Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
  • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
  • B01L3/502792—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
  • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L7/00—Heating or cooling apparatus; Heat insulating devices
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
  • G01N27/74—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids
  • G01N27/745—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids for detecting magnetic beads used in biochemical assays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/18—Means for temperature control
  • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
  • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

• the technical field generally relates to digital fluidic platforms. More specifically, the technical field relates to a digital fluidic platform that uses the electronic actuation of individual coils formed on or in a substrate to impart magnetic fields on moveable permanent magnets that are used to actuate magnetic droplets.
• the digital fluidic platform may be used to implement multiplexed and automated assays.
• the platform may also be used for pooled clinical sample testing of, for example, viruses.
• Background [0003] Over the past two decades, major epidemics (SARS, Zika, MERS, and Ebola) and pandemics (H1N1 and COVID-19) have emerged with increasingly alarming regularity. Although currently the world is grappling with the COVID-19 pandemic, the occurrence of the next wave of infectious disease outbreaks in the coming years is deemed inevitable, given the rise in population, urbanization, and global travel/trade. In that regard, large-scale population screening is the primary safeguard to contain epidemics, prevent pandemics, and mitigate their human and economic costs upon their onset.
• NAATs nucleic acid amplification tests
• the strategic pooling of samples, when most patients are expected to be negative, can lead to a dramatic reduction in resource utilization amid pandemic-induced supply chain disruptions (outweighing the marginal risk of dilution-induced false negatives).
• a swarm of individually-addressable millimeter-sized magnets were employed as mobile robotic agents (“ferrobots") that can manipulate magnetic nanoparticle-spiked droplets (“ferro-droplets”) with high precision and robustness.
• ferrobots mobile robotic agents
• the seamless integration of fluidware, hardware, and software allowed for programing and streamlining the droplet-based operations, and delivering versatile automated NAAT- centered workflows within a compact platform (e.g., here a reverse transcription loop mediated isothermal amplification, RT-LAMP was implemented).
• RT-LAMP reverse transcription loop mediated isothermal amplification
• This algorithm particularly determines the optimal testing mode and guides the operational workflow in accordance with a square matrix pooling scheme, without entailing overly burdensome sample handling procedures. Adopting this approach over the fixed individual testing approach (universally pursued) allows for significant savings over a wide viral prevalence range.
• a ferrofluidic fluid assay device includes a first substrate having a plurality of individually addressable coils formed therein or thereon; a second substrate disposed adjacent to the first substrate and separated by a gap, the second substrate containing therein one or more volumes of ferrofluid, one or more sample holding chambers or regions, one or more corrugated features for defining sub-volume(s) of the ferrofluid and/or sample, and one or more assay chambers holding an assay solution or assay reagent therein; one or more permanent magnets interposed in the gap formed between the first substrate and the second substrate, wherein the one or more permanent magnets are moveable 2023-021-2 within the gap; and a power source and control circuitry electrically connected to the individually addressable coils and configured to selectively drive current through one or more of the individually addressable coils to move the one or more permanent magnets and the one or more volumes of ferrofluid.
• a method of using the ferrofluidic fluid assay device includes the operations of: loading sample(s) into the one or more sample holding chambers or regions; and driving current through the one or more of the plurality of individually addressable coils to move the one or more permanent magnets to perform one or more unit operations on the one or more volumes of ferrofluid, wherein the unit operations comprise moving the one or more volumes of ferrofluid across a surface of the second substrate, merging the one or more volumes of ferrofluid with the sample(s), forming a plurality of sub- volumes of ferrofluid mixed with the sample with the one or more corrugated features, mixing the one or more volumes of ferrofluid and sample, pooling the one or more volumes of ferrofluid and sample, and moving the one or more volumes of ferrofluid and sample to the one or more assay chambers.
• the device is a handheld portable device that uses a microfluidic chip or cartridge that works with a first substrate or printed circuit board that is located within the device.
• the device uses one or more moveable permanent magnets to manipulate one or more volumes of ferrofluid.
• the microfluidic chip or cartridge includes one or more trench chambers including a trench recess formed therein.
• the one or more trench chambers capture magnetic beads that are carried by the one or more volumes of ferrofluid that are used to capture target species located within a sample loaded in the device.
• the target species may include nucleic acids, proteins, other biomolecules, or the like.
• FIG.1A illustrates a cross-sectional view of a ferrofluidic fluid handling device according to one embodiment.
• FIG.1B illustrates a view of the first substrate (i.e., printed circuit board (PCB)) according to one embodiment along with associated control electronics.
• PCB printed circuit board
• FIGS.1C-1I illustrate an overview of the bioanalytical swarm ferrobotic device for accessible, adaptable, and automated viral testing.
• FIG.1C illustrates spatio-temporal varying COVID-19 viral prevalence (based on the test positivity rate data from Our World in Data and California Health and Human Services Open Data Portal).
• FIG.1D illustrates the required number of tests per person to find all infected people (across different levels of local viral prevalence), based on the square matrix pooled testing strategy.
• the highlighted curve (with arrow labeled as “adaptive testing”) illustrates that maximal screening efficiency can be achieved via adaptive (prevalence-based) testing.
• FIG.1E is an illustration of the optimal testing modes and the associated ferrobotic microfluidic chips (scale bar: 1 cm) for the representative local viral prevalence levels of 25%, 10%, and 2%.
• FIG.1F is a schematic overview of the automated workflows for individual and pooled testing of 16 samples.
• FIG. 1G is an exploded schematic of a representative ferrobotic viral testing platform.
• FIG.1H are schematic illustrations of the ferrobotic equivalents of lab-based NAAT liquid handling operations, including aliquoting, merging and mixing.
• FIG.1I is an optical image of a representative ferrobotic viral testing platform for 4 2 pooled testing. [0013]
• FIGS.2A-2J illustrate ferrobotic operations enable NAAT-based testing.
• FIG.2C shows the characterization of the threshold voltage for droplet merging using different concentrations of a surfactant (PicoSurfTM) within an oil (NovecTM) environment.
• PicoSurfTM a surfactant
• FIG.2D shows the characterization of the cyclic ferrobotic operations, involving aliquoting, merging, and intermediate transportation of a parent droplet to evaluate the robustness of the ferrobotic operations (performed for > 800 cycles, scale bar: 3 mm). 2023-021-2 Parent droplet size varied by ⁇ 1% for each of the post-merging and post-aliquoting states (characterized optically).
