WO2023195977A1 - Dispositifs microfluidiques numériques dotés de substrats de luminescence à surface améliorée - Google Patents

Dispositifs microfluidiques numériques dotés de substrats de luminescence à surface améliorée Download PDF

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Publication number
WO2023195977A1
WO2023195977A1 PCT/US2022/023474 US2022023474W WO2023195977A1 WO 2023195977 A1 WO2023195977 A1 WO 2023195977A1 US 2022023474 W US2022023474 W US 2022023474W WO 2023195977 A1 WO2023195977 A1 WO 2023195977A1
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fluid
electrodes
chamber
examples
dmf
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PCT/US2022/023474
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English (en)
Inventor
Viktor Shkolnikov
Raghuvir N. SENGUPTA
Steven Barcelo
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Hewlett-Packard Development Company, L.P.
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Priority to PCT/US2022/023474 priority Critical patent/WO2023195977A1/fr
Publication of WO2023195977A1 publication Critical patent/WO2023195977A1/fr

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    • 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
    • B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • 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
    • B01L3/50273—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 the means or forces applied to move the fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • 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
    • 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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules

Definitions

  • Digital microfluidic (DMF) devices may be used to perform operations on volumes of fluid, such as the manipulation of fluid droplets to facilitate handling and testing of various fluids on a small scale.
  • DMF Digital microfluidic
  • Such devices may be used in the medical industry, for example to analyze proteins, analyze deoxyribonucleic acid (DNA), detect pathogens, perform clinical diagnostic testing, and/or for synthetic chemistry, among other types of industries and/or for other purposes.
  • sensors may form part of or couple to the DMF devices to detect the presence of certain molecules in a fluid.
  • the sensors may measure a concentration of a particular molecule.
  • Some sensors may be plasmonic sensors that are based on surface-enhanced Raman spectroscopies (SERS) or surface plasmon resonances (SPR).
  • FIGS. 1A-1G illustrate example digital microfluidic (DMF) devices, in accordance with examples of the present disclosure.
  • FIG. 2 illustrates another example DMF device, in accordance with examples of the present disclosure.
  • FIG. 3 illustrates an example apparatus including a DMF device, an optical sensing device, and coupled circuitry, in accordance with examples of the present disclosure.
  • FIGS. 4A-4H illustrate operation of an example DMF device, in accordance with examples of the present disclosure.
  • [0007JFIG. 5 illustrates an example functionalized magnetic bead and operation thereof in a DMF device, in accordance with examples of the present disclosure.
  • [0008JFIG. 6 illustrates an example method for isolating target molecules using a DMF device, in accordance with examples of the present disclosure.
  • Digital microfluidic (DMF) devices may be used to perform large numbers of chemical processing operations on different fluids in parallel by providing digitized movement of reaction fluids throughout the DMF devices.
  • the digitized movement of reaction fluids may be achieved using a plurality of electrodes that form part of or are coupled to the DMF device, and which provide an electric field to drive the flow of reaction fluids as fluid droplets.
  • the electrodes may be individually addressed by circuitry coupled to or forming part of the DMF device, sometimes herein referred to as “selective actuation”, to provide the digitized and controlled flow of picoliter to microliter sized fluid droplets of reaction fluids by drawing the fluid droplets toward an addressed electrode.
  • the addressed electrode provides an electric field within the DMF device and/or onto the reaction fluid, and due to a charge of the reaction fluid, a fluid droplet of the reaction fluid is directed along a microfluidic path.
  • the electrodes may be arranged in an array and are selectively addressable to provide a plurality of different microfluidic paths within the DMF device. For example, respective ones of the plurality of electrodes may be sequentially actuated to draw the fluid droplet of the reaction fluid along a respective microfluidic path.
  • the movement of the fluids within the DMF device may be used to move, mix, and/or split fluid droplets of reaction fluids into two respective smaller fluid droplets, among other uses, and to drive a chemical processing operation thereon.
  • DMF devices may be designed to implement biochemical reactions on chemical components within a sample carried in a fluid by performing chemical processing operations on the sample.
  • a sample fluid includes and/or refers to any biological material collected, such as from a subject or other source, and carried in a fluid.
  • the sample fluid may include a sample obtained from an organism, a liquid source, such as milk or other liquid source to be tested (e.g., public water sample, sample from a lake or other water source, ground water, milk), and/or a solid dispersed in liquid, such as a soil sample.
  • the sample fluid may contain a mixture of a plurality of different molecules of different sizes and/or with different chemical properties.
  • Example molecules include antibodies and/or other proteins, nucleic acids, amino acids, sugars, fats, cells, antibiotics, toxic chemicals, and/or non-toxic chemicals, among other molecules in the mixture.
  • a portion of the different molecules in the mixture may be of interest for further processing and/or for assessment, herein referred to as a “target molecule”.
  • Separating molecules within the sample fluid based on size and/or chemical properties may be useful for providing accurate molecule sensing using sensitive optical detection, such as with surface enhanced Raman spectroscopy (SERS) which may detect target molecules at concentrations below a threshold. Without properly isolating target molecules from the sample, the optical detection may be inaccurate.
  • SERS surface enhanced Raman spectroscopy
  • optical sensing techniques including SERS
  • SERS are unable to or may have difficulty identifying a target molecule below a threshold concentration in a mixture of a plurality of different molecules as the other non-target molecules in the mixture may be optically active (e.g., Raman active), and may obscure the optical detection results.
  • optically active e.g., Raman active
  • Various examples include a DMF device that performs sample preparation by isolating target molecules from other molecules within a sample fluid based on size and/or chemical properties using functionalized magnetic beads, and provides the target molecules to a surface-enhanced luminescence substrate for assessment by an optical sensing device.
  • the DMF device reduces the laborious process of manually handling different reaction fluids. More specifically, as sample preparation is performed on the DMF device and the surface-enhanced luminescence substrate is located on the DMF device, the target molecules may be isolated from the other molecules within a sample fluid and optically interrogated within the DMF device without or with minimal manual handling of reaction fluids and/or user manual operation of the DMF device.
  • the integrated sample preparation and isolation of target molecules may mitigate contamination risk and user errors, while allowing for sensitive detection of target molecules by a coupled optical sensing device.
  • variations in sample fluid preparations may be amplified in the optical results.
  • the DMF device allows for flexibility in the sample preparation technique, including but not limited to, adjusting parameters of the sample preparation performed based on variations in the sample. For example, a first (default) sample preparation may be performed, and based on the optical results (e.g., spectra peaks), a different sample preparation may be performed on the remaining portions of the sample fluid.
  • Such DMF device and flexibility may allow for greater control of fluid droplets of reaction fluids and exposure to different portions of the DMF device.
  • the surface-enhanced luminescence substrate may be located in a well fluidically coupled to a portion of the DMF device (e.g., a chamber) used to perform the sample preparation, which allows for implementing a wide variety of different types of surface-enhanced luminescence substrates, including different SERS substrates.
  • the DMF device may be a single use device, sometimes referred to as a consumable or disposable device, used to perform the sample preparation and isolation of the target molecule(s) in an integrated manner by digitized fluid operations and in an inexpensive manner.
  • Examples of the present disclosure are directed to a DMF device comprising a chamber disposed within a housing, fluidic inlets to input different reaction fluids, a magnetic unit disposed along the microfluidic path, and a surface-enhanced luminescence substrate.
  • the different reaction fluids may be inserted into the DMF device via the fluidic inlets and are selectively carried through a microfluidic path.
  • the plurality of reaction fluids include a sample fluid containing a sample and buffer fluids, such as a buffer fluid containing functionalized magnetic beads, wash buffer, and elution buffer.
  • the magnetic beads may be functionalized to bind to a target molecule based on size and/or chemical properties of the molecule.
  • Electrodes may be coupled to the housing to selectively move fluid droplets of the plurality of reaction fluids along the microfluidic path.
  • the selectively movement may include sequential movement of respective fluids to isolate target molecules and flow the isolated target molecules to the surface-enhanced luminescence substrate for interrogation.
  • Different types of molecules may be detected in the sample using the DMF device, such as antibiotics, toxins (e.g., polychlorinated biphenyl (PCB), polycyclic aromatic hydrocarbons, and dioxins), nucleic acids, and/or metabolites, among other molecules and uses.
  • a chamber refers to and/or includes an enclosed and/or semi-enclosed region of the DMF device, which may be formed of an etched or micromachined portion (e.g., negative space forming a conduit in a substrate or substrates) and which may be used to perform chemical processing on fluids therein.
  • an etched or micromachined portion e.g., negative space forming a conduit in a substrate or substrates
  • the fluid droplet of a reaction fluid may include a volume of about 1 microliter (pL) or less, such as a volume of between about 0.1 pL and about 1 pL, about 0.25 pL and about 1 pL, about 0.5 pL and about 1 pL, about 0.5 pL and about 0.75 pL, about 0.25 pL and about 0.75 pL, or about 0.1 pL and about 0.5 pL, among other ranges.
  • a channel refers to and/or includes a path through which a fluid or semi-fluid may pass, which may allow for transport of volumes of fluid on the order of pL, nanoliters, picoliters, or femtoliters.
  • a well such as a reaction fluid well, refers to and/or includes a column capable of storing a volume of fluid.
  • the well may store a volume of fluid that includes more than one droplet of fluid, such as at least two fluid droplets of a reaction fluid.
  • a well may store a volume of fluid in a range between about 1 pL and about several milliliters (mL) of fluid.
  • the well may store a volume of fluid between about 1 pL and about 1 mL, about 1 pL and about 500 pL, about 1 pL and about 50 pL, about 1 pL and about 10 pL, or about 1 pL and about 5 pL, among other volume ranges.
  • [0015JA reaction fluid refers to and/or includes fluid containing substances, molecules, mixtures, and/or other components used to drive a biochemical reaction, such as for isolating and/or detecting a presence of a target molecule in a sample fluid.
  • a fluid droplet of a reaction fluid refers to and/or includes a discrete portion of fluid (e.g., a liquid), which may be surrounded by a carrier fluid.
  • a carrier fluid refers to and/or includes fluid that flows through portions of the DMF device and which carries solid and/or fluid particles, such as fluid droplets of the reaction fluids.
  • an immiscible fluid such as an aqueous solution
  • Fluid droplets of reaction fluids may be formed from a fluid packet of the reaction fluid.
  • a fluid packet of the reaction fluid refers to and/or includes a volume of fluid that is larger than a fluid droplet of the reaction fluid.
  • a functionalized magnetic bead refers to and/or includes a bead having magnetic properties and/or is otherwise capable of being attracted to or repelled by a magnetic field.
  • a bead refers to and/or includes a material formed in a three-dimensional shape, such as a sphere, an ellipsoid, oblate spheroid, and prolate spheroid shapes.
  • the functionalized magnetic beads may be between 1 micrometer (pm) and 20 millimeter (mm) in diameter as non-limiting examples.
