-sensor alignment fixture that centered the ferrodroplet over the spectrophotometer pinhole. An LED was placed adjacent to the chamber to provide constant illumination. During testing, the stacked magnets 32 were programmed to drive the ferrodroplet back and forth across the top area of the pinhole, causing periodic occlusion and recovery of the light path. This motion was repeated continuously at 0.4 cycles per second for a total duration of 40,000 s, while the spectrophotometer recorded the resulting light-intensity fluctuations.

[0278] Droplet Generation with Target Volumes

[0279] Target-volume droplets were generated using two microfluidic designs, a hookbased dispenser 162 and a nozzle-based droplet generator, optimized for different volume ranges. For the hook-based dispenser 162, droplet volume was determined by the cross-sectional area of the hook structure 162. By incrementally increasing this area, daughter droplets were produced ranging from 0.5 to 9 pL. The larger hook structures were suboptimal2025-094-2due to inconsistent droplet splitting. This approach was validated for 2.5% and 5% ferrofluid formulations.

[0280] To generate larger droplets, a nozzle-based droplet generator was utilized. Using this design, the stacked magnets 32 were positioned directly above the nozzle neck 166b to draw out a portion of the parent ferrodroplet. The daughter droplet was pinched off by moving the stacked magnet 32 laterally. For this structure, droplet volume was controlled by the pause time (At) when the magnets 32 were held over the nozzle 166b. Calibration experiments were conducted for 2.5% and 5% ferrodroplets to establish the quantitative relationship between At and droplet volume. Using these calibration curves, daughter droplets were reproducibly generated ranging from 10 to 25 pL by adjusting the appropriate At.Droplet volumes for both microfluidic designs were quantified from images acquired by a digital camera. The cross-sectional area of each droplet was measured in ImageJ and converted to volume using a pre-established calibration between droplet footprint and volume. Together, the hook-based and nozzle-based designs enabled a combined dynamic droplet generation range of 0.5-25 pL.

[0281] Experimental and Indexing of Droplet Mixing

[0282] To evaluate the mixing performance on the system 10, 2 pL of dyed droplets were merged with 8 pL of 2.5% ferrodroplets. Immediately after merging, the stacked magnets 32 performed rotational stirring over the droplet. Two parameters were systematically varied: the number of rotational cycles (0, 10. 20. or 30) and the rotation radius (0. 0.5, 2.5, or 3.5 mm) with a fixed speed of 0.5 cm / s. These combinations yielded 10 experimental conditions to assess how rotational motion influences mixing. After each run, droplets were imaged using a digital camera under uniform illumination. Images were circularly cropped to isolate the droplet and converted from RGB to HSV color space for better segmentation of the dyed region. The Mixing Index (MI) was computed based on the fraction of the dyed area within the total area. Specifically, the number of pixels falling within the predefined hue range corresponding to the dye w as divided by the total number of droplet pixels. An MI approaching 1.0 indicated near-complete color homogenization, whereas lower values reflected incomplete mixing. These indices were used to compare the relative efficacy of different rotational radii and cycle counts, with the static mixed control droplet serving as the reference.

[0283] Magnetic Bead Separation2025-094-2

[0284] To evaluate the ability to separate magnetic beads (MBs) (AMPure XP # A63880, Beckman Coulter), a microfluidic cartridge 30 containing a 0.5-mm-radius semicircular chamber designed to trap MBs exclusively. 2.5% ferrodroplets were prepared with 10%, 50%, or 100% magnetic bead suspensions (~1 pm diameter) and introduced into the cartridge 30. The stacked magnets 32 guided each droplet along a predefined path and drew it against the semicircular chamber wall, where the magnetic field concentrated and immobilized the beads to form a cluster. After holding the droplet in place for several seconds, the stacked magnets 32 were quickly moved away to extract the 2.5% ferrodroplet from the chamber, leaving the trapped MB cluster in place. To quantify separation efficiency, droplets were imaged before and after MB removal using a Nikon Ti-E inverted microscope to capture 16-bit light-transmission images that were then analyzed using Image! Light-intensity values were compared with a 0% bead reference control droplet to determine the completion of MB removal; post-separation images with transparency approaching or equal to that of the reference 2.5% ferrodroplet indicated near-complete or complete separation of MBs.

