Patent Application
20230287226
Pandemics and epidemics caused by emerging infectious agents such as SARS-CoV-2, influenza viruses, Zika virus, and Ebola virus have seriously affected human health, and led to lost economic activity and challenged health-care systems. Early and accurate detection of infectious diseases, such as COVID-19, can help contain pandemics and reduce fatality rates.
Two primary approaches have been used to detect a COVID-19 infection. The first approach utilizes the genetic sequence of the virus and includes genomic sequencing, CRISPR-based test, and reverse transcription real time quantitative PCR. These approaches are relatively accurate but are generally slow because of the need for multiple sample processing steps and are dependent upon the protocol of sampling, preservation, and transportation. The second approach is to detect specific antibodies to viral antigens through serological methods, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay. These assays are based on structural proteins including spike protein (S-protein) and/or nucleocapsid protein (N-protein) and have a relatively low sensitivity. These assays are also incapable of detecting an infectious disease in the early stage (2-5 days) of infection or for someone who takes longer to develop antibodies.
Superior methods of detecting infectious diseases within days of infection are desirable, especially those that allow for detection of infectious diseases within minutes or seconds.
A method of preparing a functionalized electrode is provided. The method comprising: depositing a conductive material onto the surface of a substrate by droplet-based printing, such as aerosol jet printing, of particles comprising an electrically-conductive material, and functionalizing the surface of the conductive material with a binding reagent that binds to an analyte.
In another aspect or embodiment, an electrode for sensing an analyte is provided, comprising: a substrate comprising a droplet-based printed, and optionally sintered, conductive material, and a coating comprising a binding reagent covalently bonded to the surface of the conductive material that binds to an analyte.
In another aspect or embodiment, a microfluidic test device is provided, comprising: one or more sensing electrodes in a chamber or channel configured to receive a liquid test sample, wherein the sensing electrode comprises a substrate and an electrode array comprising a working electrode, a counter electrode, and, optionally, a reference electrode on the substrate, and the working electrode comprises a droplet-based printed, and optionally sintered conductive material, and a coating comprising a binding reagent covalently bonded to the surface of the conductive material that binds to an analyte.
In another aspect or embodiment, a method of sensing an analyte is provided, the method comprising: contacting a fluid comprising the analyte with the electrode comprising: a substrate comprising a droplet-based printed, and optionally sintered, conductive material, and a coating comprising a binding reagent covalently bonded to the surface of the conductive material that binds to an analyte.
Non-limiting aspects of the invention will now be described in the following numbered clauses:
Clause 1: A method of preparing a functionalized electrode, comprising:
Clause 2: The method of clause 1, wherein the conductive material is deposited as a plurality of protuberances onto the surface of the substrate.
Clause 3: The method of clause 2, wherein the protuberances and have a diameter of not greater than 10 millimeters, and the area of the substrate comprising the protuberances is less than or equal to 200 square millimeters (mm2) and comprises at least one protuberance per mm2.
Clause 4: The method of clause 2, wherein the area of the substrate comprising the protuberances is 4 mm2 and comprises at least 9 protuberances per mm2, at least 16 protuberances per mm2, or at least 25 protuberances per mm2 or the area of the substrate comprising the protuberances is 16 mm2 and comprises at least 4 protuberances per mm2 or at least 6.25 protuberances per mm2.
Clause 5: The method of any one of clauses 1 to 4, wherein the conductive material is deposited as an ink comprising the particles, a binder, and/or solvents.
Clause 6: The method of clause 5, wherein the particles are nanoparticles or microparticles and/or are deposited by aerosol jet printing.
Clause 7: The method of clause 6, wherein the particles are nanoparticles having a diameter of at least 4 nanometers to not greater than 1 micron.
Clause 8: The method of clause 6, wherein the particles are microparticles having a diameter of at least 1 micron to not greater than 1 millimeter.
Clause 9: The method of any one of clauses 1 to 8, wherein the particles are suspended in a non-conductive polymer solution.
Clause 10: The method of any one of clauses 1 to 9, wherein the droplets comprise a solvent, and substrate is maintained at a temperature of 50° C. or greater during the deposition of the protuberances to evaporate the solvent.
Clause 11: The method of any one of clauses 1 to 10, wherein the electrically-conductive material of the particles comprises gold, silver, platinum, nickel, rhodium, zinc, an alloy of any of the preceding, carbon, a conductive polymer, graphene, such as graphene oxide, molybdenum disulfide (MoS2), MXenes, such as titanium carbide, or any combination thereof.
Clause 12: The method of clause 11, wherein the electrically-conductive material of the particles comprises gold.
Clause 13: The method of clause 11, wherein the electrically-conductive material of the particles comprises a conductive polymer.
Clause 14: The method of any one of one of clauses 2 to 13, wherein the protuberances are individual pillars.
Clause 15: The method of clause 14, wherein the individual pillars have a height ranging from 1 micron to 1,000 microns and a diameter ranging from 0.1 microns to 500 microns.
Clause 16: The method of any one of clauses 2 to 13, wherein the protuberances form an open cell lattice.
Clause 17: The method of clause 16, wherein the open cell lattice comprises a plurality of unit cells, wherein each unit cell comprises a plurality of trusses joined at one or more joints and, together with one or more unit cells of the lattice, forming a repeated pattern of trusses defining at least a portion of the lattice.
Clause 18: The method of any one of clauses 1 to 17, further comprising sintering the deposited conductive material.
Clause 19: The method of clause 18, wherein the sintering is conducted at a temperature above 100° C. for at least 10 minutes.