  • a magnetic unit refers to and/or includes circuitry and/or a physical structure that causes or outputs a magnetic field.
  • the surface-enhance luminescence substrate refers to and/or includes a substance or material in a layer that includes a surface with metal nanostructures or a rough surface.
  • the nano-structures may include spheroidal gold, silver, platinum, or copper nanoparticles with a diameter of 30 to 100 nanometers (nm). However, other shapes, sizes, or materials, may be used.
  • the nano-structures may enhance scattering of light from molecules for detection by an optical sensing device.
  • the rough surface may include a surface roughness of between about 100 nanometers (nm) to about 1000 nm, and/or having sub-10 nm edges or gaps. In some examples, the rough surface may have between about sub-10 nm and about sub-2 nm gaps.
  • a DMF device comprising a housing that defines a microfluidic path including a chamber and with a plurality of electrodes coupled to the housing.
  • the DMF device further comprises a plurality of fluidic inlets fl uidically coupled to the chamber to input a plurality of reaction fluids including a functionalized magnetic bead and a sample fluid, a magnetic unit disposed along a portion of the microfluidic path associated with the chamber, and a surface-enhanced luminescence substrate fluidically coupled to the microfluidic path.
  • the DMF device further includes circuitry communicatively coupled to the plurality of electrodes and the magnetic unit to selectively actuate the plurality of electrodes and the magnetic unit to move fluid droplets of the plurality of reaction fluids along the microfluidic path.
  • the plurality of electrodes may be disposed on or supported by the housing in some examples. In other examples, the plurality of electrodes may be disposed on a substrate of another device coupled to the housing.
  • the DMF device may be communicatively coupled to the plurality of electrodes via an anisotropic decoupling layer of the DMF device.
  • the housing includes a base substrate, wherein the plurality of electrodes are coupled to the base substrate.
  • the housing further includes a top substrate, the chamber including a bottom surface defined by the base substrate and a top surface defined by the top substrate. And, a carrier fluid is contained within the chamber.
  • the top substrate may include or form part of a lid of the DMF device.
  • the DMF device may include a transparent lid and the plurality of fluidic inlets are disposed on and through the transparent lid.
  • the DMF device further includes a plurality of reaction fluid wells to contain the plurality of reaction fluids and fluidically coupled to the plurality of fluidic inlets.
  • the reaction fluid wells may be supported by or disposed within the housing.
  • the plurality of reaction fluid wells may be coupled to blister packs containing the plurality of reaction fluids and which provide the plurality of reaction fluids to the plurality of reaction fluid wells.
  • the DMF device includes a housing that defines a microfluidic path including a chamber, a magnetic unit disposed along the microfluidic pathway within the chamber, and a surface-enhanced luminescence substrate coupled to the chamber to isolate a target molecule in a sample fluid.
  • the circuitry is communicatively coupled to the magnetic unit and the plurality of electrodes to selectively actuate electrodes of the plurality of electrodes to move fluid droplets of a plurality of reaction fluids along the microfluidic path, the plurality of reaction fluids including the sample fluid, and selectively actuate the magnetic unit to move the functionalized magnetic bead toward the magnetic unit and facilitate bead-based separation of molecules in the sample fluid including the target molecule.
  • the optical sensing device is to interrogate the target molecule isolated on the surface-enhanced luminescence substrate.
  • the functionalized magnetic bead is enveloped by a functional layer and is porous, and is to separate the molecules in the sample fluid based on size, chemical properties, or a combination thereof.
  • the functionalized magnetic bead includes a carboxylate group, a quaternary ammonium group, or a C18 tail.
  • the surface-enhanced luminescence substrate comprises a surface enhanced Raman spectroscopy (SERS) substrate and the optical sensing device includes a Raman spectrometer.
  • a SERS substrate may include metal nano-structures or a metal rough surface, as described above, that enhance Raman scattering from molecules.
  • Raman scattering includes scatter radiation that has a frequency that is different than incident radiation.
  • the Raman scattering may be in a spectra range of about 10 centimeter (cm)' 1 to about 4000 cm -1 wavenumber shifts, about 100 cm -1 to about 4000 cm -1 , or about 100 cm -1 to about 3200 cm -1 , among other wavenumber shift ranges.
  • the SERS substrate may include metal nano-structures or a metal rough surface with sub-10 nm gaps or less, such as between about sub- 10 nm and about sub-2 nm gaps. In some examples, the SERS substrate includes metal nano-structures or a metal rough surface with about sub-2 nm gaps or less. In some examples, the SERS substrate may include metal nanostructures that are pillars. The pillars may be about 1 pm in height, about 100 nm in diameter, and are capped with an Au nano-particle approximately sphere in shape that is about 100 nm in diameter. Such pillars may be arranged in a pentamer structure, and collapse on each other due to capillary forces, when fluid is introduced to the SERS substrate. However, examples are not so limited. In some examples, the nano-structures of the SERS substrate may have a diameter between about 20 nm and about 200 nm.
  • the DMF device further includes a plurality of fluidic inlets fizid ically coupled to the chamber to input the plurality of reaction fluids including a fluid containing the functionalized magnetic bead.
  • the chamber contains a carrier fluid
  • the circuitry is to selectively actuate electrodes of the plurality of electrodes to form the fluid droplets as surrounded by the carrier fluid.
  • An example method comprises flowing a first fluid droplet of a sample fluid along a microfluidic path within a chamber of a DMF device via application of electrowetting forces by a plurality of electrodes, and merging the first fluid droplet of the sample fluid with a second fluid droplet of buffer fluid containing a functionalized magnetic bead to form a merged fluid droplet, wherein a target molecule in the sample fluid is to bind to the functionalized magnetic bead.
  • the method further includes applying a magnetic field to the merged fluid droplet via a magnetic unit disposed along the microfluidic path, and directing molecules in the sample fluid not bound to the functionalized magnetic bead to a waste reservoir.
  • the method further includes separating the target molecule from the merged fluid droplet containing the target molecule and functionalized magnetic bead, flowing the target molecule to a surface-enhanced luminescence substrate fluidically coupled to the microfluidic path, and interrogating the target molecule using an optical sensing device coupled to the surface-enhanced luminescence substrate.
  • the method further includes repeating cycles of turning off the magnetic field, allowing the functionalized magnetic bead to mix with additional fluid droplets of wash buffer fluid, and moving the additional fluid droplets of the wash buffer fluid and respective unbound molecules of the sample fluid to the waste reservoir.
  • separating the target molecule includes flowing a third fluid droplet of elution buffer fluid along the microfluidic path and merging the third fluid droplet of elution buffer fluid with the merged fluid droplet to displace the target molecule from the functionalized magnetic bead.
  • the term “merged” or a merged fluid droplet includes and/or refer first fluid droplet of a reaction fluid combining with another fluid droplet of another reaction fluid.
  • the method further includes applying a second magnetic field to the functionalized magnetic bead with the target molecule displaced to trap the functionalized magnetic bead prior to flowing the target molecule to the surface-enhanced luminescence substrate.
  • FIGs. 1A-1G illustrate example DMF devices, in accordance with examples of the present disclosure.
  • an example DMF device 100 comprises a housing 102 that defines a microfluidic path 105 including a chamber 104 and with a plurality of electrodes 106 coupled to the housing 102.
  • the housing 102 may include substrates, with the chamber 104, among other components, formed by and/or between the substrates as etched or micromachined portions.
  • the etched or micromachined portions forming the chamber 104, and optionally additional chambers, wells, and/or channels may be a height in the range of about 10 pm to about 2 mm.
  • the chamber 104 may be formed of a plurality of different materials which are in layers, e.g., layers of substrates, in stack, as further described herein.
  • the chamber 104, and optionally other chambers, wells, and channels may be formed by etching or micromachining processes in a substrate to form the various etched or micromachined portions. Accordingly, the chamber(s), wells, and/or channels may be defined by surfaces fabricated in the substrate(s) of the DMF device 100.
  • the plurality of electrodes 106 are coupled to the housing 102 and may be disposed proximal the microfluidic path 105 and the chamber 104. In some examples, the electrodes 106 are positioned along and/or exposed to the chamber 104. In other examples, the electrodes 106 are coupled to the housing 102, as further described herein.
  • Proximal as used herein (e.g., proximal to the microfluidic path 105 an/or the chamber 104), refers to and/or includes being disposed in line with a portion of the DMF device 100, such as being positioned along, above, below, and/or exposed to the portion of the DMF device 100.
  • the plurality of electrodes 106 are to actuate to selectively move a plurality of reaction fluids along microfluidic path 105.
  • Example electrodes include transparent electrodes, ring electrodes, linear electrodes, almost continuous electrodes, ground electrodes, and/or actuating electrodes, among others.
  • the plurality of electrodes 106 may be the same size or different sizes.
  • the electrodes may be formed of a conductive material, such as metal, conductive polymers, indium tin oxide (ITO), transparent conductive oxides, carbon nanotube, among other material.
  • a transparent electrode refers to and/or includes an electrode that is transparent or semi-transparent. Use of transparent electrodes, along with a transparent lid, as further described below, may allow for a user to visually view fluid flow within the DMF device 100 while chemical operations are being performed by the DMF device 100 and/or to verify proper fluid processing is occurring.
  • a ring electrode refers to and/or includes an electrode which is annulus shaped. The ring electrode(s) may be shaped to extend around a portion of the DMF device 100, such as a portion of the chamber 104.
  • a linear electrode refers to and/or includes an electrode which extends in a straight line and for a sub-portion of the DMF device 100.
  • a plurality of linear electrodes may be placed in an array along a portion of the DMF device 100 (e.g., a portion of the chamber 104), and may provide greater control of fluid flow, as compared to an almost continuous electrode, due to known electrode positions and localized resolution.
  • An almost continuous electrode refers to and/or includes an electrode which extends along a portion of the DMF device 100, such as along the bottom surface or top surface of the chamber 104 (e.g., see electrode 106-5 of FIG. 1 B).
  • An almost continuous electrode may reduce manufacturing costs, as compared to an array of linear electrodes.
  • a ground electrode refers to and/or includes an electrode that provides or establishes a connection to ground.
  • An actuating electrode refers to and/or includes an electrode that is actuated (e.g., a voltage is applied thereto by coupled circuitry), and in response, generates an electric field based on a differential between the actuating electrode (e.g., the applied voltage) and ground.
  • ground may be provided by a ground electrode, and in other examples, ground is provided by fluid within the DMF device 100.
  • Use of a ground electrode may provide greater control of fluid flow and/or formation of a fluid droplet of the reaction fluids as compared to use of fluid within the chamber 104 as ground. Using fluid as ground may reduce manufacturing costs.
  • the DMF device 100 further includes a plurality of fluidic inlets 110 fluidically coupled to the chamber 104.