• FIG.2E illustrates progressive mixing index for different actuation frequencies. Top: corresponding images of the merged droplets under mixing at different actuation frequencies for 15 s.
• FIGS.6A-6B show the characterization of the robustness of ferrobotic actuation over a range of ferro-droplet ionic strengths and chemical conditions.
• FIG.6A illustrates a schematic of the characterized ferrobotic actuation, involving cyclic actuation of 10 ferro- droplets (with differing ionic strengths and chemical conditions) by designated ferrobots (12 commuted pixels-per-cycle/ferrobot). The actuation events and commuted pixels are tracked by monitoring the current through impedance sensing electrode pairs.
• FIG. 8A illustrate a quantification of the RT-LAMP assay response by real-time absorbance measurement using a plate reader.
• FIG.8B shows the absorbance spectra and corresponding optical images (insets) of the RT-LAMP reaction products for different volumes of positive input samples (6 to 1 ⁇ L, reaction period: 30 min).
• FIG.8C shows the absorbance spectra of the RT-LAMP assay solution pre- and post-RT-LAMP reaction, showing the utility of the assay pH adjustment for optimizing the assay’s colorimetric response.
• FIGS.10A-10B illustrate the repeatability and reproducibility of ferrobotic tests with clinical samples. Ferrobotic SARS-CoV-2 RT-LAMP assay readouts corresponding to ferro-droplet sample volumes of 1 ⁇ L (FIG.10A) and 100 nL (FIG.10B) are illustrated.
• FIG.11B shows sequential optical images of the active ferrobotic sample processing operations (performed automatically).
• FIG.11C shows the timeline of the streamlined on-chip operations for automated multiplexed testing, which includes active ferrobotic sample processing operations over a time window of 2:20 min:s.
• FIG.11D illustrates optical images and corresponding on-chip readouts of the SARS-CoV-2, Influenza H1N1, and rActin (internal control, IC) RT-LAMP assays for different combinations of input samples (spiked with corresponding RNAs).
• FIG.12 illustrates and example of sample analysis and interpretation flowchart for pooled testing.
• FIGS.13A-13F illustrate microfluidic chip layouts and the ferrobot navigation plans and task assignment for pooled testing.
• FIGS.13A and 13B show schematic illustrations of the microfluidic chip layout for 3 2 (FIG.13A) and 4 2 (FIG.13B) pooled testing (key features are outlined and labeled).
• FIGS.13C and 13D illustrate an overview of the navigation plans of seven and nine ferrobots for 3 2 (FIG.13C) and 4 2 (FIG.13D) pooled testing. Each Fi represents an individual ferrobot.
• FIGS.13E and 13F illustrate the timeline of the ferrobots’ assigned tasks and status (active, standby) for 3 2 (FIG.13E) and 4 2 (FIG.13F) pooled testing.
• FIGS.14A-14C illustrate the detection of virus in diluted and undiluted clinical samples by colorimetric SARS-CoV-2 RT-LAMP assay.
• FIGS.14A, 14B show optical images of the standard assay responses in microfuge tubes (FIG.14A) and on-chip assay responses (FIG.14B) for undiluted or diluted patient samples with different Ct values.
• FIG. 14C shows corresponding optical readouts of the on-chip assay responses.
• FIG.16A illustrates optical absorbance readouts of the colorimetric RT-LAMP assay performed at 60 °C, 65 °C, and 70 °C using a SARS-CoV-2 positive control (PC, 100 cp/ ⁇ L) and negative control (NC) samples.
• FIG.16B shows the characterization of the local temperature set by an on-board resistive heater for different input current at different surrounding temperatures ( ⁇ 15 °C, 25 2023-021-2 °C, 35 °C, generated by a Peltier module).
• FIG.16C shows the required input current through the on-board resistive heater to maintain a local temperature of 65 °C for different surrounding temperatures (derived from the results shown in FIG.16B).
• FIG.17 illustrates images and optical absorbance readouts of the RT-LAMP assay performed in ferro-droplets containing: negative control, negative control spiked with E. coli, SARS-CoV-2 positive control RNA, and SARS-CoV-2 positive control RNA spiked with E. coli (PC: 10,000 cp/ ⁇ L; E. coli: 10,000 CFU/ ⁇ L).
• FIG.18 shows sequential images of the active ferrobotic sample processing operations in a small sample volume device (dispensed sample ⁇ 100 nL; scale bar: 5 mm).
• FIGS.19A-19D illustrate pooled testing using 100 nL sample volumes.
• FIGS. 19A, 19B show readouts obtained from ferrobotic pooled testing of two groups of 9 clinical samples using the 3 2 pooling chip.
• the negative assay A response indicated no infected sample was present among the first group of samples in (FIG.19A).
• the positive assay A response along with the positive assay R1 and C2 responses led to the identification of the infected sample (located at the 1 st row/2 nd column) among the second group of samples (FIG. 19B).
• FIGS.19C and 19D show readouts obtained from ferrobotic pooled testing of two groups of 16 clinical samples using the 4 2 pooling chip.
• the negative assay A response indicated no infected sample was present among the first group of samples in (FIG.19C).
• FIG.20 illustrates the maximum ferro-droplet velocity within different oil environments is plotted as a function of oil viscosity.
• FIG.21A illustrates a schematic of the relevant pressure mechanisms acting at the interface of two oppositely charged emulsion droplets during coalescence.
• FIG.21B shows the estimated threshold disjoining pressure across different surfactant concentrations.
• FIGS.22A-22C illustrate a comparison of conventional PCB electromagnetic coil- based actuation vs. ferrobotic actuation.
• FIG.22A shows a simulated magnetic flux density 2023-021-2 induced by a standalone electromagnetic coil.
• FIG.22B shows simulated magnetic flux density induced ferrobotically.
• FIG.22C illustrates magnetic flux density enhancement factor due to the presence of the field-amplifying ferrobot.
• the x axis is the vertical distance from the center of the magnetic source.
• FIG.23A illustrates the experimental setup for the standard ferrobotic system including a power supply, laptop, an chicken microcontroller (MCU) module, and the ferrobotic control unit.
• FIG.23B illustrates a battery-operable handheld ferrobotic unit with 7.4 V- and 3.7 V-lithium-ion batteries and MCU module integrated on its backside. These components are placed next to a quarter-dollar coin for comparison.