Clause 20: The method of any one of clauses 1 to 19, further comprising coating the deposited conductive material with an electrically active material.
Clause 21: The method of clause 20, wherein the electrically active material comprises graphite, hard carbon, synthetic graphite, carbon black, graphene, such as graphene oxide, carbon nanotubes, gold, molybdenum disulfide (MoS2), MXenes, such as titanium carbide, or any combination thereof.
Clause 22: The method of clause 21, wherein the electrically active material comprises graphene.
Clause 23: The method of any one of clauses 1 to 22, further comprising coating the deposited conductive material and/or electrically conductive material with a linking molecule comprising a first portion, a second portion, and a linking portion, wherein the first portion of the linking molecule comprises a functional group for attachment of the linking molecule to the surface of the protuberance, the second portion comprises a functional group for attachment of the linking molecule to the binding reagent, and the linking portion of the molecule extends between the first portion and the second portion.
Clause 24: The method of clause 23, wherein the linking molecule is (3-aminopropyl)triethoxysilane (APTES), L-Cysteine, thioglycolic acid, poly(ethylene glycol), N-hydroxysuccinimide esters, 11-mercaptoundecanoic acid, 12-mercaptodeodecanoic acid, or any combination thereof.
Clause 25: The method of any one of clauses 20 to 24, further comprising reacting the electrically active material or the second portion of the linking molecule with the binding reagent, to link the binding reagent to the deposited conductive material.
Clause 26: The method of any of one of clauses 1 to 25, wherein the binding reagent comprises: a protein, such as a lectin; an antibody or an antibody fragment; an epitope-containing polypeptide, an antigen; an aptamer, an affimer, a nucleic acid or any combination of the preceding.
Clause 27: The method of clause 26, wherein the binding reagent comprises an antigen or epitope of a pathogen, such as a virus, a bacteria, a fungus, or a parasite, such as a protein of a coronavirus, such as SARS-CoV-2, ebola virus, human immunodeficiency virus (HIV), influenza virus, herpes virus, zika virus, Escherichia coli, or Mycobacterium tuberculosis.
Clause 28: The method of clause 27, wherein the antigen is a SARS-CoV-2 spike protein antigen.
Clause 29: An electrode, comprising:
Clause 30: The electrode of clause 29, wherein the deposited conductive material comprises gold, silver, platinum, nickel, rhodium, zinc, alloys of any of the preceding, carbon, a conductive polymer, graphene, such as graphene oxide, molybdenum disulfide (MoS2), MXenes, such as titanium carbide, or any combination thereof.
Clause 31: The electrode of claim 29 or 30, wherein the conductive material is deposited as a plurality of protuberances onto the surface of the substrate.
Clause 32: The electrode of clause 31, wherein the area of the substrate comprising the protuberances is less than or equal to 200 square millimeters (mm2) and comprises at least 1 protuberance per mm2.
Clause 33: The electrode of clause 31, wherein the area of the substrate comprising the protuberances is 16 mm2 and comprises at least 4 protuberances per mm2 or at least 6.25 protuberances per mm2 or the area of the substrate comprising the protuberances is 4 mm2 and comprises at least 9 protuberances per mm2, at least 16 protuberances per mm2, or at least 25 protuberances per mm2.
Clause 34: The electrode of any one of claims 31 to 33, wherein the protuberances are individual pillars.
Clause 35: The electrode of clause 34, wherein the individual pillars have a height ranging from 1 micron to 1,000 microns and a diameter ranging from 0.1 microns to 500 microns.
Clause 36: The electrode of any one of clauses 31 to 33, wherein the protuberances form an open cell lattice.
Clause 37: The electrode of clause 36, wherein the open cell lattice comprises a plurality of unit cells, wherein each unit cell comprises a plurality of trusses joined at one or more joints and, together with one or more unit cells of the lattice, forming a repeated pattern of trusses defining at least a portion of the lattice.
Clause 38: The electrode of any of one of clauses 29 to 37, wherein the binding reagent comprises: a protein, such as a lectin; an antibody, an antibody fragment, or an engineered antibody, e.g., an scFv; an epitope-containing polypeptide, an antigen; an aptamer, a nucleic acid, or any combination of any of the preceding.
Clause 39: The electrode of clause 38, wherein the binding reagent comprises an antigen or epitope of a protein of a virus, a bacteria, a fungus, or a parasite, such as a protein of a coronavirus, such as SARS-CoV-2, ebola virus, human immunodeficiency virus (HIV), influenza virus, herpes virus, zika virus, Escherichia coli, or Mycobacterium tuberculosis.
Clause 40: The electrode of clause 39, wherein the antigen or epitope is a SARS-CoV-2 spike protein antigen or epitope.
Clause 41: A microfluidic test device comprising:
Clause 42: The microfluidic test device of clause 41, wherein the conductive material is deposited as a plurality of protuberances.
Clause 43: The microfluidic test device of clause 41 or 42, comprising:
Clause 44: The microfluidic test device of any one of clauses 41 to 44, wherein the sensing electrode comprises a plurality of leads attached independently to the counter electrode, the working electrode, and, when present, to the reference electrode, and a module configured to send an electrical signal to and/or receive an electrical signal from the sensing electrode.
Clause 45: The microfluidic test device any one of clauses 41 to 44, wherein the test device comprises at least two sensing electrodes.
Clause 46: The microfluidic test device of any one of clauses 41 to 44, wherein the chamber or channel comprises soda-lime glass, polydimethylsiloxane, polymethyl methacrylate, sapphire, or any combination thereof.