  • a fluidic inlet refers to and/or includes an inlet port, e.g., an aperture, that is fluidically coupled to the chamber 104.
  • the plurality of fluidic inlets 110 may be used to input a plurality of reaction fluids into the DMF device 100.
  • the plurality of reaction fluids may include a functionalized magnetic bead and a sample fluid.
  • the plurality of reaction fluids include the sample fluid and a plurality of buffer fluids.
  • the buffer fluids may include a buffer fluid containing the functionalized magnetic bead, a wash buffer fluid, and an elution buffer fluid.
  • the buffer fluid and/or fluid droplet of the buffer fluid contains a plurality of functionalized magnetic beads.
  • Buffer fluids refer to and/or include fluids which assist in maintaining a pH within the fluids, such as mitigating or resting pH changes and/or maintaining the pH within a range.
  • Example buffer fluids include a solution with a weak base or acid, such as a solution containing citrate, acetate, or phosphate salts.
  • Example buffer fluids containing the functionalized magnetic beads include a phosphate buffered saline or a carbonate-bicarbonate buffer, among other buffer fluids.
  • buffer fluids include fluids containing salt such as 2-(N- morpholino)ethanesulfonic acid (MES), Bis-tris methane, 2,2’,2"-Nitrilotriacetic acid (ADA), Bis-tris propane, and/or piperazine-N,N'-Bis(2-ethanesulfonic acid) (PIPES).
  • salt such as 2-(N- morpholino)ethanesulfonic acid (MES), Bis-tris methane, 2,2’,2"-Nitrilotriacetic acid (ADA), Bis-tris propane, and/or piperazine-N,N'-Bis(2-ethanesulfonic acid) (PIPES).
  • MES 2-(N- morpholino)ethanesulfonic acid
  • ADA 2,2’,2"-Nitrilotriacetic acid
  • PPES piperazine-N,N'-Bis(2-ethanesulfonic acid)
  • buffer fluids include fluids containing tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2- hydroxyethyl)amino)acetic acid (Bicine), tris(hydroxymethyl)aminomethane, or 2-amino-2-(hydroxymethyl)propane-1 ,3-diol) (Tris), N- [tris(hydroxymethyl)methyl]glycine (Tricine), 3-[N- tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-[[1 ,3-dihydroxy-2- (hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N- morpholino)propanesulfonic acid (MOPS), 2-Hydroxy-3- morpholinopropanesulfonic acid (M
  • the functionalized magnetic bead(s) may have functionalized surfaces to selectively bind to a target molecule in the sample fluid and not bind to other molecules in the sample fluid.
  • the functionalized magnetic beads may be used to segregate the target molecules from the other molecules in the sample fluid based on size, chemical properties, and/or combinations thereof, sometimes herein referred to as “bead-based separation of molecules”.
  • the functionalized magnetic bead(s) may be of a size such that the beads are capable of moving through the chamber 104.
  • the functionalized magnetic bead(s) may be between 1 pm and 20 mm in diameter as non-limiting examples.
  • the functionalized magnetic bead(s) may be formed of, for example, glass, polymer, silica, alumina, silicon carbide, tungsten carbide iron oxide steel, silica coated metal, boron nitride, or other suitable material which is magnetic, is made with magnetic atoms, and/or includes a core with a magnetic coating.
  • example functionalized magnetic bead(s) may consist essentially of iron oxide, a soft ferrite, a ferromagnetic material, a ferrimagnetic material, and/or combinations thereof.
  • Non-limiting example compositions of functionalized magnetic bead(s) include iron oxide (Fe2Os), soft ferrites ranging from spinel-type ferrites (MeFe2O4) to manganese-zinc ferrite (MnaZn ⁇ i-a)Fe2O4), nickel-zinc ferrite (NiaZn ⁇ i-a)Fe2O4), and a nickel-iron alloy (Ni-Fe (80:20)), among others.
  • the functionalized magnetic bead(s) may be spherical, such as beads, or may not be spherical, such as disk-shaped, rock or gravel-like, or other suitable shapes.
  • the functionalized magnetic bead(s) may be monodispersed or poly-dispersed.
  • the functionalized magnetic bead(s) may include a core formed of a non-magnetic material and a magnetic coating, such as a tungsten carbide core and an iron oxide coating as a non-limiting example.
  • the core may increase the density of the functionalized magnetic bead(s) and the magnetic coating provides the magnetic properties.
  • Example wash buffer fluid includes deionized water or another buffer fluid, such as those described above. As further described herein, the wash buffer fluid may be used to wash away molecules that are not bound to the functionalized magnetic bead.
  • Example elution buffer fluid includes acetonitrile, ethanol, and hexane. In some examples, the elution buffer fluid includes acetonitrile. The elution buffer fluid may be used to elute and/or displace the bound target molecule from the functionalized magnetic bead.
  • the DMF device 100 further includes a magnetic unit 108 disposed along a portion of the microfluidic path 105.
  • the magnetic unit 108 may include a magnet that provides a magnetic field and electrical connects which may couple to circuitry, such as the circuitry 103 illustrated by FIG. 1 B.
  • the magnet of the magnetic unit 108 includes an electromagnet which is selectively actuated to output a magnetic field used to attract the functionalized magnetic bead, as further described below.
  • the electromagnet of the magnetic unit 108 may be actuated in response to an electrical signal (e.g., a voltage) applied thereto, and in response, outputs the magnetic field.
  • the magnetic field may be subsequently deactivated or removed by removing the electrical signal.
  • the magnet of the magnetic unit 108 includes a permanent magnet, and the magnetic unit 108 further includes or is coupled to a stage or other movable hardware that moves the permanent magnet to different positions.
  • the permanent magnet may be moved positions to provide a magnetic field within the chamber 104 that is sufficient to attract the functionalized magnetic bead and then moved to a position that the magnetic field within the chamber 104 is insufficient to attract the functionalized magnetic bead.
  • the DMF device 100 further includes a surface-enhanced luminescence substrate 112 fluidically coupled to the microfluidic path 105.
  • the surface-enhanced luminescence substrate 112 may include a surface with a plurality of nano-structures or a rough surface.
  • the surface-enhanced luminescence substrate 112 includes the plurality of nano-structures with diameters between about 1 pm and about 20 mm.
  • the target molecule may be isolated from the other molecules of the sample fluid and flown to the surface-enhanced luminescence substrate 112 for subsequent interrogation.
  • the interrogation as further described below, may include illuminating the surface-enhanced luminescence substrate 112 with a light source and measuring a response.
  • the nano-structures may include metal particles that may amplify optical response, such as amplifying a Ramen spectral response.
  • the nano-structures may be spheroidal gold, silver, platinum, or copper nanoparticles with a diameter of between about 3 nm to 100 nm.
  • other shapes, sizes, or materials may be used.
  • the shape, size, and material of the nano-structures may affect the resonant frequency of the nano-structures. Thus, variations in these values may cause variations in the spectral response observed during testing.
  • the DMF device 100 illustrated by FIG. 1A may include various variations, some of which are illustrated by FIGs. 1 B-1G.
  • Each of the DMF devices of FIGS. 1 B-1G include an implementation of the DMF device 100 of FIG. 1A, including at least some of the same features and components, as illustrated by the common numbering. The common features and components are not repeated for ease of reference.
  • FIG. 1 B-1C illustrate an example implementation of the DMF device 100 of FIG. 1A.
  • FIG. 1 B is a cross-sectional view of the chamber 104 of the example implementation of the DMF device 100
  • FIG. 1C is a top view of the example implementation.
  • the housing 102-1 , 102-2 of the DMF device 100 includes a base substrate 102-1 and a top substrate 102-2.
  • the top substrate 102-2 may form or include a lid of the DMF device 100.
  • the top substrate 102-2, or a portion thereof, may be transparent.
  • the top substrate 102-2 may be transparent, and, in other examples, both the top and base substrates 102-1 , 102-2 are transparent.
  • a transparent substrate(s) (and optionally electrodes) may allow for optical monitoring of fluid flow and/or chemical operations within the DMF device 100 by a user, which may be used to visually verify the DMF device 100 is functioning properly.
  • the chamber 104 include a bottom surface 109 defined by the base substrate 102-1 and a top surface 107 defined by the top substrate 102-2.
  • a bottom surface of the chamber refers to and/or includes a floor or lower surface of the chamber with respect to gravity.
  • a top surface of the chamber refers to and/or includes a ceiling or overhead surface of the chamber with respect to gravity.
  • the plurality of electrodes 106-1 , 106-2, 106-3, 106-4, 106-5 of the DMF device 100 may be coupled to the base substrate 102-1 .
  • the plurality of electrodes 106 are disposed on or within the base substrate 102-1.
  • the plurality of electrodes 106 may extend level with or extrude above the bottom surface 109 of the chamber 104 as defined by the base substrate 102-1 , such that the electrodes 106 may be in contact with fluids contained in the chamber 104.
  • the plurality of electrodes 106 may be disposed within the base substrate 102-1 and may not be exposed to fluids in the chamber 104, may have a coating disposed on the plurality of electrodes 106, and/or may be disposed in another substrate, such as substrate 111 illustrated by FIG. 1G.
  • the plurality of electrodes 106 include actuating electrodes 106-1 , 106-2, 106-3, 106-4 and a ground electrode 106-5.
  • the actuating electrodes 106-1 , 106-2, 106-3, 106-4 may be disposed on or within the base substrate 102-1 and the ground electrode 106-5 may be disposed on or within the top substrate 102-2.
  • Use of a ground electrode 106-5 with plurality of actuating electrodes 106-1 , 106-2, 106-3, 106-4 may allow for greater control of fluid flow and/or formation of fluid droplets of the reaction fluids as compared to using fluid control without the ground electrode 106-5.
  • the charge from the actuating electrodes 106-1 , 106-2, 106-3, 106-4 may go to ground. Without the use of the ground electrode 106-5, a stray charge may accumulate in the DMF device 100, which produces an electric field and causes forces on fluid therein.
  • FIG. 1 B illustrates the ground electrode 106-5 as a single electrode (e.g., an almost continuous electrode), examples are not so limited.
  • a plurality of ground electrodes may be disposed on or within the top substrate 102-2.
  • all of the plurality of electrodes 106 are actuating, and no ground electrodes are used.
  • respective electrodes of the plurality of electrodes 106 may be floating or set at ground, such as when the respective electrodes are not being used to draw the fluid along the microfluidic path.
  • the DMF device 200 may include a plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4.
  • the plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4 may contain the plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4 and are fluidically coupled to the plurality of fluidic inlets 210-1 , 210-2, 210-3, 210-4.
  • the plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4 may be disposed within the housing 202, such as between the substrates (e.g., between the top substrate 102-2 and the base substrate 102-1 as illustrated by FIG. 1 B or otherwise contained by the base substrate 102-1 and side substrates 102- 3,102-4 as illustrated by FIG. 1 D).