• FIGS.24-1, 24-2, 24-3, 24-4, 24-5, 24-6 include a table showing clinical sample results.
• FIG.25A illustrates another embodiment of the digital ferrofluidic fluid assay device. This is a self-contained, handheld device that is able to perform assays such as viral assays.
• FIG.25B illustrates an exemplary workflow for the trench chamber illustrated in FIG.25A.
• FIG.25C illustrates a three-dimensional (3D) view of a single droplet of ferrofluid and reagents located above a ferrobot traveling over a PCB containing addressable coils.
• FIG.26A illustrates the trench recess in the trench chamber acts as a barrier to magnetic beads and traps the same inside the trench chamber as the ferrofluid volume or droplet is moved past the trench chamber.
• FIG.26B illustrates a graph showing separation efficiency for different magnetic bead concentrations. High separation efficiencies are achieved over a range of magnetic bead concentrations.
• FIG.1A illustrates a cross-sectional view of a digital ferrofluidic fluid assay device 10 according to one embodiment.
• the ferrofluidic fluid assay device 10 is “digital” in the sense that it creates, manipulates, and operates on discrete volumes or droplets of ferrofluid 100 contained within the ferrofluidic fluid assay device 10.
• a ferrofluid 100 is a liquid fluid 2023-021-2 that is magnetic due to the presence of small (e.g., nanometer-sized to tens of nanometer- sized) magnetic particles 102 suspended in a carrier fluid.
• the volumes or droplets of ferrofluid 100 may also contain a package 104 that may be sample, reagent, or the like.
• the package 104 may be a biological material resulting in a bio-package 104.
• the ferrofluidic fluid assay device 10 includes a first substrate 12 that has a plurality of individually addressable coils 14 formed therein or thereon.
• the individually addressable coils 14 operate as an electromagnet (EM) when actuated and current is driven through the addressable coils 14.
• This first substrate 12 acts as a navigation floor for permanent magnets 34 which act or operate as “ferrobots” as explained herein.
• the first substrate 12 may, in one preferred embodiment, be a printed circuit board (PCB) that includes the plurality of individually addressable coils 14 formed therein.
• PCB printed circuit board
• the first substrate 12 is formed from a multi-layer PCB where the plurality of individually addressable coils 14 are formed as spirals with different layers of the PCB 12 containing additional spirals of the coil structure (e.g., three different layers for the spiral structure).
• FIG.1B illustrates a view of the navigation floor or first substrate 12 (e.g., PCB) according to one embodiment.
• the plurality of individually addressable coils 14 are formed as an array or matrix on the first substrate 12 with individual addressable coils 14 formed in rows and columns, although other configurations may be used.
• the number of individual addressable coils 14 may vary depending on the size of the overall ferrofluidic fluid assay device 10, size of individual addressable coils 14, pitch between adjacent coils 14, etc.
• each individual addressable coil 14 had a three-turn configuration with a size of 1.5 x 1.5 mm stacked in three layers in the PCB making up the first substrate 12 (FIGS.7A, 7B, 7E-7F, FIG.9B). Adjacent coils 14 were separated by a gap of 0.1 mm.
• the first substrate 12 includes, in one embodiment, a first IC switch 16A and a second IC switch 16B.
• the first and second IC switches 16A, 16B may be directly integrated on the first substrate 12.
• the IC switches 16A, 16B are used to select and actuate or power individual addressable coils 14.
• the first IC switch 16A may be used for row selection (e.g., MAX14662 (Maxim Integrated, CA, USA)) while the second IC switch 16B is used for column selection (e.g., MC33996 (NXP semiconductor, Netherlands).
• the first and second IC switches 16A, 16B are connected to and controlled by a microcontroller unit (MCU) 18 which acts as the control circuitry for actuating coils 14 (see also FIG.7E).
• MCU microcontroller unit
• a serial peripheral interface (SPI) may connect the first and second IC switches 16A, 16B to the MCU 18.
• SPI serial peripheral interface
• the coils 14 can be sequentially and/or simultaneously activated to perform the desired unit operation or task as described herein.
• the MCU 18 may be located on-board the PCB that makes up the first substrate 12 or it may be located separate from the first substrate 12.
• the MCU 18 may itself be operably connected to a computing device 20 (e.g., personal computer, laptop, tablet PC, mobile phone) using, for example, a serial communication.
• the computing device 20 includes software 22 executed by one or more processors 24 that are used to program the sequencing and timing of actuation of the individually addressable coils 14.
• Target coordinates i.e., target coils 14
• SPI commands i.e., SPI commands
• a second substrate 30 is disposed adjacent to the first substrate 12.
• the second substrate 30 is, in one preferred embodiment, a microfluidic chip that contains the volumes of ferrofluid 100 that are manipulated as described herein.
• the second substrate 30 is disposed adjacent to the first substrate 12 and separated by a gap G. Spacers 32 are optionally used to control the gap G distance.
• the second substrate 30 generally lies in a plane that is substantially parallel to the plane of the first substrate 12.
• One or more moveable permanent magnets 34 are interposed in the gap region G formed between the first substrate 12 and the second substrate 30.
• the permanent magnets 34 preferably comprise rare earth magnets but may also include metallic materials or composite magnetic materials (e.g., ceramic or ferrite), or other materials commonly used for permanent magnets.
• the dimensions of the permanent magnets 34 may vary depending on the particular ferrofluidic fluid assay device 10 but are generally millimeter-sized permanent magnets. In experiments conducted herein, the permanent magnets 34 had a height or thickness of 0.8 mm and 2.54 mm diameter (cylindrically shaped). In some embodiments, the width or diameter of the permanent magnets 34 may be about the same or less than the width or diameter of a single coil 14. In other embodiments, the width or diameter of the permanent magnets 34 may be larger than the width or diameter of a single coil 14 thus overlapping multiple coils 14.
• the second substrate or microfluidic chip 30 contains the working area of the ferrofluidic fluid assay device 10 and contains the volumes of ferrofluid 100 where the digital operations take place.
• the volumes of ferrofluid 100 are preferably in the form of droplets 100.
• the droplets 100 have volumes in the range of 100 nL to 1 ⁇ L. Less preferably the droplets 100 have volumes in the range of 10 nL to 100 ⁇ L.
• the volumes of ferrofluid 100 contain therein magnetic particles 102.
• the magnetic particles 102 are preferably biocompatible and, in some embodiments, are nanoparticles.