Clause 47: The microfluidic test device of any one of clauses 41 to 46, wherein the substrate comprises glass, silicon, silicon dioxide, or any combination thereof.
Clause 48: The microfluidic test device of any one of clauses 41 to 47, wherein the microfluidic channel is configured to separate plasma from blood.
Clause 49: The microfluidic test device of any one of clauses 41 to 48, wherein the binding reagent comprises an antigen or epitope of a protein of a virus, a bacteria, a fungus, or a parasite, such as a protein of a coronavirus, such as SARS-CoV-2, ebola virus, human immunodeficiency virus (HIV), influenza virus, herpes virus, zika virus, Escherichia coli, or Mycobacterium tuberculosis.
Clause 50: The microfluidic test device of clause 49, wherein the antigen or epitope is a SARS-CoV-2 spike protein antigen or epitope.
Clause 51: The microfluidic test device of any one of clauses 41 to 50, comprising two different working electrodes, each having different binding reagents.
Clause 52: The microfluidic test device of clause 51, wherein a first working electrode comprises a SARS-CoV-2 antigen or epitope, and a second working electrode comprises an influenza antigen or epitope.
Clause 53: A method of sensing an analyte, the method comprising:
Clause 54: The method of clause 53, wherein the fluid is plasma.
Clause 55: The method of clause 53, wherein the analyte is a binding partner to the binding reagent.
Clause 56: The method of clause 55, wherein the analyte is an antibody.
Clause 57: The method of any one of clauses 53 to 56, wherein the binding reagent comprises an antigen or epitope of a virus, a bacteria, a fungus, or a parasite, such as a protein of a coronavirus, such as SARS-CoV-2, ebola virus, human immunodeficiency virus (HIV), influenza virus, herpes virus, zika virus, Escherichia coli, or Mycobacterium tuberculosis.
Clause 58: The method of clause 57, wherein the antigen or epitope is a SARS-CoV-2 spike protein antigen or epitope.
Clause 59: The method of any one of clauses 53 to 58, comprising two different working electrodes, each having different binding reagents.
Clause 60: The method of clause 59, wherein a first working electrode comprises a SARS-CoV-2 antigen or epitope, and a second working electrode comprises an influenza antigen or epitope.
These and other features and characteristics of the present disclosure, as well as the methods of operation and functions of the related elements of structures and the combination of parts and economies of manufacture, will become more apparent upon consideration of the following description and the appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals designate corresponding parts in the various figures. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limit of the invention.
Other than in the operating examples, or where otherwise indicated, the use of numerical values in the various ranges specified in this application are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. Further, as used herein, all numbers expressing dimensions, physical characteristics, processing parameters, quantities of ingredients, reaction conditions, and the like, used in the specification and claims are to be understood as being modified in all instances by the term “about”. Moreover, unless otherwise specified, all ranges disclosed herein are to be understood to encompass the beginning and ending range values and any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less, e.g., 1 to 3.3, 4.7 to 7.5, 5.5 to 10, and the like.
As used herein “a” and “an” refer to one or more. The term “comprising” is open-ended and may be synonymous with “including”, “containing”, or “characterized by”. The term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. The terms “a” and “an” are intended to refer to one or more.
As used herein, spatial or directional terms, such as “left”, “right”, “inner”, “outer”, “above”, “below”, “over”, “under”, and the like, relate to the invention as it is shown in the drawing figures are provided solely for ease of description and illustration, and do not imply directionality, unless specifically required for operation of the described aspect of the invention. It is to be understood that the invention can assume various alternative orientations and, accordingly, such terms are not to be considered as limiting.
A “group” or “functional group” is a portion of a larger molecule comprising or consisting of a grouping of atoms and/or bonds that confer a chemical or physical quality to a molecule. A “residue” is the portion of a compound or monomer that remains in a larger molecule, such as a polymer chain, after incorporation of that compound or monomer into the larger molecule. A “moiety” is a portion of a molecule, and can comprise one or more functional groups.
To prepare electrode assemblies and a three-dimensional (3D) electrode array as described herein, a plurality of protuberances are deposited onto the surface of a substrate by a droplet-based printing method. In the droplet-based printing method, such as aerosol jet (AJ) printing, an electrically-conductive material is dispersed in a liquid medium, such as a solvent, and is deposited in a suitable pattern on a suitable substrate. An “array” refers to, in the context of the electrodes described herein, a substrate with multiple protuberances deposited on the surface of a substrate, e.g., by aerosol jet printing.
As used herein, “aerosol jet printing” or “AJ printing”, also referred to as Maskless Mesoscale Materials Deposition or M3D, involves atomization of ink, e.g., by ultrasound or by pressurized gas, and entraining the ink droplets into a stream of gas for delivery to a print head that focuses the gas stream, for example using a gas sheath. An aerosol jet printer, or AJ printer is a device or system used for aerosol jet printing. Aerosol jet printing is capable of producing and accurately-depositing ink particles of 10 microns (μm, or micrometers) or less. As such, aerosol jet printing is capable of producing structures/features 10 μm or greater in size. Aerosol jet printing is capable of delivering suitably-sized particles, such as particles having a diameter of not greater than 10 millimeters (mm), for preparation of electrode structures as described herein.
The particles may be nanoparticles or microparticles. By “nanoparticle(s)” it is meant particles in a size range, either absolute or statistically defined (e.g., average or median), of from 1 nm to 1000 nm, or more typically from 1 nm to 100 nm, which may be defined according to any standard, e.g., ultrafine particles or as defined under ISO/TS 80004. The particles may be nanoparticles having a diameter of at least 4 nanometers (nm) to not greater than 1 μm. The particles may be microparticles having a diameter of at least 1 μm to not greater than 1 mm.