  • FIG. 1 B illustrates a respective reaction fluid well 120-1 that is disposed between the top substrate 102-2 and the base substrate 102-1.
  • the plurality of reaction fluid wells may fluidically couple to the chamber 104, as illustrated by the reaction fluid well 120-1 fluidically coupling to the chamber 104 and the fluidic inlet 110-1.
  • the DMF device 100 may further include a plurality of blister packs.
  • the plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4 may couple to the plurality of blister packs which contain the plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4.
  • the blister packs may provide the plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4 to the reaction fluid wells 220-1 , 220-2, 220-3, 220-4.
  • the plurality of blister packs may be disposed on the top substrate of the DMF device 200 (e.g., top substrate 102-2 of FIG.
  • each blister pack couples to a respective reaction fluid well of the plurality 220-1 , 220-2, 220-3, 220-4 through a fluidic inlet of the plurality of fluidic inlets 210-1 , 210-2, 210-3, 210-4.
  • the plurality of blister packs may be disposed within the plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4 or within other wells on the DMF device 200 that fluidically couple to the reaction fluid wells 220-1 , 220-2, 220-3, 220-4.
  • the plurality of blister packs may include wells, which in response to being pierced (as further described below), pressure within the blister pack equilibrates with atmospheric pressure such that the DMF device 200 may pull fluid from the blister pack.
  • FIG. 1 D illustrates an example of a blister pack 116 coupled to the DMF device 100 illustrated by FIGs. 1 B-1C, as illustrated by the top substrate 102-2 in FIG. 1 D.
  • a blister pack refers to and/or includes a chamber containing fluid, sometimes referred to as “blister”, and a layer of breakable material coupled to the chamber.
  • the chamber 118 may be formed of a flexible material.
  • Breakable material refers to and/or includes material which may be pierced, torn, or otherwise broken.
  • the breakable material 124 may include aluminum foil, plastic, and other types of materials which may be pierced and/or otherwise break. More particularly, FIG.
  • 1 D illustrates a respective fluidic inlet 110-1 of the plurality of fluidic inlets coupled to the blister pack 116.
  • reaction fluid 115 Prior to breaking the layer of breakable material 124, reaction fluid 115 is contained within the blister pack 116 and the blister pack 116 is coupled to the fluidic inlet 110-1 of the DMF device 100.
  • the blister pack 116 may be coupled to the fluidic inlet 110-1 of a plurality of fluidic inlets, and the fluidic inlet 110-1 is coupled to a reaction fluid well fluidically coupled to the chamber of the DMF device 100, such as the reaction fluid well 120-1 and the chamber 104 of the DMF device 100 of FIG. 1 B.
  • a force 119 may be applied to the chamber 118 and the layer of breakable material 124 to cause the blister pack 116 to fluidically couple to the DMF device 100, such as the reaction fluid well 120-1 and/or to the chamber 104 of the DMF device 100 of FIG. 1 B.
  • the chamber 118 of the blister pack 116 is formed of a flexible material, such that a force 119 (e.g., pressing) on the flexible material causes pressure on the layer of breakable material 124 via the reaction fluid 115 filled therein and causes the layer of breakable material 124 to break.
  • piercing structures 123 may be located below the layer of breakable material 124 to assist with breaking the breakable material 124.
  • the reaction fluid 115 from the chamber 118 of the blister pack 116 flows to a channel 125 that is coupled to the fluidic inlet 110-1 the DMF device 100.
  • a piercing structure includes and/or refers to an object with a sharp point or edge.
  • the blister pack 116 may be pierced manually by a user and/or by a piercing structure of an instrument that the DMF device 100 is inserted into.
  • a carrier fluid 114 may be contained between the bottom surface 109 and the top surface 107 of the chamber 104 of the DMF device 100. As noted above, the carrier fluid 114 may be used to flow the plurality of reaction fluids, as fluid droplets, through the chamber 104.
  • the plurality of reaction fluids may include aqueous fluids and the carrier fluid 114 may include an oil fluid.
  • the carrier fluid 114 may include an oil.
  • the carrier fluid 114 may include a silicon oil or fluorinated oil, such as FC-40 or FC-3283.
  • Non-limiting examples of the carrier fluid 114 include FC-40, FC-43, FC-77, fluorophoroheptane (FC-84), FC- 3283, perfluoro-n-octane, perfluorodecalin, perfluorophenanthrene, perfluorohexyloctane, octofluoropropane, decafluorobutane, perfluoropentane, perfluorohexane, perfluorooctane, decafluoropentane, perfluoro(2-methyl-3- pentaone), perfluoro- 15-crown-5-ether, bis-(perfluorobutyl) ethane, perfluorobutyl tetrahydrofuran, bi-perfluorohexyl ethane, perfluoro-n-hexane, perfluorooctyl bromide, perfluorotributylamine, perfluorotrip
  • the carrier fluid 114 may include a non-fluorinated oil, such as polyphenylmehtylsiloxane, polydimethylsiloxane, hexadecane, tetradecane, octadecane, dodecane, mineral oil, isopar, or squalene.
  • a non-fluorinated oil such as polyphenylmehtylsiloxane, polydimethylsiloxane, hexadecane, tetradecane, octadecane, dodecane, mineral oil, isopar, or squalene.
  • examples are not so limited and may include other types of carrier fluids and reaction fluids that are immiscible.
  • the DMF device 100 may further include or be coupled to circuitry 103.
  • the circuitry 103 may be communicatively coupled to the plurality of electrodes 106 and the magnetic unit 108 to selectively actuate the plurality of electrodes 106 and the magnetic unit 108 to move fluid droplets of the plurality of reaction fluids along the microfluidic path.
  • the circuitry 103 may be supported by the housing 102-1 , 102-2. In other examples, the circuitry 103 may be supported by another device and is couplable to the DMF device 100. For example, the circuitry 103 may be external to the housing 102-1 , 102-2 and/or the DMF device 100.
  • Example circuitry includes a processor and memory, as further described below.
  • the circuitry 103 includes an anisotropic conductive layer (e.g., an anisotropically decoupling layer 103-1 and the plurality of electrodes 106 illustrated by FIG. 1G) of the DMF device 100 which may conduct electricity in one direction and is coupled to the plurality of electrodes 106 and is couplable to external circuitry, such as an external processor and/or memory.
  • Using a conductive layer on the DMF device 100 may reduce costs of the DMF device 100, which may be disposable.
  • Use of processor and/or memory may allow for greater control of fluid flow as compared to use of external processor and/or memory.
  • FIG. 1C illustrates a top view of the DMF device 100.
  • the plurality of fluidic inlets 110-1 , 110-2, 110-3, 110-4 are disposed on and through the top substrate 102-2.
  • the top substrate 102-2 may be a lid with the ground electrode 106-5 disposed thereon.
  • the lid and ground electrode 106-5 may be transparent to allow for viewing of fluid flow and/or chemical operations within the DMF device 100.
  • the circuitry 103 may be communicatively coupled to the plurality of electrodes 106 to selectively actuate the plurality of electrodes 106 and, in response, to cause application of electrowetting forces on the plurality of reaction fluids to form fluid droplets of the plurality of reaction fluids and to drive the selective fluid flow of the fluid droplets of the reaction fluids within the chamber 104.
  • fluid droplets of the plurality of reaction fluids may be formed by drawing fluid from the blister pack and/or reaction fluid wells into the chamber 104.
  • the reaction fluids may be contained within the blister packs and/or reaction fluid wells and drawn into the chamber 104 to form the fluid droplets of the reaction fluids using the plurality of electrodes 106. Electrowetting forces may be generated by the electrodes 106 to split fluid packets of reaction fluids into fluid droplets of the reaction fluids, such as splitting a fluid packet 115-1 of the respective reaction fluid 115-1 , 115- 2 into the fluid droplet 115-2 of the reaction fluid 115-1 , 115-2 as illustrated by FIG. 1 B.
  • [0068JFIG. 1 B illustrates a respective reaction fluid well 120-1 fluidically coupled to the fluidic inlet 110-1 and is used to describe an example operation for forming a fluid droplet 115-2 of the reaction fluid 115-1 , 115-2.
  • the reaction fluid 115-1 , 115-2 may be inserted to the DMF device 100 and forms a fluid packet 115-1 , which comprises a finite number of separate fluid droplets of the reaction fluid 115-1 , 115-2, and which may be moved together within the reaction fluid well 120-1.
  • Respective electrodes 106-1 , 106-2, 106-5 of the plurality may be located in the reaction fluid well 120-1 and used to form the fluid droplet 115-2 of the respective reaction fluid 115-1 ,115-2 from the fluid packet 115-1.
  • the reaction fluids may be inserted to the reaction fluid wells via a pipette or other object containing a volume of the reaction fluid and via the plurality of fluidic inlets.
  • the reaction fluid 115-1 , 115-2 is inserted into the fluidic inlet 110-1 and, in response, the fluid packet 115-1 of the reaction fluid 115-1 , 115-2 forms in the reaction fluid well 120-1 .
  • Electrowetting forces split the fluid packet 115-1 into the fluid droplet 115-2 of the reaction fluid 115-1 , 115-2. The electrowetting forces are generated by applying an electric field via the electrodes 106, and which cause individual fluid droplets of the reaction fluid 115-1 , 115-2 to pull off from the fluid packet 115-1.
  • the electric field may cause a change in conductivity and permittivity at the interface between the reaction fluid 115-1 , 115-2 and carrier fluid 114, and produces an electric force on the interface.
  • the electric force may cause stress on the interface, which may be referred to as a Maxwell stress, or when integrated over the area of the interface, this may be referred to as the Maxwell force.
  • the reaction fluid 115-1 , 115-2 may be pulled into a shape that contains a neck 117 via electrowetting forces, and then pulled further by the electrowetting forces, with the neck 117 breaking off to form a fluid droplet 115-2 of the reaction fluid 115-1 , 115-2.
  • At least two of the electrodes of the reaction fluid well 120-1 may provide electrowetting forces on the fluid packet 115-1 of the reaction fluid 115-1 , 115-2 to form the neck 117 and break off the neck 117 to form the fluid droplet 115-2 of the reaction fluid 115-1 , 115-2 that is smaller than the fluid packet 115-1.
  • the circuitry 103 may selectively actuate the plurality of electrodes 106 and the magnetic unit 108 to provide electrowetting forces and magnetic forces on fluids within or proximal to the microfluidic path of the chamber 104 and to draw the fluids along the microfluidic path while selectively trapping functionalized magnetic beads to separate and isolate target molecules, as further described herein.
  • FIG. 1 E-1 F illustrate an example implementation of the DMF device 100 of FIG. 1A, and which is similar implementation to FIGs. 1 B-1C but without a top substrate 102-2.
  • FIG. 1 E is a cross-sectional view of the chamber 104 of the example implementation of the DMF device 100
  • FIG. 1 F is a side view of the example implementation.