• the second substrate or microfluidic chip 30 includes one or more microfluidic features 50 thereon.
• the microfluidic features 50 may include microfluidic channels, walls, flow path(s), holding chambers or regions, blocks that define rows and columns that allow for movement of volumes of ferrofluid or droplets 100.
• these may include may include ferrofluid holding chambers 110 for holding ferrofluid to be used in the volumes of ferrofluid or droplets 100, one or more sample holding chambers or regions 112 (e.g., for loading or holding samples), one or more corrugated features 28 that are used to define and split off sub-volumes of ferrofluid 100 as they move across the corrugated structure, one or more assay chambers 114, a waste or disposal chamber 64 (e.g., FIGS.13A-13B), a reagent chamber, and the like.
• ferrofluid holding chambers 110 for holding ferrofluid to be used in the volumes of ferrofluid or droplets 100
• sample holding chambers or regions 112 e.g., for loading or holding samples
• corrugated features 28 that are used to define and split off sub-volumes of ferrofluid 100 as they move across the corrugated structure
• assay chambers 114 e.g., FIGS.13A-13B
• a waste or disposal chamber 64 e
• the holding chamber 110 is used to hold the initial volume or droplet of ferrofluid 100 as well as hold the waste residue and operates as a disposal chamber 64 as seen in FIG.3C.
• the second substrate or microfluidic chip 30 may include a central region that includes an open area that permits easy lateral (e.g., planar) travel of the volumes or droplets of ferrofluid 100 across the surface of the second substrate or microfluidic chip 30. This allows volumes or droplets of ferrofluid 100 to move between different physical locations of the second substrate 30 or microfluidic 2023-021-2 chip.
• volumes or droplets of ferrofluid 100 can move between different physical locations where dedicated operations are performed (i.e., merging with sample, aliquot of sub-volume of sample, merging or pooling of volumes or droplets of ferrofluid 100, heating a volume or droplet of ferrofluid 100 containing a sample, cooling a volume or droplet of ferrofluid 100/sample, imaging a volume or droplet of ferrofluid 100 containing a sample, electrochemically measuring a sample, performing an assay, or waste removal).
• the volumes or droplets of ferrofluid 100 are surrounded by a filler fluid 106.
• the second substrate or microfluidic chip 30 may be formed as a laminate structure that is formed by multiple layers 56 of a polymer that are adhered to each other using an adhesive or tape 58 with adhesive backing.
• a polymer that are adhered to each other using an adhesive or tape 58 with adhesive backing.
• PET polyethylene terephthalate
• Additional materials such as plastics or polymer materials or glass may be used with manufacturing processes known in the art, such as hot embossing, injection molding, 3D printing and the like.
• the physical features on the second substrate or microfluidic chip 30 can be created using laser-cutting.
• electrodes 36 may be deposited or patterned prior to assembly.
• the second substrate or microfluidic chip 30 includes a top surface 38 and a bottom surface 40.
• FIGS.1G-1I illustrates a representative ferrobotic testing platform, which includes two modules (entirely constructed by low-cost components): 1) a disposable oil-filled microfluidic chip 30 with passive and active actuation interfaces that hosts input sample(s) and ferrofluid/assay reagents and 2) a printed circuit board (PCB) as the first substrate 12, 2023-021-2 featuring 2D arrayed coils 14 (“navigation floor”), which can be independently activated to electromagnetically direct individual ferrobots 34.
• modules include two modules (entirely constructed by low-cost components): 1) a disposable oil-filled microfluidic chip 30 with passive and active actuation interfaces that hosts input sample(s) and ferrofluid/assay reagents and 2) a printed circuit board (PCB) as the first substrate 12, 2023-021-2 featuring 2D arrayed coils 14 (“navigation floor”), which can be independently activated to electromagnetically direct individual ferrobots 34.
• PCB printed
• FIG.2A illustrates the precise and tunable ferrobotic sample aliquoting capability in the optimized NovecTM oil environment.
• droplet merging is useful for adding reagent(s) to the input sample(s) and combining multiple input samples for pooling.
• a relatively low voltage ⁇ 0.3 V - 1.5 V, depending on the surrounding oil surfactant composition
• droplet merging in less than a few seconds can be achieved.
• Droplet 100 merging can also be achieved without application of voltage by bringing droplets 100 in contact to each other using a ferrobot magnet 34 in a surrounding environment without surfactant.
• cyclic aliquoting, merging, and intermediate transportation of a parent volume or droplet of ferrofluid 100 over 800 cycles was performed with ⁇ 1% variation in the corresponding size of the parent droplet post-aliquoting and post-merging (FIG.2D).
• the permanent magnet 34 can be oscillated to induce chaotic fluid motion within the merged volume or droplet of ferrofluid 100 by alternatively activating the neighboring coils.
• Colorimetric detection is based on the generated hydrogen ions, causing a color change of an incorporated pH indicator (phenol red) from red- orange to yellow (optimization experiment results shown in FIGS.8A-8C).
• the color change allows for the binary interpretation of the test, above or below a threshold as positive or 2023-021-2 negative, respectively.
• This color change can be optically interrogated visually (FIG.2I) by the naked eye, or electronically by integrating the response of an optical sensing module 44 (FIG.2J and FIGS.7B, 7D), without the absorbance of the ferrofluid affecting the readout interpretations.
• the optical sensing module 44 includes a light source 46 and a light or optical sensor 48.
• the assay was also successfully performed by using microfluidic structures of reduced height ( ⁇ 150 ⁇ m) to aliquot a 10-fold smaller ferro-droplet volume (100 nL; reagent volume 1.9 ⁇ L; FIG.8E), which is below the volume that can be accurately pipetted using robotic liquid handlers, but useful for minimizing reagent use.
• the characterization results also verified the reliability of the assay in the presence of temperature variations of a few degrees C° (FIG. 16A) and in the presence of biological interferents (FIG.17).
• a disposable microfluidic chip including the first substrate 12, second substrate 30, permanent magnets 34, addressable coils 14 and other features of the device 10 described herein was customized to host the input sample, associated reagents, and dedicated aliquoting/merging components (FIG.3A, FIG.7B).
• a PCB module was used as the first substrate 12 containing the navigation coils 14, resistive heaters 42, and colorimetric sensing circuitry. By programming the PCB 12 at the software-level, a ferrobotic instruction set was installed to seamlessly execute the assay.