An AJ printer creates an aerosol mist of the droplets of ink comprising the particles comprising an electrically-conductive material from a reservoir by using either a pneumatic or an ultrasonic atomizer and utilizes an aerodynamic focus to deposit aerosolized materials onto the substrate. Pneumatic atomization is used for the printing of thicker liquids such as polymers. The aerosol jet printing may be carried out with an atomizer gas flow rate of 1-30 sccm (standard cubic centimeters per minute) and a sheath gas flow rate of 1-70 sccm, which varies with particular liquid media and viscosities.
The AJ printing “ink” comprises particles suspended in a solvent. The solvent may be any suitable solvent, for example and without limitation: deionized water, ethylene glycol, toluene, hexane, 2-methoxyethanol, glycerol, 2-amino-2-methyl-1-propanol (AMP), tetradecane, or a combination of two or more of the preceding liquids. The solution may comprise a rheology modifier, such as ethylene glycol, N-vinylpyrrolidone, or hydrophobically modified ethylene oxide urethane (HEUR), or a combination of two or more rheology modifiers. The solvent and rheology modifier may be the same, as is the case of ethylene glycol. The ink may also comprise a binder or binding agent. Useful binding agents for metal particles, such as in the context of conductive ink include, without limitation: polyalkylene carbonates, acrylic resins, or 2-methoxyethanol, or a combination of two or more binding agents.
The particles for deposition by aerosol jet printing may be electrically-conductive materials such as a metals, carbon black, carbon allotropes (e.g., conductive carbon allotropes, such as graphite, carbon nanotubes, graphene, or fullerenes), graphene, such as graphene oxide, molybdenum disulfide (MoS2), MXenes, such as titanium carbide, ceramics, conductive polymers, or any combination thereof. The particles may be metal, such as gold, silver, platinum, nickel, rhodium, zinc, an alloy of any of the preceding, or a combination of any of the preceding. The particles may be coated with a polymer, such as poly(ethylene glycol) (PEG), polyethylenimine, thiols, or amines, or a combination of any of the preceding, to inhibit agglomeration in the dispersion. The electrically-conductive particles may be suspended in a non-conductive polymer. The electrically-conductive particles may be droplets of a conductive polymer, such as, without limitation, a polyaniline, a polyacetylene, a polythiophene, a poly(p-phenylene sulfide), a poly(p-phenylene vinylene), a polyindole, or a polypyrrole, or a combination of any of the preceding.
Each protuberance of an array may be of the same or different materials or a combination of materials. If the printed protuberances are of different materials, the material is either mixed prior to printing, or printed independently, e.g., sequentially.
The protuberances may be formed by depositing, for example and without limitation by aerosol jet printing, onto a surface of a substrate a plurality of layers of the electrically conductive material. Non-limiting examples of suitable substrates include flexible or rigid polymers, metals, alumina, ceramics, silicon structures, glass, diodes, integrated circuit, or a circuit board such as a printed circuit board (PCB). Examples of flexible polymers include but are not limited to polydimethylsiloxane (PDMS), Kapton® (polyimide), or Poly(lactic acid) (PLA). The substrate also may include an electrical path, such as conductive leads, conductive traces, or conductive pads, for the electrodes to an external circuit. The protuberances may be deposited on or in electrical contact with a conductive lead, trace, or pad. Conductive traces, leads, or pads may be formed on a non-conducting substrate by methods such as physical vapor deposition or chemical vapor deposition with a thin layer of conducting material, such as a metal or a conducting polymer, to provide the electrical path. For example, the substrate may be a glass, coated with chromium and/or gold. Examples of suitable metal substrates include stainless steel, copper, aluminum, silver, gold, chromium, and tin. Metal substrates may optionally be coated or patterned with an additional, different conducting material and/or insulating layers, such as parylene, to form suitable electrical paths for the individual electrodes. For example, the substrate may be stainless steel, optionally with a 50 nm chromium coating or a thin poly(3,4-ethylenedioxythiophene) (PEDOT) coating. The substrate may be a component of a sensing electrode or other structure or device the array is to be incorporated into. For an electrode assembly, the substrate shape, and configuration or arrangement of the electrodes and 3D electrode arrays may be varied, depending on the ultimate structure of the assembly and any associated microfluidics.
The protuberances may be printed on a substrate that is planar or non-planar. The substrate may be selected to withstand heating to a sintering temperature of the material(s) forming the protuberances.
The area of the substrate having the printed protuberances may be less than or equal to 200 square millimeters (mm2), less than or equal to 100 mm2, less than or equal to 50 mm2, or less than or equal to 16 square millimeters (mm2), for example 100 mm2, 75 mm2, 50 mm2, 25 mm2, 20 mm2, or 16 mm2, or any increment in the stated ranges. The printed protuberance may have any suitable density, for example and without limitation, of at least one protuberance per mm2, such as at least 2.25 protuberances per mm2. For example, the area of the substrate having the protuberances may be 16 mm2 and may have at least 4 protuberances per mm2 or at least 6.25 protuberances per mm2. The area of the substrate having the protuberances may be 4 mm2 and may have at least 9 protuberances per mm2, such as at least 16 protuberances per mm2, or such as at least 25 protuberances per mm2. Suitable densities of the protuberances may be optimized based on the thickness of the protuberances, providing sufficient gaps between the protuberances for liquid flow, for example and without limitation to maximize liquid flow through the array, and surface area of the protuberances. Suitable densities of the protuberances on the substrate may be optimized to control the limit of detection (LOD) and the electrical response of the 3D electrode array and can be tailored by one having ordinary skill in the art.