  • the housing of the DMF device 100 includes a base substrate 102-1 with side substrates 102-3, 102-4, and without a top substrate 102-2 as illustrated by FIG. 1 B.
  • FIG. 1 B does not illustrate side substrates, the implementation of the DMF device 100 of FIG. 1 B may include side substrates.
  • the chamber 104 includes a bottom surface 109 defined by the base substrate 102-1 and a top surface 107 defined by a carrier fluid 114 disposed within the chamber 104.
  • the carrier fluid 114 may be contained by the base substrate 102-1 and the side substrates 102-3, 102-4.
  • a top substrate By not including a top substrate, a user may more easily view fluid operations within the chamber 104 and fabrication may be simplified.
  • including a top substrate 102-2 may allow for more integrated fluid flow and prevent contamination, fluid spill, and/or other errors.
  • the plurality of fluidic inlets 110-1 , 110-2, 110-3, 110-4 are disposed on and through the side substrate 102-4. Similar to the implementation illustrated by FIGs. 1 B-1C, the electrowetting forces generated by the electrodes 106, via selective actuation by the circuitry 103 may draw the reaction fluids into the chamber 104 to form the fluid droplets of the reaction fluids, as illustrated by the example fluid droplet 115 of the reaction fluid, and may further drive selective flow of the fluid droplets of the reaction fluids within the chamber 104. Similarly, the magnetic unit 108 may be selectively actuated to trap the functionalized magnetic bead(s) and to separate target molecules from other components within in sample fluid of the plurality of reaction fluids.
  • a reaction fluid well may be located between the fluidic inlet 110-1 and the chamber 104, similar to the reaction fluid well 120-1 illustrated by FIG. 1 B.
  • a blister pack may couple to the fluidic inlet 110-1 , similar to the blister pack 116 illustrated by FIG. 1 D.
  • the electrodes 106 may not be disposed on the base substrate 102-1 of the DMF device 100.
  • FIG. 1G illustrates an example implementation of the DMF device 100 of FIG. 1A. More particularly, FIG. 1G is a partial view of the chamber 104 of the DMF device 100 and does not illustrate all components of the DMF device 100.
  • the electrodes 106-1 , 106-2, 106-3 are disposed on or within another substrate 111 which is coupable to the base substrate 102-1 .
  • the other substrate 111 may form part of another device 127 which includes the circuitry 103-2.
  • the other device 127 may include an instrument that the DMF device 100 is inserted into and which couples the electrodes 106 and the circuitry 103-2 to the DMF device 100 via circuitry 103-1 of the DMF device 100.
  • the DMF device 100 may be a consumable device which may be used once and then discarded. Having the electrodes 106 and (external) circuitry 103-2 separate from and couplable to the DMF device 100 may reduce manufacturing costs. As shown by FIG.
  • the circuitry 103-1 of the DMF device 100 may include an anisotropically decoupling layer which couples the electrodes 106 and external circuitry 103-2 (e.g., a processor and/or memory) to the DMF device 100 to move fluid droplets of the reaction fluids along the microfluidic path.
  • external circuitry 103-2 e.g., a processor and/or memory
  • the DMF device 100 illustrated by any of FIGs. 1A-1 G may further include a waste reservoir.
  • the DMF device 200 may further a waste reservoir 440 fluid ical ly coupled to the chamber 204.
  • the waste reservoir 440 may be located within the housing 202 or off device, in some examples.
  • FIG. 2 illustrates another example DMF device, in accordance with examples of the present disclosure.
  • the DMF device 200 of FIG. 2 may comprise at least some of substantially the same features and components as DMF device 100 as illustrated by any of FIGs. 1A-1G, as shown by the similar numbering.
  • the DMF device 200 includes a housing 202, a chamber 204, a plurality of fluidic inlet 210-1 , 210-2, 210-3, 210-4 (herein generally referred to as the “plurality of fluidic inlets 210” for ease of reference), a magnetic unit 208, and a surface-enhanced luminescence substrate 212.
  • the DMF device 100 include a lid and the fluidic inlets 210 are disposed in and through the lid. The common features and components are not repeated for ease of reference.
  • the DMF device 200 includes or is coupled to a plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4 which fluidically couple to the fluidic inlets 210 and to the chamber 204.
  • the plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4 contain or store the plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4.
  • the plurality reaction fluid wells 220-1 , 220-2, 220-3, 220-4 are disposed within the housing 202.
  • the reaction fluid wells 220-1 , 220-2, 220-3, 220-4 may each be coupled to a respective blister pack (not illustrated by FIG.
  • the plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4 may be disposed within the housing 202 and may fluidically couple to the chamber 204.
  • Each of the fluidic inlets 210 may be fluidically coupled to a different respective reaction fluid well, with each of the reaction fluid wells 220-1 , 220-2, 220-3, 220- 4 being fluidically coupled to the chamber 204.
  • each of the plurality of blister packs may couple to a respect one of the plurality of reaction fluid wells 220-1 , 220-2, 220-3, 220-4 through a respective one of the fluidic inlets 210.
  • the blister packs may be contained in the reaction fluid wells 220-1 , 220-2, 220-3, 220-4 or in other wells coupled to the reaction fluid wells 220-1 , 220-2, 220-3, 220-4.
  • Respective fluids may be inserted into the reaction fluid wells 220-1 , 220- 2, 220-3, 220-4, for example, via pipette or other fluid source.
  • the reaction fluids 222-1 , 222-2, 222-3, 222-4 may be self-contained in the blister packs and/or reaction fluid wells 220-1 , 220-2, 220-3, 220-4.
  • a plurality of blister packs may disposed within or on the housing 202 and couple to a plurality of reaction fluid wells, as previously illustrated by FIG. 1 D.
  • the blister packs may be pierced by another instrument, such as by inserting the DMF device 200 into an instrument containing a piercing structure.
  • FIG. 1 D a plurality of blister packs
  • the structure may be located in the instrument proximal to where the DMF device 100 is disposed or inserted in, and in response to inserting the DMF device 100 into the instrument, the breakable material 124 of the blister pack 116 is pierced.
  • the blister packs may be pierced manually by a user using the piercing structure, such as mechanical plunger with a sharp end.
  • the fluid flow may be caused by the electrodes 206 and in response to the reaction fluids 222-1 , 222-2, 222-3, 222-4 being input to the DMF device 200, such as via a pipette, and with or without including the use of blister packs.
  • the DMF device 200 further includes an additional well 221 that contains or holds the surface-enhanced luminescence substrate 212.
  • the well 221 may be fluidically coupled to the chamber 204, such as via a fluidic outlet 224 fluidically coupled to the chamber 204 and the well 221 .
  • a fluidic outlet refers to and/or includes an outlet port, e.g., an aperture, that is fluidically coupled to the chamber 204.
  • the plurality of electrodes 206 may be arranged in array, which may be used to provide localized resolution of the electric field to provide fluid droplet formation and selective control of fluid flow of the fluid droplets of the reaction fluids.
  • the plurality of electrodes 206 are arranged in an array that includes rows and columns of electrodes forming a rectangular shape.
  • examples are not so limited and other shaped arrays may be formed.
  • the electrodes 206 may have a variety of different arrangements and sizes.
  • the electrodes 206 may be arranged in linear arrays, two dimensional arrays, and/or may include ring electrodes, and may include more or less electrodes than illustrated. [0081JFIG.
  • the apparatus 330 comprises a DMF device 200, a plurality of electrodes 206, a functionalized bead (e.g., as contained in one of the plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4 contained in one of the reaction fluid wells 220-1 , 220-2, 220-3, 220-4) to selectively bind to a target molecule, circuitry 203, and an optical sensing device 332.
  • a functionalized bead e.g., as contained in one of the plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4 contained in one of the reaction fluid wells 220-1 , 220-2, 220-3, 220-4
  • the DMF device 200 may include the DMF device illustrated by FIG. 2, and may comprise an example implementation of, or comprise at least some of substantially the same features and components as any one of the examples DMF as described in association with any of FIGs. 1 A-2.
  • the DMF device 200 includes a housing 202 that defines a microfluidic path including a chamber 204, a magnetic unit 208 disposed along the microfluidic path and within the chamber 204, and a surface-enhanced luminescence substrate 212 coupled to the chamber 204 to isolate a target molecule from a sample fluid.
  • the DMF device 200 may include a plurality of fluidic inlets 210 fluidically coupled to the chamber 204 to input the plurality of reaction fluids including a fluid containing the functionalized magnetic bead.
  • the details of the common features and components are not repeated for ease of reference.
  • a plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4 may be contained in blister packs and/or the reaction fluid wells 220-1 , 220-2, 220-3, 220-4 that couple to the chamber 204.
  • the DMF device 200 includes an additional well 221 that contains or holds the surface-enhanced luminescence substrate 212.
  • the plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4 may include a sample fluid and a plurality of buffer fluids, with a buffer fluid of the plurality of buffer fluids containing a functionalized magnetic bead, such as containing a plurality of functionalized beads.
  • the functionalized magnetic bead(s) may be enveloped by a functional layer and/or may be porous, and may separate the molecules in the sample fluid based on size, chemical properties, and/or combinations thereof.
  • the functionalized magnetic bead(s) includes a carboxylate group, a quaternary ammonium group, or a C18 tail on a surface of the bead(s).
  • the functionalized magnetic bead(s) include a carboxylate group, a quaternary ammonium group, or a C18 tail and a porous surface.
  • the plurality of electrodes 206 are coupled to the housing 202.
  • the plurality of electrodes 206 are disposed within or on a substrate of the housing 202.
  • the electrodes 206 may form part of another device, such as instrument containing the electrodes 206 and the circuitry 203 that the DMF device 200 is inserted into.
  • the apparatus 330 further includes circuitry 203.
  • the circuitry 203 is coupled to or forms part of the DMF device 200, and may track and/or control operation of the plurality of electrodes 206 and the magnetic unit 208. Such operations may comprise activation or actuation, deactivation, and other settings, such as setting to ground or floating and timings associated with the same.
  • the circuitry 203 may coordinate operations of the DMF device 200 including the flow of fluid and/or electrowetting-caused manipulation of fluid droplets of the reaction fluids 222-1 , 222-2, 222-3, 222-4 within the DMF device 200, such as moving, merging, and/or splitting, respectively. Such manipulation may include causing fluid droplets of the reaction fluids 222-1 , 222-2, 222-3, 222-4 to move along the chamber 204 within the DMF device 200 to isolate target molecule(s) from other molecules in a sample fluid and to move the target molecule(s) to the surface-enhanced luminescence substrate 212.
  • the various examples operations of the circuitry 203 may be operated interdependently and/or in coordination with each other, in at least some examples.