• the instruction set charts the navigation plan of a dedicated permanent magnet 34 “ferrobot” and details the electrode 36 excitation conditions for merging and heating, while accounting for a 5 min-heat lysis and a 30-min RT-LAMP reaction period (FIG.3B).
• the active ferrobotic operations take place over a period of 1.75 minutes (FIGS.3B-3C).
• a volume or droplet of ferrofluid 100 is first magnetically 2023-021-2 transported to, then merged and mixed with an introduced sample droplet, in order to make the sample amenable for ferrobotic manipulation.
• the next steps in the sequence are aliquoting the ferro-sample using, for example, the corrugated features 28, disposing the ferro-sample residue, and delivering the aliquot (1 ⁇ L) to the assay chamber 114 (containing the assay reagents).
• the RT-LAMP process initiates, and after 30 min, the assay readout is colorimetrically quantified, rendering the test result in a sample-to-answer manner (FIGS.11A-11B).
• a similar workflow was implemented using microfluidic chips with reduced height (in second substrate 30) to achieve smaller ferro- droplets ( ⁇ 100 nL) for analysis with reduced reagents (FIG.18).
• the accuracy of the device 10 was assessed with real world samples by testing one hundred clinical samples with the ferrobotic RT-LAMP chip, and comparing the on-chip readouts with the corresponding readouts obtained from the standard RT-PCR and RT-LAMP assays (summarized in FIG.3D, detailed in FIGS.24-1 through 24-6).
• the collected samples were based on nasopharyngeal swabs from SARS-CoV-2 infected or uninfected patients.
• the viral on-chip detection threshold (710 a.u.) was derived from receiver operating characteristic (ROC) analysis (aliquoted sample volume: 1 ⁇ L).
• FIGS.13A, 13B illustrates the corresponding layouts of the 3 2 and 4 2 microfluidic chips.
• the expanded layouts especially include arrays of sample aliquoting interfaces using corrugated features 28 and assay chambers 114 (containing SARS-CoV-2 RT-LAMP assay solutions), orthogonal corridors for intra-chip sample aliquot transport, and extended merging interfaces.
• the microfluidic chip 30 2023-021-2 contains a series of blocks 60 that define columnar and row flow paths within the microfluidic chip 10. The perimeter of the blocks 60 contain structural features to perform one or more operations.
• aliquoting structures in the form of a corrugated surface 28 is positioned on one side of the blocks 60.
• Sample inlets 62 are located on another surface of the block 60 where sample is loaded into the microfluidic chip 30. Sample inlets 62 may be located adjacent to sample holding regions 112 although sample holding regions 112 may also be located in areas away from the sample inlets 62.
• Disposal chambers 64 are also located on surfaces of some of the blocks 60 which act as chambers to store waste or unwanted fluid(s).
• Mixing regions 66 are located along the rows and columns defined by the blocks 60 and are used to mix the ferrofluid volumes or droplets 100.
• Droplet holders 68 are provided on a surface of the blocks 60 and are used to hold droplets of ferrofluid 100 and/or sample.
• FIG.13B illustrates a similar microfluidic chip 30 as disclosed in FIG.13A with the difference that rather than a 3 x 3 construction, a 4 x 4 construction is used for holding 16 samples. Additional N x N sample pooling follows a similar structure except for N rows and N columns and N 2 samples (here N is a number greater than 4). The footprint of the device increased geometrically, but the construction otherwise remains the same.
• FIG.4B illustrates the sequence of the operations performed by a swarm of nine permanent magnets 34 or ferrobots to deliver a representative 4 2 pooled testing workflow (FIG.13B also illustrates the nine permanent magnets 34 used in the swarm operations).
• the demonstrated sequence involves: 1) making three aliquots of each input sample with the aid of four permanent magnets 34 or ferrobots.
• the aliquots were ferrobotically collected, merged, mixed, and then dispensed as a droplet with a metered volume (1 ⁇ L).
• the final volume may be delivered to an assay chamber 114 for visualization as explained herein.
• the overview of the navigation plan and the detailed timeline of the task sequence executed by each ferrobot 34 (in coordination with the other ferrobots 34) are illustrated in FIGS.13C-13F.
• the dilutive effect of sample pooling was evaluated on the assay detection capability (using positive nasal swab samples).
• Table 1 provides a detailed account of the number of droplet actuation and ferrobotic operations that were reliably carried out to achieve pooled testing. 2023-021-2 Table 1 [0080] Table 1: Breakdown of the ferrobotic operations and commuted pixels for 4 2 pooled testing. [0081] This was achieved by harnessing the competitive advantages of the ferrobotic technology that overcomes performance limits (in terms of reliability, scalability, reagent use, portability, etc.) and cost barriers of alternative microfluidics approaches.
• Table 2 Comparison of the cost of key materials of a low-cost EWOD chip vs. a generic ferrobotic chip. Abbreviations: polyvinyl alcohol (PVA), indium tin oxide (ITO), and polyethylene terephthalate (PET).
• PVA polyvinyl alcohol
• ITO indium tin oxide
• PET polyethylene terephthalate
• Table 3 Table 3: The list and quantity of constituent hardware components and estimated cost of a ferrobotic platform with N testing channels. ⁇ ⁇ denotes the ceiling function.
• a second substrate 30 in the form of disposable microfluidic chip or cartridge is disposed adjacent to the first substrate 12 as described previously and one or more permanent magnets 34 or ferrobots are controlled to manipulate volumes or droplets of ferrofluid 100 to perform one or more operations withing the microfluidic chip 30.
• a single permanent magnet 34 can be used to perform all of the device operations although it should 2023-021-2 be appreciated that a plurality of such permanent magnets 34 could also be employed.
• the device 10 is battery-operated and miniaturizes, integrates, and automates magnetic bead- based sample preparation and multiplexed nucleic acid amplification workflows that are currently performed manually in standard lab settings.
• the device 10 includes an optional display 70 that can be used to show test results to the user.
• the device 10 uses bead-based sample preparation and multiplexed nucleic acid amplification workflows to enable seamless low-cost testing against a panel of pathogens.
• the beads are magnetic beads that are functionalized to a target or target class of molecules or species.
• the target species may include nucleic acids, proteins, biomolecules, and the like.
• these magnetic beads differ from the magnetic particles 102 that are nanometer-scale particles within the droplets 100. These magnetic beads are larger in size such that a larger magnetic force can be applied to concentrate the beads to a smaller volume or location in the droplet 100. This enables the beads to enrich and concentrate target molecules from a sample.