The protuberances can be made using other suitable additive manufacturing methods such as, but not limited to, inkjet printing, gravure printing, extrusion printing, and 2-photon lithography. In one example, the protuberances may be made using manufacturing methods such as, but not limited to, lithographic methods.
The ink from which the protuberance structures are fabricated may be dispensed, e.g., using an AJ printer, in multiple layers. The process of printing a 3D electrode array may involve deposition of one layer or droplet(s) of the particle solution followed by the use of heat or other form of energy to remove (evaporate) the solvent of the ink. The substrate, and therefore the protuberances may be heated to a temperature sufficient to remove the solvent by either heating the substrate to a suitable temperature, or by directing a laser at the site of particle solution deposition. The substrate temperature may be maintained in a range of from, for example and without limitation, 25° C. to 150° C. or increments there between, such as 80° C., 100° C., 110° C., or 125° C. After deposition and removal of solvent, a layer is formed, which by itself or in combination with other layers, e.g., in a layer of a protuberance, forms a solid base to receive the next printed layer. The next printed layer may have the same or different composition as the previously deposited layer. This process is repeated as desired, e.g., according to a predetermined protuberance configuration, to produce high aspect ratio, and high surface area electrodes, for example as shown in
The protuberances may be deposited as individual pillars across an area of the substrate to form a 3D electrode array. By individual, it is meant that the pillars do not connect or bridge aside from their connection via the substrate, for example as compared to an open-cell matrix or lattice as described herein. The individual pillars may be solid. The individual pillars may be hollow (see, e.g.,
For example, the array dimension may be 10×10, the area of the substrate having the individual pillars may be 4 mm2 and may have at least 25 individual pillars per mm2.
Alternatively, at least a part of the protuberances may be deposited in the form of irregular or regular three-dimensional structures of trusses, plates, open cell lattice, or other shapes on the substrate. An open cell lattice comprises a plurality of unit cells, where each unit cell comprises a plurality of trusses joined at one or more joints. Together with one or more unit cells of the lattice, a repeated pattern of trusses defines at least a portion of the lattice. Exemplary lattice structures are shown in
The trusses can be any suitable shape, so long as the arrangement thereof in the lattice permits flow of liquid throughout the lattice. Non-limiting shapes include: a rod, a cylinder, a column shape, a cylindroid, a scutoid, a conical shape, a polyhedron, a sphere, a spheroid, an ovoid, a spiral, or a helix. Shapes can be combined in order to make the lattice. The open lattice structures can be made of unit cell or cells of arbitrary shapes and sizes comprising two or more trusses joined at a joint or node in a geometric configuration. The smallest group of trusses in at least a portion of the lattice is a unit cell. The open lattice can have repeating or non-repeating unit cells. The number of repeating unit cells of the lattice structure may be any number, such as in the range of 1 to 100,000 in the X-dimension, Y-dimension, and/or Z-dimension.
Non-limiting examples of suitable repeated unit geometries include: square, rectangular, triangular, hexagonal, octahedral, rhomboidal, icosahedral, spherical, or any other regular or irregular shape and/or pattern of joined trusses. In one example, the open lattice has an octahedral unit cell comprising eight trusses joined to produce an eight-sided structure, formed from eight trusses joined at six nodes or joints. As would be understood by a person of ordinary skill, the stated geometric designation for any unit cell describes the arrangement of the trusses, and due to the three-dimensional shapes of the trusses, does not define absolutely a resultant 3D structure created by the trusses, for example because the trusses are cylindrical and therefore cannot form a perfect geometric edge (a line) for a shape such as an octahedron. Further, the trusses do not necessarily define every edge of a structure. The open lattice may have a combination of two or more unit cell geometries. For example, the open lattice can have a combination of hexagonal and octahedral unit cells. The open lattice may have different unit cells of the same or different sizes, where the cell size of the open lattice controls the porosity, in terms of voids and open channels. The cells may have a periodicity (average distance between centers of, or like features of adjacent unit cells) ranging from 1 μm to 1 mm, such as from 2 μm to 500 μm, from 10 μm to 1000 μm, or from 100 μm to 300 μm. The structure of the lattice may be such that individual cells are indistinguishable in a dimension, e.g., forming a tubular, or elongated cell, and periodicity may be only in one or two dimensions.
The trusses may be deposited perpendicular (normal), at a 90 degree (90°) angle to, a plane of the substrate, or at any other angle to the substrate between 0° and 90°, including any increment there between, such as 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, or 85°. One or more of the trusses may be deposited normal to the substrate. One or more of the trusses may be deposited at an angle between 10° and 90°, such as between 30° and 40°, to the substrate. The trusses can be 3D printed straight, curved or any other shape suitable for the end use. The trusses can have different shapes, even within a single unit cell.
The open cell lattice comprises trusses joined at nodes, or joints, between truss structures, such as rods. Each lattice truss ranges from 1 μm to 1 mm in a major dimension (length, that is distance between nodes or joints), depending on the geometry of the lattice, to yield suitable cell periodicity in the range of from 2 μm to 500 μm, and in a lattice structure, trusses can be of the same length or a combination of lengths. For example, the lattice structure may contain trusses that are a single length throughout the lattice structure, or different lengths, to produce certain geometries. Lattice structures may have a length ranging from 5 μm to 5 centimeters (cm), such as from 100 μm to 1 cm or from 200 μm to 500 μm. The overall size and shape of the lattice may be selected to fit within the area of the electrode. The truss diameter, e.g., the diameter of a spherical truss, or a non-major dimension, such as a diameter of a rod, with a circular or ovoid cross-section, may range from 0.1 μm to 500 μm, such as 2 μm to 500 μm, such as 20 μm to 50 μm, such as 1 μm to 100 μm, and can be selected to optimize surface area and liquid flow through the matrix of the open cell lattice.