  • the circuitry 203 is communicatively coupled to the plurality of electrodes 206 and the magnetic unit 208 to selectively actuate electrodes of the plurality of electrodes 206 to move fluid droplets of a plurality of reaction fluids along the microfluidic path, and selectively actuate the magnetic unit 208 to move the functionalized magnetic bead toward the magnetic unit 208 and facilitate bead-based separation of molecules in the sample fluid.
  • the chamber 204 may contain a carrier fluid
  • the circuitry 203 is to selectively actuate electrodes of the plurality of electrodes 206 to form the fluid droplets of the plurality of reaction fluids as surrounded by the carrier fluid. The fluid droplets of the plurality of reaction fluids are then sequentially moved, as further illustrated by FIGs. 4A-4H.
  • the apparatus 330 further includes an optical sensing device 332 to interrogate the target molecule(s) isolated on the surface-enhanced luminescence substrate 212.
  • the optical sensing device 332 may illuminate the surface-enhanced luminescence substrate 212 at a test spot 213 using a light source and collects light in response to the illumination and from the surface- enhanced luminescence substrate 212.
  • the light source may include a laser light or a light emitting diode (LED).
  • Some example light sources include semiconductor lasers, helium-neon lasers, carbon dioxide lasers, LEDs, incandescent lamps, and other examples radiation emitting sources.
  • the light source may emit illumination light in a wavelength range between about 350 nm and about 1000 nm.
  • FIGs. 2-4H illustrate a single test spot 213, examples may include a plurality of test spots which the optical sensing device 332 may sample, and measure the response (e.g., Raman response) at each of the plurality of test spots.
  • the average of the responses may be used to detect the target molecule(s) and which may be used to provide greater sensitivity as compared to one test spot.
  • the surface-enhanced luminescence substrate 212 may be pre-spotted with a reference target molecule, which is isolated from respective reaction or other fluids flown to the surface-enhanced luminescence substrate 212.
  • the reference target molecule is a sample of the target molecule, and may be used by the optical sensing device 332 as a reference for calibration of the signal and/or quantification. Use of a reference target molecule may increase accuracy of the results.
  • the surface-enhanced luminescence substrate 212 may include reference material used by optical sensing device 332 as a reference for calibration. Use of a reference material may allow for a simplified design of the surface-enhanced luminescence substrate 212, as respective reaction or other fluids flown to the surface- enhanced luminescence substrate 212 may not be isolated from the reference material while still providing the calibration.
  • the optical sensing device 332 may provide plasmonic sensing, such as Raman spectroscopy.
  • Raman spectroscopy is a technique for determining the chemical make-up of a target molecule by measuring the spectral response of the target molecule to electromagnetic radiation provided, for example, by a laser beam or other light source. More particularly, Raman spectroscopy may be used to study transitions between molecular energy states when photons interact with molecules, which results in the energy of the scattered photons shifting, referred to a Raman scattering.
  • the Raman scattering of a target molecule may be described as follows.
  • the target molecule which is at a certain energy state, is first excited into another energy state (virtual or real) by incident or excitation photons, which is ordinarily in the optical frequency domain (e.g., the illumination light output by light source 331 of the optical sensing device 332).
  • the excited target molecule than radiates as dipole source under the influence of the environment in which it sits at a frequency that may be lower (e.g., Stokes scattering) or higher (e.g., anti-Stokes scattering) than the excitation photons.
  • the Raman spectrum of different molecules or matter have characteristic peaks which may be used to identify the species of molecule. Raman spectroscopy may be applied to a variety of different chemical and/or biological interrogation and detection applications.
  • the intrinsic Raman scattering process of a target molecule is inefficient, and the Raman scattering processes may be enhanced using the surface-enhanced luminescence substrate 212.
  • the enhancement of the Raman scattering using nano-structures and/or rough surface of the surface-enhanced luminescence substrate 212 is sometimes referred to as SERS.
  • the surface-enhanced luminescence substrate 212 may comprise a SERS substrate and the optical sensing device 332 includes a Raman spectrometer.
  • the SERS substrate may include metal nano-structures and/or a metal rough surface that enhances (e.g., amplifies) Raman scattering from a target molecule when exposed to illumination light.
  • the metal nano-structures and/or metal rough surface of the SERS substrate may enhance the Raman signal or are otherwise capable of increasing a number of Raman scattered photons when the target molecule is located proximal to a respective metal nano-structure or metal rough surface and when the target molecule and SERS substrate are subjected to electromagnetic radiation by the optical sensing device 332.
  • the SERS substrate may increase the number photons inelastically scattered by the target molecule positioned near or adjacent to a metal nano-structure or gap in the metal rough surface.
  • the metal nano-structures and/or rough surface may be formed of silver, gold, platinum or copper, although examples are not so limited.
  • the optical sensing device 332 and/or the Raman spectrometer includes a light source 331 to illuminate the test spot 213, and an optical detector 333 to measure the Raman scattering light in response.
  • the optical detector 333 may receive and detect Raman scattered photons in response to illuminating the test spot 213.
  • An optical detector includes and/or refers to circuitry used to collect light, such as Raman scattering, which may be dispersed by other components.
  • the optical detector 333 includes a charged coupled device (CCD) detector.
  • CCD charged coupled device
  • the optical detector 333 includes a monochromator (or other device that determines the wavelength of the Raman scattered photons) and a device that determines the quantity, e.g., intensity, of the Raman scattered photons, such as a photomultiplier.
  • the light source 331 may include a laser light, among other light sources as previously described.
  • the optical sensing device 332 and/or the Raman spectrometer may further include a grating, filters, and/or other non-illustrated optical components to separate Raman scattering light from other light (e.g., Rayleigh signal and reflected laser light), and which may be disposed between the test spot 213 and the optical detector 333.
  • additional non-illustrated optical components may be disposed between the light source 331 and the test spot 213 to collimate, filter, and/or focus the illumination light prior to impinging on the surface-enhanced luminescence substrate 212.
  • the Raman spectrometer (or other optical sensing device 332) may be controlled by the circuitry 203 to interrogate target molecules deposited on the test spot 213 of the surface-enhanced luminescence substrate 212 after an amount of time sufficient to allow the test spot 213 to dry.
  • the circuitry 203 controls the Raman spectrometer to perform measurements and collect test data.
  • the test process may include illuminating the test spot 213 by laser light and measuring the spectral response of the test spot 213.
  • the spectral response may be an emission intensity of the light deflected by the test spot 213 over a wavelength range of interest associated with a target molecule.
  • the resulting data may be collected, stored, and/or displayed by the circuitry 203, such as by using a display communicatively coupled to the circuitry 203.
  • the circuitry 203 may process the spectral data from the test spot 213 and based on the results, identifies the presence (or not) of the target molecule based on the resulting Raman spectrum and/or characteristic peaks present in the spectral data.
  • the DMF device 200 and/or apparatus 330 may be used to perform hyper-Raman spectroscopy.
  • a small number of photons may be scattered at frequencies corresponding to the higher order harmonics of the excitation radiation, such as the second and third harmonic generations (e.g.,, twice or three times the frequency of the excitation radiation).
  • Some of these photons may have a frequency that is Raman-shifted relative to the frequencies corresponding to the higher order harmonics of the excitation radiation.
  • These higher order Raman scattered photons may provide information about the target molecule that cannot be obtained by first order Raman spectroscopy.
  • Hyper-Raman spectroscopy involves the collection and analysis of these higher order Raman scattered photons.
  • SERS is a plasmonic sensing technique in which Raman scattering is enhanced by a plasmonic material, such as a rough surface or metal nano-structures.
  • a plasmonic material such as a rough surface or metal nano-structures.
  • the inclusion of the plasmonic material amplifies the Raman scattering response of the target, resulting in a much more sensitive test that may detect a small number of target molecules or even a single target molecule.
  • the target molecule is brought in contact with or close proximity to metal nano-structures or a metal rough surface, which increases the intensity of the Raman scattering and results in a more sensitive test.
  • Example are not limited to Raman spectrometry and/or SERS, and may include other types of optical sensing devices.
  • Other suitable types of an optical sensing device 332 include a hyperspectral camera, a line scanning spectrophotometer, and others. Further, the optical sensing device 332 may also include optical objectives to collect light emitted (e.g. scattered) from the test spot 213 on the substrate 212 and direct that light to the spectrometer.
  • Example DMF devices and/or apparatuses may include variations from that illustrated by FIGs. 1A-3. As noted above, such variations may include, but are not limited to, the number of fluidic inlets and/or fluidic inlets, the number of electrodes, and/or arrangement of electrodes, among others.
  • FIGs. 4A-4H illustrate operation of an example DMF device, in accordance with examples of the present disclosure.
  • the operation may be implemented using the example DMF device 200 illustrated by FIG. 2 and/or the apparatus 330 illustrated by FIG. 3, and may comprise an example implementation of, or comprise at least some of substantially the same features and components as, any one of the examples DMF as described in association with any of FIGs. 1A-3.
  • the common features and components are not repeated for ease of reference.
  • the DMF device 200 is set to an initial state by drawing fluid droplets 442, 444, 446, 448 of the plurality of reaction fluids 222-1 , 222-2, 222-3, 222-4 into the chamber 204 from the reaction fluid wells 220-1 , 220-2, 220-3, 220-4.
  • the initial state is caused by inserting the DMF device 200 into an instrument which may cause piercing of the blister packs.
  • a user may cause actuation of select electrodes by pressing a button on the instrument or otherwise instructing the circuitry 203 to being the operation sequence.
  • the fluid droplets 442, 444, 446, 448 of the plurality of reaction fluids 222-1 , 222-2, 222- 3, 222-4 are drawn into the chamber 204 and held at or near the initial position until further selective movement is activated.
  • select electrodes of the plurality of electrodes 206 are actuated to move (e.g., pull) a first fluid droplet 442 of the sample fluid 222-1 and a second fluid droplet 444 of a reaction fluid 222-2 (e.g., buffer fluid) containing functionalized magnetic beads toward one other such that the first and second fluid droplets 442, 444 mix to form a merged fluid droplet 450 containing the sample fluid and the functionalized magnetic beads.
  • Sufficient time is allowed for a target molecule to bind to a functionalized surface of a functionalized magnetic bead.
  • the remaining fluid droplets 446, 448 are held at or near the initial position.
  • the merged fluid droplet 450 is moved toward the magnetic unit 208.
  • the merged fluid droplet 450 may be moved by selective actuation of electrodes of the plurality 208 to move the merged fluid droplet 450 toward the region of the chamber 204 containing the magnetic unit 208, followed by actuation of the magnetic unit 208 to draw and/or trap the functionalized magnetic beads on or near the magnetic unit 208.
  • a third fluid droplet 446 of wash buffer fluid 222-3 may be drawn from the initial position and/or formed from wash buffer fluid 222-3 contained in the respective reaction fluid well 220-3.
  • portions 452 of the merged fluid droplet 450 that are not bound to the functionalized magnetic beads may be moved from (e.g., pulled off) the merged fluid droplet 450 and moved toward a waste reservoir 440 coupled to the chamber 204.