[0285] Automated Serial Dilutions

[0286] Automated serial dilution experiments were conducted using three variations of a microfluidic design, each containing dispenser geometries matched to the target dilution factor. For 2x dilutions, the cartridge 30 incorporated paired 5 pL buffer and 5 pL sample dispensers; for 5x dilutions, 8 pL buffer and 2 pL sample dispensers; and for 10x dilutions, 9 pL buffer and 1 pL sample dispensers. To optimize the visualization of colorimetric changes and ensure that the dynamic range covered the dilution series, the initial dye concentration was adjusted for each experiment: l / 64x stock for 2x dilutions, l / 10x stock for 5x dilutions, and undiluted stock for 10x dilutions. For each dilution protocol, the system 10 was programmed to dispense the required buffer droplet, aliquot the sample droplet using the corresponding dispenser, merge the droplets via contactless electrofusion, and mix the merged droplet using magnetic stirring before proceeding to the next dilution cycle. Each workflow produced four sequential dilution cycles, as shown in FIGS. 18B-D.

[0287] Following completion of the automated dilution sequence, droplets from all dilution steps, including the starting sample droplets, were imaged using a digital camera under consistent illumination. Colorimetric quantification was performed by measuring the 1 ~R ratio, where R ratio denotes the ratio of the droplet's measured intensity7to that of the reference control droplet. Log-transformed (1-7? ratio) values were used to plot dilution2025-094-2curves for each protocol. Parallel 10x dilution experiments were conducted using 2.5% and 10% ferrofluid to evaluate the impact of ferrofluid concentration on optical properties.

[0288] Automated Colorimetric LAMP-on-Chip

[0289] Colorimetric loop-mediated isothermal amplification (LAMP) assays were carried out on the system 10 using the WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs). LAMP primer sets were designed using the NEB LAMP Primer Design Tool (v 1.4.1) and synthesized by Thermo Fisher Scientific. Before conducting on-chip amplification, the system's thermal behavior was calibrated by measuring the cartridge’s temperature as the 3D printer bed setpoint (stage 14) was incrementally adjusted (5 °C increments from 25 °C to 110 °C). A strong linear relationship between the programmed bed temperature and the cartridge temperature was established, enabling precise temperature control. Temperature stability at 65 °C was assessed across 10 independent trials, and the coefficient of variation (CV) was calculated to validate consistent isothermal incubation.

[0290] For on-chip LAMP reactions, droplets containing LAMP master mix, five candidate primer sets, positive DNA templates, and negative controls were preloaded into separate chambers on the microfluidic cartridge 30. On the system 10, droplets were magnetically transported, merged, and assembled into five parallel LAMP reactions, each containing a positive reaction and a negative control. Following 30 min of isothermal incubation at 65 °C, endpoint colorimetric images were acquired using a digital camera. Amplified droplets exhibiting a pink-to-yellow transition were classified as successful reactions, while droplets remaining pinkish contained anon-amplifying or inefficient primer set candidate. To confirm amplification outcomes, droplets from each reaction were collected and subjected to conventional gel electrophoresis. Gel images were used to verify the presence or absence of expected LAMP amplicons and to validate the on-chip classification of primer sets as efficient or inefficient.

[0291] Gel Electrophoresis Validation of LAMP Products

[0292] Verification of colorimetric LAMP reactions was performed using agarose gel electrophoresis. LAMP generates a mixture of stem-loop DNA structures and concatenated repeats of the target sequence, producing a characteristic ladder-like banding pattern on agarose gels (FIG. 19C). This method was used to validate BRCA1 amplification outcomes from on-chip reactions. End-point ferrodroplets containing LAMP products were analyzed on 1% agarose gels stained with GelRed nucleic acid dye. For each reaction, a 10 pL ferrodroplet was mixed with 6x TnTrack DNA loading dye and loaded into individual wells2025-094-2alongside a GeneRuler DNA ladder for size reference. Electrophoresis was performed at 130 V for 40 min, and gels were imaged using a Bio-Rad Universal Hood II imaging system. The presence of a distinct multi-band ladder pattern confirmed successful amplification, whereas its absence indicated inefficient or no detectable amplification.