The as-printed protuberances may be sintered or joined together by some energy source, such as laser, UV light, or thermal heat. In some examples, the printed protuberance structures are not sintered, leaving the protuberance structures in a particle format. Sintering is the process of forming a solid mass of material by heat or pressure without melting it to the point of liquefaction. Sintering occurs naturally in mineral deposits, or as a part of a manufacturing process used with metals, ceramics, plastics, and other materials. In the context of the deposited particles described herein, particularly the metal particles, sintering is a heat treatment applied to a particle powder structure in order to impart strength and integrity. The temperature used for sintering is below the melting point of the major metal constituent of the particle material. After printing, neighboring powder particles are held together by cold welds, which give the compact sufficient “green strength” to be handled. At sintering temperature, diffusion processes cause necks to form and grow at these contact points. As a consequence of the sintering process, water and other liquid medium or solvents, rheology modifiers, and anti-agglomeration coatings are removed by evaporation and burning, and any surface oxides are reduced.
The particles comprising an electrically-conductive material, such as metal particles, printed on the surface of the substrate as a protuberance, may be sintered by raising the temperature to a temperature below the melting point of the metal particles. During the sintering process, the temperature may be raised at any rate. The temperature may be raised at a rate of less than 5° C. per minute, such as 1° C. per minute. Once the sintering temperature is reached, the sintering process may be stopped by reducing the temperature at any suitable rate, or the maximum sintering temperature may be maintained for any suitable time period, such as for 5 minutes (5′), 10′, 20′, 30′, one hour, 2 hours, or longer, including increments there between. The particles comprising an electrically-conductive material may be sintered by heating to temperatures that varies with the material. For example, sintering for gold nanoparticles occur in a temperature range of from about 200° C. to 500° C. Optimal maximum, sintering temperatures, temperature ramp rates, or durations may be empirically determined depending on the composition and structure of the particles.
Sintering the printed protuberances introduces hierarchical porosity. The hierarchical porosity can be controlled by varying the sintering temperature, duration, and temperature ramping rates. For example, the porosity of the protuberances may be from 20% to 30%.
An electrically active material may be deposited over at least a portion of the protuberances to form an outer shell. The protuberances may be coated, at least in part, with an electrically active material through an appropriate deposition method such as electroplating, atomic layer deposition (ALD), sputtering, physical vapor deposition, or chemical vapor deposition. Non-limiting examples of electrically active materials include conductors, semiconductors, insulators, or any combination thereof, such as, for example and without limitation: graphite, hard carbon, synthetic graphite, carbon black, graphene flakes (planar or 2D graphene, which alternatively may be referred to simply as graphene), carbon nanotubes, graphene oxide, MoS2, MXenes, such as titanium carbide, or gold. For example, the protuberances may be gold and may be coated with graphene, e.g., graphene flakes. For example, the protuberances may be silver and may be coated with gold.
A linking molecule having a first portion, a second portion, and a linking portion may be deposited over at least a portion of the protuberances to link a binding reagent or binding partner to the protuberances. The protuberances may be in any form or composition such as the protuberances described above, e.g., the sintered protuberances or the protuberances coated with an electrically active material. The linking molecule having a first portion, a second portion, and a linking portion may have the following formula I:
X-Y-Z (I)
where X is the first portion, Z is the second portion, and Y is the linking portion that extends between the first portion and the second portion. X and Z can be functional groups that may be used to link a protein, an enzyme, a nucleic acid, or another suitable composition to the protuberances. For example and without limitation, X may be a thiol (—SH), an amine, such as a primary amine (—NH2), a carboxylic acid (—C(O)OH), an aldehyde (—C(O)H), a hydroxyl (—OH), a phosphate (PO4−), a sulfonic acid (—S(O)2—OH), an ester (—C(O)O—), a cysteine moiety, or any functional group or any combination thereof that reacts with the electrically active material. Z may be, without limitation, a carboxylic acid group, a hydroxyl group, an alkene, an alkyne, a ketone, an azido, or a primary amine group, or any combination thereof. The second portion may be selected based on available and/or reactive groups or moieties, such as amine or carboxyl groups, of the binding reagent. Y may be an alkyl or hydrocarbyl chain, having a sufficient number of carbon atoms to provide spatial or steric separation between the first portion and the second portion of the molecule. For example, the alkyl chain of the linkage portion may be long enough, e.g., with 5-20 carbon atoms, so that the molecules of the binding reagent have sufficient space to bind to the second portion of the molecule. The linkage portion may be an oligomer of any suitable monomer, such as an acrylate, styrene, or olefin monomer, and may be prepared using a controlled polymerization method for low polydispersity, such as controlled radical polymerization methods, e.g., atom-transfer radical polymerization.
“Alkyl” refers to straight, branched chain, or cyclic hydrocarbon groups, or combinations thereof, including from 1 to about 20 carbon atoms, for example and without limitation C1-3, C1-6, C1_10, C1-C12, C1-18 groups, for example and without limitation, straight, branched chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like.