  • the waste reservoir 440 may form part of the DMF device 200 or may be separate from the DMF device 200, and/or may fl uidically couple to the chamber 204 via a fluidic outlet.
  • select electrodes of the plurality 206 associated with a path to the waste reservoir 440 may be actuated to draw fluids containing the portions 452 while actuating the magnetic unit 208 to attract the functionalize magnetic beads of the merged fluid droplet 450, such that the portions 452 are flown to the waste reservoir 440 and the functionalized magnetic beads with bound target molecules remain at or near the magnetic unit 208.
  • the portions 452 may include proteins, polar molecules, and other components of the sample fluid and the buffer fluid.
  • the third fluid droplet 446 of wash buffer fluid 222-3 is moved toward the magnetic unit 208 and over the merged fluid droplet 450 via selective actuation of respective electrodes of the plurality of electrodes 206.
  • the magnetic field output by the magnetic unit 208 may then be deactivated to allow for the merged fluid droplet 450 to mix with the third fluid droplet 446 of wash buffer fluid 222-3 to wash the functionalized magnetic beads.
  • the magnetic field is again actuated to draw and/or trap the functionalized magnetic beads.
  • Select electrodes of the plurality of electrodes 206 are actuated to draw the third fluid droplet 446 of wash buffer fluid 222-3, which may contain other components, to the waste reservoir 440.
  • the operations illustrated by FIGs. 4E and 4F may be repeated a plurality of times to remove molecules larger than a target size from the chamber 204.
  • a fourth fluid droplet 448 of elution buffer fluid 222-4 is moved toward the magnetic unit 208 and over the merged fluid droplet 450 via selective actuation of respective electrodes of the plurality of electrodes 206.
  • the magnetic field output by the magnetic unit 208 may be deactivated to allow for the merged fluid droplet 450 to mix with the fourth fluid droplet 448 of elution buffer fluid 222-4 to wash the functionalized magnetic beads.
  • the elution buffer fluid 222-4 may cause target molecules bound to functionalized magnetic bead to unbind and separate from the functionalized magnetic beads, and to merge with fluid in the fourth fluid droplet 448.
  • the fourth fluid droplet 448 containing the elution buffer fluid 222-4 and the eluted target molecule(s) are moved toward the surface-enhanced luminescence substrate 212.
  • the fourth fluid droplet 448 containing the elution buffer fluid 222-4 and the eluted molecule(s) may be isolated from the functionalized magnetic beads and moved toward the surface- enhanced luminescence substrate 212 via selective actuation of respective electrodes of the plurality of electrodes 206 associated with a path to the surface-enhanced luminescence substrate 212, and with concurrent actuation of the magnetic unit 208 to output an magnetic field and draw or trap the functionalized magnetic beads at or near the magnetic unit 208.
  • the optical sensing device 332 may interrogate the test spot 213 by illuminating the test spot 213 with illumination light 454 and sensing light emitted back in response.
  • a carrier fluid is contained in the chamber 204.
  • the carrier fluid may include an oil, which may cause detection issues for the optical sensing device 332.
  • the fourth fluid droplet 448 containing the elution buffer fluid 222-4 and the eluted target molecule(s) may be pulled out from the carrier fluid and into air.
  • the eluted target molecule(s) may be pulled out from the carrier fluid by selectively actuating the electrodes 206 of the DMF device 200 to move the fourth fluid droplet 448 and leave the carrier fluid (e.g., oil phase), such that the eluted target molecule(s) enter an air phase.
  • circuitry 203 may selectively actuate the electrodes 206 and/or magnetic unit 208 to generate electric fields and/or magnetic fields.
  • FIG. 5 illustrates an example functionalized magnetic bead and operation thereof in a DMF device, in accordance with examples of the present disclosure.
  • FIG. 5 may include a close-up view of a chamber 104, 204 of any of the DMF device 100, 200 illustrated by FIGs. 1A-3, the common features and components not being repeated.
  • the operation 560 illustrated by FIG. 5 may occur via the selective movement of fluids and activations of magnetic fields, as illustrated by the operations of FIGs. 4A-4H.
  • Functionalized magnetic beads may be used to separate molecules in a sample fluid based on size and/or chemical properties. Example chemical properties include charge, hydrophobicity, and binding affinities, among others.
  • the functionalized magnetic beads may have a functional layer or other composition to separate molecules in the sample.
  • the functional layer may include a porous external surface, such that molecules smaller than the pores of the porous surface may be incorporated inside the functionalized magnetic beads.
  • the functional layer may include functional groups or other compositions on a surface of the functionalized magnetic beads that bind to particular classes of molecules.
  • the functionalized magnetic beads may contain a surface with a carboxylate group, a quaternary ammonium group, and/or a C18 tail. The surface may be an exterior surface and/or an interior surface of the beads.
  • the functionalized magnetic beads have multiple functional layers or functionalities, sometimes herein referred to as a “multifunctionalized magnetic bead”.
  • the functionalized magnetic beads may include a porous external surface, and a functional group or other composition on a surface, such as a carboxylate group, a quaternary ammonium group, and/or a C18 tail on an interior surface and/or exterior surface of the bead.
  • An interior surface of a bead refers to and/or includes a surface accessible through a pore of the porous surface or otherwise not exposed to the environment with a solid or non-porous bead.
  • an exterior surface of bead refers to and/or includes a surface exposed to the environment, and which may be exposed to molecules of any size.
  • the target molecules may be incorporated inside the bead and then prevented and/or mitigated from escaping prematurely.
  • the functionalized magnetic beads may include a magnetic core-mesoporous shell with a CI S-functional interior surface. C18 may bind to antibiotics, for example, and the pores may be smaller than a size of proteins in the sample, although examples are not so limited.
  • the functionalized magnetic beads may include a magnetic core-mesoporous shell with quaternary ammonium groups on a surface which may allow for a strong anion exchange.
  • Molecules that possess a net negative charge may bind to quaternary ammonium groups.
  • Example molecules with a net negative charge include DNA and acids with acid dissociate constants (pK a s) that are less than the pH at neutral pH, such as penicillin G, e.g., pK a of about 2.
  • pK a s include a quantitative measure of a strength of an acid in solution.
  • the functionalized magnetic beads may include a magnetic core- mesoporous shell functionalized with a carboxylate group on a surface which may be used for weak cation exchange.
  • Molecules that possess a net positive charge may bind to carboxylate groups.
  • Example molecules with a net positive charge include bases with pK a s that are greater than the pH at neutral pH, such as neomycin, e.g., pKa of about 12.9 at neutral pH.
  • the carboxyl group may be further functionalized via 1 -Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC)-mediated coupling to produce derivatives, such as sulfonic acid derivatives, for strong cation exchanges, as well as derived for weak anion exchange, such as aminoethyl derivatives.
  • EDC Ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • the operation 560 may include mixing a fluid droplet containing a multi-functionalized magnetic bead 563 with a fluid droplet of a sample fluid containing molecules 565, 567.
  • the multi-functionalized magnetic bead 563 includes a porous surface and functionalized interior surface that includes a functional group.
  • FIG. 5 illustrates a functionalized magnetic bead within the fluid droplet, the fluid droplet may contain a plurality of functionalized magnetic beads in various examples.
  • the target molecules that are smaller than the pore size of the porous surface may travel through the pores to the interior surface of the multi-functionalized magnetic bead 563 and may bind with the functional group on the interior surface.
  • the interior surface may include C18 tails that bind to the target molecule 565 that is hydrophobic. Molecules that are larger than the pores, such as the labeled molecule 567, may remain outside the multifunctionalized magnetic bead 563.
  • a magnetic field is applied using the magnetic unit 508 to attract the multi-functionalized magnetic bead 563. While the magnetic field is applied, a fluid droplet of wash buffer fluid is flown to and mixed with the sample fluid to discard fluid containing unbound molecules.
  • the magnetic field is removed and the various fluid is mixed with a fluid droplet of elution buffer fluid to elute out the target molecules, such as the illustrated target molecule 565.
  • a magnetic field is applied using the magnetic unit 508 to attract the multi-functionalized magnetic bead 563, while the elution buffer fluid with the eluted target molecules are flow to the surface-enhanced luminescence substrate 512 for interrogation by an optical sensing device.
  • FIG. 6 illustrates an example method for isolating target molecules using a DMF device, in accordance with examples of the present disclosure.
  • the method 680 may be implemented using any of the above-described DMF devices and apparatuses.
  • the method 680 includes flowing a first fluid droplet of a sample fluid along a microfluidic path within a chamber of a DMF device via application of electrowetting forces by a plurality of electrodes.
  • the microfluidic path may include a plurality of microfluidic paths within the chamber which are provided by the electrodes arranged in an array.
  • the method 680 includes merging the first fluid droplet of the sample fluid with a second fluid droplet of buffer fluid containing a functionalized magnetic bead to form a merged fluid droplet, wherein a target molecule in the sample fluid is to bind to the functionalized magnetic bead.
  • a plurality of functionalized magnetic beads may be contained in the second fluid droplet of the buffer fluid.
  • the method 680 includes applying a magnetic field to the merged fluid droplet via a magnetic unit disposed along the microfluidic path, and directing molecules in the sample fluid not bound to the functionalized magnetic bead to a waste reservoir.
  • the method 680 includes separating the target molecule from the merged fluid droplet containing the target molecule and the functionalized magnetic bead, and at 690, flowing the target molecule to a surface-enhanced luminescence substrate fluidically coupled to the microfluidic path.
  • the method 680 includes repeating cycles of turning off the magnetic field, allowing the functionalized magnetic bead to mix with additional fluid droplets of wash buffer fluid, and moving the additional fluid droplets of wash buffer fluid and respective unbound molecules of the sample fluid to the waste reservoir.
  • separating the target molecule includes flowing a third fluid droplet of elution buffer fluid along the microfluidic path and merging the third fluid droplet of elution buffer fluid with the merged fluid droplet to displace the target molecule from the functionalized magnetic bead.
  • the method 690 includes applying a second magnetic field to the functionalized magnetic bead with the target molecule displaced to trap the functionalized magnetic bead prior to flowing the target molecule to the surface-enhanced luminescence substrate.
  • the method 680 further includes interrogating the target molecule using an optical sensing device coupled to the surface-enhanced luminescence substrate.
  • the optical sensing device may illuminate a test spot of the surface-enhanced luminescence and measure the response, and based on the response, identify and/or detect the target molecule in the sample fluid.
  • the method 680 and/or the DMF devices and apparatuses described herein may be used to perform sample preparation and detection of target molecules in a sample.
  • the method 680 may be used to isolate a target molecule and verify the molecule is isolated, such as for isolation of products following a chemical or biochemical reaction.
  • Different types of target molecules may be detected, such as molecule adulterants in food (e.g., antibiotics in milk), toxic chemicals in soil or other samples, among detection of drugs, pesticides, and adulterants in other types of samples.