• the beads are functionalized to bind to nucleic acids.
• the workflows are based on those currently performed manually following standard viral diagnostic practices and using commercially available reagents.
• bead-based sample preparation enhances the limit of detection (LoD) and allows for reliable analysis in multiple complex biomatrices including blood, saliva, and oral swab in viral transport media.
• LiD limit of detection
• 25A illustrate the second substrate or microfluidic chip 30 that includes a sample inlet 72 which may include a self-healing septum (e.g., vial stopper).
• a chamber 74 for storage of lysis buffer is provided along with wash buffer chambers 76 and an elution buffer chamber 78.
• a disposal buffer chamber 80 is provided for waste fluid storage.
• One or more assay chamber(s) 114 are located in the second substrate or microfluidic chip 30 and contain assay reagents therein.
• the one or more assay chambers 114 contain RT- LAMP reagents (e.g., primers) and/or buffers. Different assay chambers 114 may contain different RT-LAMP reagents and/or buffers specific to different targets.
• the different assay chambers 114 may contain test reagents or buffers specific to different viruses (e.g., Ebola Zaire (Filovirus), Ebola Sudan (Filovirus), Marburg (Filovirus), and 2023-021-2 Lassa Fever Virus (Arenavirus).
• the nucleic acid amplification workflow is based on the established LAMP methods allowing for simplifying the assay workflow and hardware requirements while delivering PCR-level performance. If needed PCR or other nucleic acid amplification workflows can also be miniaturized, integrated, and automated within the device 10.
• the magnetic beads are attracted to the magnetic field of the underlying permanent magnet 34 and are accumulated at the bottom surface and are retained in the trench recess 84 of the trench chamber 82 as the ferrofluid is pulled out of the trench chamber 82.
• other non-magnetic beads which are configured to bind biomolecule targets are introduced in the droplet 100.
• These non-magnetic beads comprise beads with a diameter that is larger than a gap size in the trench chamber 82, leading to accumulation and concentration of the non-magnetic beads, based on their larger size upon the movement of the ferrofluid droplet 100 through the trench chamber 82.
• FIG.25C illustrates a 3D view of a volume or droplet of ferrofluid 100 that contains the ferrofluid and reagents therein. Aliquoting is performed using the corrugated features 28. Fluorescent or colorimetric detection takes place in the assay chambers 114 using optical sensor(s) 48 as described herein.
• Single stranded RNA (ssRNA) fragments of SARS-CoV-2 (10 8 copies/ ⁇ L) were purchased from Sigma-Aldrich. Living E.coli K-12 strain (3 ⁇ 10 5 CFU/ ⁇ L) in liquid nutrient broth was purchased from Carolina Biological Supply (NC, USA).
• a SARS-CoV-2 Rapid Colorimetric RT-LAMP Assay Kit was purchased from New England Biolabs (NEB, MA, USA) and stored at -20°C.
• the Viral Transport Media (VTM) was purchased from BD (NJ, USA).
• the UCLA Clinical Microbiology Laboratory performed RT-PCR using the following assay: TaqMan COVID-19 RT-PCR Assay (ThermoFisher Scientific, Carlsbad, CA, USA).
• a microfluidic device 30 for merging and mixing was fabricated and assembled, with patterned electrocoalescence electrodes 36 (1 mm width, spaced 2 mm apart, thicknesses of 20 nm of Cr and 100 nm of Au) on PET substrate.
• the underlying ferrobot 34 was directed to induce chaotic fluid motion within the merged droplet 100 with different frequencies (0.2, 1, 3, 5 Hz).
• a video recording was taken for the mixing process, and the droplet homogenization rate was calculated through image processing.
• the video frames were imported into a MATLAB, and the pixel data (in grayscale) at the droplet region were extracted.
• a mixing index is defined, as expressed below: 00113] Mixing ⁇ 1 ⁇ ⁇ ⁇ ⁇ ⁇ [ ⁇ ⁇ ⁇ ⁇ ⁇ 2023-021-2 [00114] where ⁇ , ⁇ ⁇ , and ⁇ ⁇ are the total number of pixels, the grayscale values at pixel ⁇ , and the average grayscale values over ⁇ pixels, respectively. [00115] Characterization of long-term cyclic ferrobotic operations [00116] A microfluidic device 30 that contains two chambers and a connection channel in between was fabricated and assembled. The connection channel contains a corrugated wall feature 28 and a pair of merging electrodes 36 deposited on the PET substrate.
• the ferrobot 34 manipulated the ferro-droplet 100 periodically: dispense the droplet 100 into mother and daughter droplets 100 when transporting from the right chamber to the left chamber, and merge the mother droplet 100 with the dispensed droplet 100 when transporting from the left chamber to right chamber. These actions were repeatedly performed for more than 800 cycles. Images were taken during the whole process, and the dynamic variation of the droplet size was measured through image analysis.
• RT-LAMP characterization To detect RNA, RT-LAMP assays were conducted at room temperature.
• compositions of the assay are: 10 ⁇ L WarmStart Colorimetric RT-LAMP 2X master mix, 2 ⁇ L guanidine hydrochloride, 2 ⁇ L target RNA primer mix, 5 ⁇ L nuclease-free water and 1 ⁇ L input sample.
• the input sample contained ssRNA fragments of SARS-CoV-2 in various concentrations (0, 25, 100, 1000 copies/ ⁇ L) and 13% of ferumoxytol.
• the RT-LAMP assays were loaded in the assay chamber 114 and incubated at 65 °C for 30 minutes. The incubation process of the assay was recorded by video. After the RT-LAMP reaction, the color was quantitatively measured by the optical sensors 48.
• Each microfluidic chip 30 was preloaded with a ferrofluid droplet 100 (50% ferumoxytol) in the ferrofluid chamber 110 and RT-LAMP assay solution in the assay chamber 114 (reagent volume: 1.9 ⁇ L and 19 ⁇ L for analysis of 100-nL and 1 ⁇ L-aliquoted samples, respectively).
• the starting sample was pipetted into the microfluidic chip 30 at the sample holding chamber or region 112 (i.e., input chamber via the designated sample inlet). Specifically, 0.52 ⁇ L and 5.2 ⁇ L of starting samples were correspondingly used for subsequent aliquoting/analysis of 100-nL and 1 ⁇ L droplets.