[0293] While embodiments of the present invention have been shown and described, various modifications may be made without departing from the scope of the present invention. The invention, therefore, should not be limited, except to the following claims, and their equivalents.

Claims

2025-094-2What is claimed is:

1. A system for liquid handling and droplet manipulation comprising:a multidimensional positioning instrument comprising a multi-axis manipulator having a manipulation head mounted thereon and containing one or more tools secured to the manipulation head;a stage disposed adjacent to the multi-axis manipulator, wherein the stage is configured to receive one or more cartridges and wherein the multi-axis manipulator and / or stage are configured for relative motion along three orthogonal axes; anda computing device comprising software configured to control the relative motion of the multi-axis manipulator and / or stage to place the one or more tools over or in contact with selected regions of the one or more cartridges as part of series of pre-programmed operations or steps.

2. The system of claim 1, wherein the one or more tools are selected from the group comprising: a camera, an optical sensor, one or more permanent magnets, an electromagnet, a pipettor, an electrofusion head, a syringe plunger holder, and a contact member.

3. The system of claim 1, further comprising a display having a Graphical User Interface (GUI).

4. The system of claim 3, wherein the GUI displays a grid on the display that approximates the position of the stage along with position(s) of the one or more cartridges.

5. The system of claim 3, wherein the series of pre-programmed operations or steps are programed or executed through the GUI.

6. The system of claim 1, wherein the one or more cartridges comprise magnetic droplets contained on or within a surface or volume of the one or more cartridges.

7. The system of claim 6, wherein the magnetic droplets comprise droplets of ferromagnetic fluid or droplets including magnetic beads.2025-094-28. The system of claim 1, wherein the one or more tools comprises a plurality of permanent magnets arranged in a stack or an electromagnet.

9. The system of claim 1, wherein the one or more cartridges comprises a top flexible membrane.

10. The system of claim 1, wherein the stage comprises one or more heaters and / or one or more coolers therein.

11. The system of claim 1, wherein a plurality of cartridges are disposed on the stage.

12. The system of claim 1, further comprising a syringe holder secured to the stage and a syringe plunger holder secured to the manipulation head.

13. The system of claim 1, wherein the manipulation head comprises a pipette adaptor configured to receive a pipette tip.

14. The system of claim 1, wherein the one or more cartridges comprise electrode pair(s) disposed therein and wherein the manipulation head comprises low-voltage contacts configured to electrically connect with the electrode pair(s).

15. The system of claim 1, wherein the one or more tools are removably secured to the manipulation head.

16. A method of performing liquid handling and droplet manipulation using the system of any of claims 1-15 comprising:loading reagents or a sample in the one or more cartridges;executing the series of pre-programmed operations or steps to perform one or more operations including droplet generation, droplet transport, droplet merging, droplet mixing, droplet reaction, droplet incubation, or droplet splitting.2025-094-217. The method of claim 16, wherein the droplet(s) comprise magnetic droplet(s).

18. The method of claim 17, wherein the operations are performed using one or more permanent magnets or an electromagnet secured to the manipulation head.

19. The method of claim 16. wherein the operations are performed using a contact member secured to the manipulation head that contacts an upper flexible membrane of the one or more cartridges.

20. The method of claim 16, wherein the multi-axis manipulator is configured for motion in three orthogonal axes and the stage is stationary’.

21. The method of claim 16, wherein the multi-axis manipulator is configured for linear motion and the stage is a moveable stage configured for 2D motion.

22. The method of claim 16, wherein the one or more tools comprise a camera and one or more permanent magnets.

Images