Suitable linker molecules having a first portion, a second portion, and a linking portion that may be used include, but are not limited to, glutaraldehyde, (3-aminopropyl)triethoxysilane (APTES), L-Cysteine, thioglycolic acid, poly(ethylene glycol), N-hydroxysuccinimide esters, 11-mercaptoundecanoic acid, 12-mercaptodeodecanoic acid, or any combination thereof. Either of the first or second portion of the linking molecule may comprise the reaction product of two or more additional compounds. For example, as shown in
For example, the protuberance may be a gold protuberance with 11-mercaptoundecanoic acid deposited thereover. The thiol group of the 11-mercaptoundecanoic acid molecule binds to the surface of the protuberance and the carboxylic acid group is available to react with the reactive groups or moieties of the binding reagent (
The binding reagent may covalently react with an electrically active material deposited over the surface of the protuberances, such that the binding reagent is covalently bonded to the surface of the protuberances (
The term “binding reagent” refers to a compound having a binding moiety that binds specifically and non-covalently to a binding moiety of another compound, such as a specific target or analyte, referred to as its binding partner. The binding reagent and its binding partner bind non-covalently to form a binding pair. The strength of binding between members of a binding pair may be referred to as avidity. Non-limiting examples of binding pairs, include, but are not limited to: antigen/antibody, epitope/paratope (antigen binding site, ABS), lectin/carbohydrates, complementary nucleic acids, or aptamer/aptamer target.
The binding reagent on the 3D electrode array described herein may be a protein, an antibody, an antibody fragment, an epitope-containing polypeptide, an antigen, an aptamer, a nucleic acid, an affimer, or a combination of any of the preceding. The binding reagent may be an antigen or epitope of a protein of a coronavirus, such as SARS-CoV-2, ebola virus, human immunodeficiency virus (HIV), influenza virus, a herpes virus, zika virus or a bacteria, such as E. coli or M. tuberculosis. For example, the binding reagent may be SARS-CoV-2 spike S1 protein or SARS-CoV-2 spike receptor binding domain (RBD) protein. The binding reagent may be an antiviral antibody or antibody fragment, such as an antibody targeting an antigen of a coronavirus such as SARS-CoV-2, an ebola virus, a human immunodeficiency virus (HIV), influenza virus, a herpes virus or a zika virus. For example, the binding reagent may be an anti-SARS-CoV-2 spike S1 antibody or an anti-SARS-CoV-2 spike RBD antibody.
The binding reagent may comprise an antigen. The term “antigen” refers to a compound, composition, or substance that is produced by pathogen that stimulates an immune response in an animal, including, without limitation, proteins, polysaccharides, and glycoproteins. An antigen may be an oligopeptide or polypeptide comprising one or more epitopes of a protein of a pathogen. For example, the antigen may be a protein or an enzyme that is present on the surface of the pathogen. As used herein, “pathogen” refers to an infectious agent or pathogen, such as a bacteria, fungus, parasite, or a virus. For example, the binding reagent may be an antigen of a bacteria, such as Escherichia coli or Mycobacterium tuberculosis. The binding reagent may be an antigen of a virus, such as a coronavirus such as SARS-CoV-2, an influenza virus, a herpes virus, a human immunodeficiency (HIV) virus, an ebola virus, or a zika virus. Examples of suitable antigens include proteins of a pathogen that commonly elicits antibodies, such as neutralizing antibodies, including, without limitation: a coronavirus spike protein, an influenza virus hemagglutinin or neuraminidase protein, an ebola virus glycoprotein, or a zika virus envelope protein, as are broadly-known. The entire protein, or antigenic portions or fragments of an antigenic protein may be used in the structures, devices, and methods described herein. For example, the S1 portion of SARS-CoV-2, or E80 ectodomain of the zika envelope protein may be used. Useful antigens, such as fragments of larger proteins may be identified by their ability to specifically bind (for example) antibodies generated by a patient. Antigenic proteins are broadly-known in the art and typically are described in public databases, such as GenBank and UniProt. For example, SARS-CoV-2 proteins are provided in NCBI Reference Sequence: NC_045512.2, UniProtKB—PODTC2 (SPIKE_SARS2), and UniProtKB—PODTC9 (NCAP_SARS2, nucleocapsid), and fragments of the spike protein, e.g., S1 and S2 also may be used in the devices and methods provided herein. Determining appropriate antigens for use in binding antibodies found in patient's blood is well within the abilities of those of ordinary skill in the relevant arts.
The binding reagent may be an antibody or an antibody fragment, including engineered antibody fragments. The term “antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. As such, the antibody operates as a ligand for its cognate antigen, which can be virtually any molecule. Natural antibodies comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site capable of specifically binding an antigen (e.g., a paratope). The term “VH” refers to a heavy chain variable region of an antibody. The term “VL” refers to a light chain variable region of an antibody. Antibodies may be derived from natural sources, or partly or wholly synthetically produced. Many antibodies and fragments thereof are available from commercial sources. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
The term “antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments, but are not limited to, Fab, Fab′, F(ab′)2, Fv, Fd, dsFv, scFv, diabody, triabody, tetrabody, di-scFv (dimeric single-chain variable fragment), bi-specific T-cell engager (BiTE), single-domain antibody (sdAb), or antibody binding domain fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, or it may be recombinantly or synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multi-molecular complex. A functional antibody fragment may consist of at least about 50 amino acids or at least about 200 amino acids. Antibody fragments also include miniaturized antibodies or other engineered binding reagents, such as scFvs, that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding (e.g., paratope) sequences and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition (see, e.g., Nelson, A L, “Antibody Fragments Hope and Hype” (2010) MAbs 2(1):77-83).