• a microfluidic device 30 with a sample holding chamber or region 112 (i.e., input chamber), a ferrofluid chamber 110, an array of assay chambers 114, two pairs of merging electrodes 36 (patterned at the sample holding chamber or region 112 and across the array of assay chambers 114), and a dispenser array was fabricated and assembled.
• the dispenser array was 2023-021-2 formed by corrugated features 28 that function to create defined volumes or droplets 100 and/or sample.
• Each microfluidic chip 30 was preloaded with a ferrofluid droplet 100 in the ferrofluid chamber 110 and three 19- ⁇ L RT-LAMP reaction solutions, containing primers for SARS-CoV-2, H1N1 (Thermo Fisher Scientific, MA, USA), and internal control (NEB, MA, USA) respectively, in the array of assay chambers 114.
• a blank sample or negative nasal swab sample either with or without target was loaded into the microfluidic chip 30 at the sample holding chamber or region 112.
• Inactivation/lysis was then performed on the PCB 12 for 5 minutes by powering a 20-Ohm resistive heater 42 with 0.14 A DC current. Thereafter, a ferrobot 34 performed the sample processing steps of transportation, merging, mixing, aliquoting, disposal, and delivery to the assay chambers 114.
• Each RT-LAMP assay solution ended up receiving a 1 ⁇ L ferro-sample. Then, the on-chip RT-LAMP reaction (at 65 °C) continued for 30 min. The readout for each assay was measured by the optical sensing module 44.
• Ferrobotic pooled clinical sample testing For pooled tests of clinical samples, microfluidic devices 30 with a matrix array of sample holding chamber or regions 112, dispensers (formed using corrugated features 28), two arrays of assay chambers 114 and five pairs of merging electrodes 36 (patterned across the array of assay chambers 114 and mixing regions) were fabricated and assembled.
• the assay chambers 114 were preloaded with RT-LAMP assay solutions (reagent volume: 1.9 ⁇ L and 19 ⁇ L for 100-nL and 1 ⁇ L-aliquoted samples, respectively). A number of 3.5- ⁇ L heat- inactivated starting ferro-samples were loaded into the input chambers (9 for 3 2 , 16 for 4 2 pooling testing).
• ferrobots 34 performed the sample processing steps of several rounds of aliquoting, transportation, merging, mixing, and delivery to the corresponding assay chambers 114.
• the navigation planning of the ferrobots 34 accounted for the maintenance of an inter-ferrobot distance of 10 mm to avoid inter-ferrobot magnetic interference.
• the on-chip RT-LAMP reaction took place for 30 min (at 65 °C).
• the assay readout was measured by the optical sensing module 44.
• RT-LAMP validation in diluted clinical sample Five nasal swab samples (originally obtained from COVID-19 infected donors, pre-characterized via RT-PCR) with various Ct values (11, 15.7, 21.16, 24.97, and 28.95) were diluted in PBS with different dilution rates (4, 9, 16, and 25). Then, all the diluted and undiluted samples were tested by both standard off-chip RT-LAMP and on-chip RT-LAMP 2023-021-2 testing. The reaction products of standard off-chip RT-LAMP were visually recorded in tubes. The reaction products of on-chip individual RT-LAMP were visually recorded in the microfluidic chips 30, then quantitatively measured by the optical sensing module 44.
• the number of formed groups ( ⁇ equals to: ⁇ ⁇ ⁇ ⁇ (1) ⁇ ⁇ [00143] Random variables and their [00144] For a viral prevalence of ⁇ , one can assume the probability of a sample being positive is the same for each sample and equals to ⁇ , likewise the probability of one sample being negative equals to 1 ⁇ ⁇ . This situation can be modeled as a binomial trial (or Bernoulli trial), wherein each sample has two possible outcomes: “positive” or “negative”.
• a random variable ⁇ is defined as the number of positive samples in the pooled group (with corresponding possible values ⁇ ⁇ ⁇ 0, 1, 2, ... , ⁇ ).
• a random variable ⁇ was also defined which represents whether all the positive samples are in the same row/column or not ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ ⁇ ⁇ ).
• the probability of all samples in the pooled group of ⁇ samples being negative i.e., the number of positive samples is equal to zero
• the probability of at least one sample being positive in the pooled group can be expressed as: 2023-021-2 P ⁇ ⁇ ⁇ 0 ⁇ ⁇ 1 ⁇ ⁇ 1 ⁇ ⁇ (3)
• the probability of ⁇ being equal to a given value of ⁇ follows the binomial distribution: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 1 ⁇ ⁇ ⁇ ⁇ ⁇ (4) ⁇ ⁇ ⁇ [00148]
• ⁇ First round The samples in each group will be pooled as one sample, which is then tested by a dedicated assay (determining whether all samples are negative, or at least a positive sample is present). Since the first round is necessary for all groups, the number of tests in the first round ( ⁇ ⁇ ) always equals to: ⁇ ⁇ ⁇ ⁇ 1 (7) [00152] ⁇ Second round: The prerequisite for this round is at least one positive sample exists in the pooled group of samples ( ⁇ ⁇ 0). In this round, samples are pooled along ⁇ rows and ⁇ columns, leading to 2 ⁇ sample aggregates for testing.
• the number of tests in the second round ( ⁇ ⁇ ) can be expressed as: ⁇ ⁇ ⁇ ⁇ ⁇ 0, ⁇ ⁇ 0 ⁇ ⁇ ⁇ ⁇ (8) [00153] Then, the expected calculated as: E ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (9) ⁇ ⁇ ⁇ ⁇ ⁇ 0 ⁇ ⁇ ⁇ ⁇ 0 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 0 ⁇ ⁇ ⁇ ⁇ 0 ⁇ ⁇ ⁇ 0 ⁇ [00154] According to (2), (3) and (8): 2023-021-2 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 2 ⁇ ⁇ 1 ⁇ ⁇ 1 ⁇ ⁇ ⁇ (10) [00155] ⁇ Third round: The prerequisite for this round is ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ .
• the maximum number of tests in the third round ( ⁇ ⁇ ⁇ ) can be expressed as: ⁇ ⁇ 0, ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ min ⁇ ⁇ , ⁇ , ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (11) [00156]
• the expected number of tests in the third round ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ) can be approximated based on the derived expression for the maximum number of required tests in this round: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) [00159] [00160] The expected total number of tests: [00161] In the scope of testing the samples in each group, the expected total number of tests is the summation of the expected number of tests for each round of pooling: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ (14) 2023-021-2 ⁇ ⁇ 1 ⁇ 2 ⁇ 1 ⁇ ⁇ 1 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
• droplet kinematics are determined by three forces: a magnetic body force ⁇ ⁇ acting on the ferrofluid droplet 100, a friction force between the droplet and channel surface ⁇ ⁇ , and a drag force on the droplet in an oil environment ⁇ ⁇ .
• a magnetic body force ⁇ ⁇ acting on the ferrofluid droplet 100 a friction force between the droplet and channel surface ⁇ ⁇
• a drag force on the droplet in an oil environment ⁇ ⁇ Droplet deformation by shear was ignored because of the small capillary number (an indicator of the relative strength of viscous forces in the presence of surface tension; here ⁇ 0.01, Table 5).
• the magnetic body force can be expressed as: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (17) permeability of free space, and ⁇ is the magnetic flux density.
• ⁇ is the magnetic 2023-021-2 susceptibility (proportional to ferrofluid concentration), which can be equivalently expressed as ⁇ ⁇ ⁇ ⁇ ⁇ ( ⁇ : volume ratio of ferrofluid; ⁇ ⁇ : magnetic susceptibility of 100% ferumoxytol).
• the frictional force between the ferrofluid droplet and channel surface is on the order of: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 18 ⁇ [00169] where ⁇ ⁇ is the friction constant, ⁇ ⁇ is the radius of the contact area between ferrofluid droplet and channel surface, ⁇ ⁇ is the viscosity of the oil lubrication layer, and ⁇ is the velocity of the carrier (assuming that viscous drag in the vicinity of the contact line is significant).
• drag force is on the order of: ⁇ ⁇ 3 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 19 ⁇ ⁇ ⁇ ⁇ ⁇ [00171]
• the driving force ⁇ ⁇ is counteracted by the restraining forces ⁇ ⁇ and ⁇ ⁇ , establishing an upper-bound velocity (i.e., terminal velocity) for the droplet motion, which can be expressed as: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ [00173] Rearranging maximum velocity and the viscosity of the surrounding oil.
• the interface between two water-phase droplets in the oil (containing surfactant) are determined by three pressures (FIG.21A): an electric compression pressure ⁇ ⁇ and a Laplace pressure ⁇ ⁇ that squeeze the water-oil interfaces and induce merging, as well as a repulsive disjoining pressure ⁇ (originating from surfactant molecules aggregating at the interface, countering the direct contact of the droplets and merging).
• the electric compression pressure at the water-oil interface can be expressed as: ⁇ ⁇ ⁇ ⁇ ⁇ (23) [00181] is the applied voltage, h is the thickness of the oil film.
• the Laplace pressure at the water-oil interface can be expressed as: ⁇ ⁇ ⁇ ⁇ ⁇ 24 ⁇ [00183] where ⁇ is the radius of the droplet, ⁇ is the surface tension of the interface. 2023-021-2 [00184]
• the electrocoalescence of two droplets occurs when the combined electric compression pressure ⁇ ⁇ and Laplace pressure ⁇ ⁇ exceed the upper bound of the disjoining pressure ⁇ (i.e., threshold disjoining pressure, ⁇ ⁇ ⁇ : ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 25 ⁇ [00185]
• the effect of Laplace pressure can be neglected here, given that it is much smaller than the electric compression pressure in the experiment setting: ⁇ ⁇ / ⁇ ⁇ ⁇ 183 (assuming ⁇ ⁇ ⁇ 5.8, characteristic applied voltage ⁇ ⁇ 1 V, characteristic oil film thickness h ⁇ 100 nm, surface tension ⁇ ⁇ 7 mN/m, characteristic droplet radius ⁇ ⁇ 1 mm).
• the lower bound of applied voltage ⁇ i.e., threshold voltage, ⁇ ⁇
• ⁇ ⁇ threshold voltage
• the threshold disjoining pressure ⁇ ⁇ and correspondingly ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , increase with the increasing concentration of the introduced surfactant. This is in line with the trend observed in the experimental results shown in FIG.2C.
• the automated platform was adapted to simultaneously test for the presence of SARS- CoV-2, Influenza H1N1, and rActin (an endogenous housekeeping gene, practically serving as an internal control).
• the disposable microfluidic chip 30 layout was gloryd. Specifically, the microfluidic chip 30 was expanded to house three aliquoting interfaces 2023-021-2 formed by the corrugated features 28 (e.g., three such corrugated features 28 formed in a wall) and three assay chambers 114 (FIG.11A). In this customized microfluidic chip 30, the assay chambers 114 were pre-filled with the RT-LAMP assay solutions containing the primer sets specific to the assigned targets.
• the assay reagents may also be provided in dry form in some embodiments.
• the instruction set was updated to accommodate for the ferrobotic production of three aliquots from a single sample and delivery of each of the aliquots to a designated assay chamber 114 (FIGS.11B, 11C).
• the suitability of the platform was validated for multiplexed testing, by successfully differentiating different combinations of spiked input samples (FIG.11D).
• FIG.11D [00191]
• Competitive advantages of Ferrobotics [00192] The competitive advantages of the ferrobotic technology are rooted in the electronically programmable nature of the platform, strong, contactless magnetic droplet actuation mechanism that it uniquely employs for liquid handling (which is in principle, battery-operable; FIG.23B).
• this technology bypasses the fundamental limitations of magnetic droplet microfluidics approaches that either 1) use complex translational stages (requiring robotic arms for automation) and bulky magnets that are not scalable/portable or 2) use standalone electromagnetic coils to directly actuate the droplets, thus lacking the ample driving forces necessary to execute fluid operations in a rapid and robust manner (leading to two orders of magnitude weaker actuation forces compared to the ferrobotic equivalent; FIGS.22A-22C).
• the employed contactless magnetic actuation mechanism of the ferrobotic technology allows bypassing the reliability issues encountered in EWOD approaches (including surface breakdown, electric charging, and surface hydrophobicity loss caused by ionic droplets).
• the ferrobotic technology uses electronically-driven PCBs 12 and millimeter-sized magnets 34.
• Considerations for operational scalability and miniaturization For the demonstrated application herein, scaling up to larger 2D array sizes for manipulating a higher number of samples was limited by sample over-dilution.

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