  • the method 680 may be used to test for antibiotic residues in milk, which may have adverse health impacts for mammals.
  • adverse health impacts include antibiotic resistance, allergies, reactions, cancers, and/or mutations, among other disturbances.
  • DMF devices and methodologies may be used to separate out the antibiotic molecules from other larger molecules, such as proteins, and to detect and/or identify the molecules.
  • the DMF device may manipulate fluid droplets that are smaller than 1 mL, which is compatible with various optical sensing techniques, such as SERS which use 10-240 pL of liquid.
  • An example method of manufacturing may include forming a housing defining a microfluidic path including the chamber and to support a plurality of electrodes, a magnetic unit, and a surface-enhanced luminescence substrate, and disposing the plurality of electrodes and the magnetic unit along the microfluidic path.
  • the method may further include including positioning circuitry for support by the housing for actuating the plurality of electrodes and/or the magnetic unit.
  • any of the above and below described DMF devices may be formed of a variety of material formed in a stack.
  • a housing may formed of a plurality of different materials which are in layers, e.g., layers of substrates, in a stack.
  • the different material layers may include a top (transparent) substrate material layer and/or a base substrate material layer, with etched or micromachined portions between that form the reaction fluid wells and the chamber, among other components.
  • at least one of the substrate layers may have electrodes formed thereof.
  • the top (transparent) substrate material and/or the base substrate layer may have a low energy coating (e.g., a polytetrafluoroethylene (PTFE), such as TeflonTM, fluorosilane, a polyamide, such as Kapton® FN, fluoroalkylsilane, 1 H,1 H,2H,2H- Perfluorodecyltriethoxysilane, trichloro(1 H,1 H,2H,2H-perfluorooctyl)silane)) proximal to and/or in contact with the chambers, wells, and/or channels of the DMF device and the electrodes, and/or a dielectric coating (e.g., a polyimide, such as Kapton®, Ethylene tetrafluoroethylene (ETFE), paralyne, alumina, silica, silicon nitride, aluminum nitride, aluminum oxide) proximal to and/or in contact
  • a low energy coating includes and/or refers to a layer formed of a material having surface free energy less than 30 milliNewton/meter (mN/m).
  • the low energy coating may have a free energy of 20 mN/m, and/or may provide a contact angle hysteresis of less than about 10 degrees.
  • the stack may additionally include a planarization layer with a thickness that is proportional to the electrodes, which may be formed of SU-8, paralyne, Polydimethylsiloxane (PDMS), acrylates, among other materials.
  • the planarization layer may have a thickness between the same thickness as the electrodes (e.g., in wells and/or primary chamber) to plus 100 percent of the thickness of the electrodes (e.g., two times the thickness of the electrodes).
  • the planarization layer has a thickness of between the same thickness as the electrodes and plus 10 percent of the thickness of the electrodes, or the same thickness of the electrodes and plus 50 percent of the thickness of the electrodes, among other ranges.
  • the carrier fluid e.g., an inert filler fluid
  • the chambers, wells, and/or channels may be a height in the range of about 10 pm to about 2 mm.
  • the various electrodes may be a length of about 40 pm to about 3 mm.
  • the low energy coating is formed of PTFE.
  • the dielectric coating may be formed of a polyimide (e.g., Kapton®) for ease of deposition.
  • the dielectric coating may be formed of silicon nitride.
  • the planarization layer may be formed of the same material as the dielectric coating, such as a polyimide, and which may reduce the number of fabrication steps.
  • the stack may include a low energy coating formed of PTFE, a dielectric coating formed of a polyimide (e.g., Kapton®), and a planarization layer formed of the polyimide (e.g., Kapton®).
  • control the flow of fluid within the wells and/or the primary chamber of any of the described DMF devices may be provided via ion emitters of the DMF device, instead of and/or by the electrodes.
  • a charge applicator may be brought into charging relation to a plate of the DMF device, whereby the charge applicator is to apply (e.g., deposit) charges onto the plate to cause an electric field which induces electrowetting movement of fluid within and through the DMF device.
  • the charge applicator is an addressable airborne charge depositing unit which may be brought into charging relation to the plate of the DMF device to deposit airborne charges onto the plate.
  • the charge applicator may be brought into releasable contact with, and charging relation to, the plate.
  • the charge applicator may generate and apply the charges having a first polarity and/or an opposite second polarity, depending on whether the charge applicator is to build charges on anisotropic decoupling layer of the DMF device or is to neutralize charges.
  • the first polarity may be positive or negative depending on the particular goals, while the second polarity is the opposite of the first polarity.
  • example DMF may omit the electrodes, which would otherwise be used to cause microfluidic operations such as moving, merging, and/or splitting droplets within the DMF device.
  • Charge refers to and/or ions (+/-) or free electrons.
  • any of the above described device and/or substrates may include an anisotropic decoupling layer (e.g., 103-1 of FIG. 1G).
  • the anisotropic decoupling layer may decouple the working areas of the DMF device (e.g., the chamber) from electronics of the DMF device, such as the plurality of electrodes and/or the magnetic unit.
  • the anisotropic decoupling layer and the electrodes may be referred to as an anisotropic conductive layer, which facilitates migration of charges across the base substrate by providing lower resistivity across or through the base substrate and a higher lateral resistivity along the plane through which the base substrate extends.
  • the decoupling may allow for the working areas of the of the DMF device, which contain fluids, to be inexpensive and consumable.
  • the anisotropic decoupling layer may be formed of metal microparticles or nanoparticles aligned to form chains in one direction and encased in a polymer matrix (e.g., polymethylacrylate).
  • Circuitry such as the circuitry 103, 203 of FIGs. 1 B and 2, may include a processor and a memory. Circuitry may comprise a processor and associated memories, and optionally communication circuitry. Example circuitry includes a processor electrically coupled to, and in communication with, memory to generate control signals to direct operation of a DMF device, as well as the particular portions, components, operations, instructions, and/or methods, as described herein. Example control signals include instructions stored in memory to direct and manage microfluidic operations.
  • the circuitry may be referred to as being programmed to perform the above-identified actions, functions, etc.
  • the circuitry 103, 203 may include an anisotropic conductive layer, such as the above-described anisotropic decoupling layer and a plurality of electrodes which are used to provide a plurality of microfluidic paths, which couples to electrodes of an external device.
  • the circuitry generates control signals as described above.
  • the circuitry may be embodied in a general purpose computing device and/or incorporated into or associated with at least some of the example DMF devices, as well as the particular portions, components, electrodes, fluid actuators, operations, instructions, and/or methods, etc. as described herein.
  • Processor includes and/or refers to a presently developed or future developed processor that executes machine readable instructions contained in a memory or that includes circuitry to perform computations. Execution of the machine readable instructions, such as those provided via memory of the circuitry, may cause the processor to perform the above-identified actions, such as circuitry to implement operations via the various examples.
  • the machine readable instructions may be loaded in a random access memory (RAM) for execution by the processor from their stored location in a read only memory (ROM), a mass storage device, or some other persistent storage (e.g., non- transitory tangible medium or non-volatile tangible medium), as represented by memory.
  • the machine readable instructions may include a sequence of instructions, a processor-executable machine learning model, or the like.
  • memory comprises a computer readable tangible medium providing non-volatile storage of the machine readable instructions executable by a processor of circuitry.
  • the machine readable tangible medium may be referred to as, and/or comprise at least a portion of, a computer program product.
  • hard wired circuitry may be used in place of or in combination with machine readable instructions to implement the functions described.
  • circuitry may be embodied as part of at least one application-specific integrated circuit (ASIC), at least one field- programmable gate array (FPGA), and/or the like.
  • ASIC application-specific integrated circuit
  • FPGA field- programmable gate array
  • the circuitry not limited to any specific combination of hardware circuitry and machine readable instructions, nor limited to any particular source for the machine readable instructions executed by the circuitry.
  • the circuitry may be implemented within or by a stand-alone device, such as a microprocessor.
  • the circuitry may be partially implemented in interface devices and partially implemented in a computing resource separate from, and independent of, the example interface devices but in communication with the example interface devices.
  • the circuitry may be implemented via a server accessible via the cloud and/or other network pathways.
  • the circuitry may be distributed or apportioned among multiple devices or resources
  • the plurality of reaction fluids may include a sample fluid and buffer fluids.
  • the sample fluid may include an aqueous solution or fluid containing a sample, in solid or fluid form, and/or reagents.
  • a sample fluid refers to and/or any material, collected from a subject, such as biologic material.
  • Example samples include, but are not limited to, whole blood, blood plasma, and other body fluids, as well as tissue cell cultures obtained from humans, plants, or animals.
  • Such samples may contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles.
  • Such biological material may comprise all types of mammalian and non-mammalian animal cells, plant cells, algae including blue-green algae, fungi, bacteria, protozoa, etc.
  • samples include whole blood and blood-derived products such as plasma, serum and buffy coat, urine, feces, cerebrospinal fluid or any other body fluids, tissues, cell cultures, cell suspensions, etc.
  • Other samples include fluids containing the functionalized magnetic beads to which a portion of a biologic sample or other particles are attached.
  • Sample fluids may contain an analyte of interest, such as a substance (e.g., molecule, particle, protein, nucleic acid, antigen) of interest for a chemical process or test.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

L'invention concerne un dispositif microfluidique numérique (DMF) donné à titre d'exemple comprenant un boîtier définissant un trajet microfluidique comprenant une chambre et une pluralité d'électrodes couplées au boîtier. Le dispositif DMF comprend en outre une pluralité d'entrées fluidiques couplées à la chambre pour introduire une pluralité de fluides de réaction, y compris une bille magnétique fonctionnalisée et un fluide échantillon, une unité magnétique disposée le long d'une partie du chemin microfluidique associé à la chambre, et un substrat de luminescence à surface améliorée couplé fluidiquement au chemin microfluidique.
PCT/US2022/023474 2022-04-05 2022-04-05 Dispositifs microfluidiques numériques dotés de substrats de luminescence à surface améliorée WO2023195977A1 (fr)

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PCT/US2022/023474 WO2023195977A1 (fr) 2022-04-05 2022-04-05 Dispositifs microfluidiques numériques dotés de substrats de luminescence à surface améliorée

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130252262A1 (en) * 2006-04-13 2013-09-26 Advanced Liquid Logic Inc. Droplet-based affinity assays
WO2021041709A1 (fr) * 2019-08-27 2021-03-04 Volta Labs, Inc. Procédés et systèmes de manipulation de gouttelettes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130252262A1 (en) * 2006-04-13 2013-09-26 Advanced Liquid Logic Inc. Droplet-based affinity assays
WO2021041709A1 (fr) * 2019-08-27 2021-03-04 Volta Labs, Inc. Procédés et systèmes de manipulation de gouttelettes

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