As used herein, the term “epitope” refers to a physical structure or moiety on a molecule that interacts with an antibody or antibody fragment.
“Lectins” are a group of proteins from non-immune origins that bind carbohydrates and agglutinate animal cells. They exhibit extremely high binding affinities for specific sugars, and can be used to target specific cell types expressing their binding partner or analytes, including carbohydrates, polysaccharides, glycoproteins, and glycolipids. Lectins can agglutinate cells and/or precipitate complex carbohydrates and, as such, have served as a powerful tool for biomedical research and clinical utility, including, carbohydrate studies, fractionation of cells and other particles, lymphocyte subpopulation studies, mitogenic stimulation, blood group typing, and histochemical studies. They are isolated from a wide variety of natural sources, both plant and animal Concanavalin A (Con A) is a broadly-studied lectin that binds α-D-mannosyl and α-D-glucosyl residues. Peanut agglutinin targets Galβ1-3GalNAcα1-Ser/Thr and, e.g., inhibits T-cell activity and can be used to distinguish lymphocyte subsets. Many other lectins are broadly-known and characterized, and can be obtained from commercial sources.
“Aptamers” are oligonucleotides or peptides that are selected to bind specifically to a desired molecular structure. Peptide aptamers are also referred to as “affimers”. Aptamers typically are the products of an affinity selection process similar to the affinity selection of phage display (also known as in vitro molecular evolution). The process involves performing several tandem iterations of affinity separation, e.g., using a solid support to which the desired immunogen is bound, followed by polymerase chain reaction (PCR) to amplify nucleic acids that bound to the immunogens. Each round of affinity separation thus enriches the nucleic acid population for molecules that successfully bind the desired immunogen. In this manner, a random pool of nucleic acids may be “educated” to yield aptamers that specifically bind to aptamer target binding partners. Nucleic acid aptamers typically are RNA, but may be DNA or analogs or derivatives thereof, such as, without limitation, peptide nucleic acids and phosphorothioate nucleic acids.
The binding reagent may be a nucleic acid or an analog thereof, e.g., a single-stranded nucleic acid or analog thereof, for binding complementary nucleic acid sequences found in a sample. “Complementary” refers to the ability of a single-stranded nucleic acid to hybridize to another single-stranded nucleic acid under assay conditions by Watson-Crick, or Watson-Crick-like base pairing of complementary bases. As such, free nucleic acids in a biological sample may be detected using the described electrode assembly where a binding partner of the free nucleic acid, that is, a complementary nucleic acid, is linked to the electrode array as described herein.
Also provided herein is a microfluidic test device.
The substrate of the electrode assembly may be glass, silicon, silicon dioxide, plastic, or any combination thereof. The substrate may be patterned with a layer of a conductor, such as chromium, gold, silver, platinum, carbon, nickel, or indium tin oxide to form the electrode traces for the reference electrode, the counter electrode, and the working electrode. The electrode traces may be deposited in any useful pattern.
To form the reference electrode traces on the substrate, a conductive material may be coated onto the base of the reference electrode to form the reference electrode by methods known in the art, such as electrodeposition, dip coating, spray coating, curtain coating, or doctor (or draw-down) blade coating. A shadow mask may be used to apply the reference electrode material onto the substrate. The reference electrode material may be an ink containing silver/silver chloride (Ag/AgCl), gold, chromium, platinum, carbon, nickel, indium tin oxide, or any combination thereof. The reference electrode may also be a Calomel electrode, a pseudo silver electrode, or a combination thereof.
The counter electrode may be formed on the substrate in the same manner as the reference electrode and may comprise any suitable conductive material, e.g., as described above. A suitable material may be coated onto the counter electrode trace.
The 3D working electrode array is deposited onto the conductive working electrode trace. As with the reference and counter electrodes, the trace of the working electrode may be any suitable conductor, such as, without limitation: gold, chromium, platinum, silver, copper, or PEDOT. The 3D electrode array is AJ printed directly onto the working electrode trace. Alternatively, the 3D electrode array may be printed on a separate substrate and the substrate having the 3D electrode array may be adhered to the base of the working electrode using an electrically-conductive adhesive, such as an electrically-conductive glue, paste, or tape.
The portion of the working electrode having the 3D electrode array is in direct contact with the microfluidic channel (
Alternatively, droplet-based printing may be used to deposit the conductive material in a two-dimensional, but not necessarily flat pattern over or in contact with a conductive trace or lead on a substrate, without protuberances. The conductive material may be sintered as described above, resulting in a uniquely roughened surface amenable to modification. As above, for the protuberances, the surface may be then coated with another electrically-conductive material as described above, such as a reduced graphene oxide. Then the surface of the electrode may be functionalized with any binding reagent as described above. A benefit to this two-dimensional electrode is that it may be useful in detection of analyte, as described herein, where the sensitivity and high density functionality of the protuberances may be unnecessary or may be contraindicated. Also, a mixed electrode surface comprising two-dimensional portions and portions comprising protuberances may be provided, by which means sensitivity of the electrode may be tailored for the binding reagent/analyte combination.
The electrode may also include additional leads, or wires, attached independently to the reference electrode, the counter electrode, and the working electrode. The leads are then connected to a controller system. The module may send electrical signals to the leads. The module may receive an electrical signal from the electrode assembly. The controller system includes any required elements for powering the system, such as batteries, e.g., rechargeable batteries and/or a power supply, such as a DC power supply for powering other local components of the system, signal amplifier(s), microprocessors, non-transient memory such as read-only memory, hard drives, or flash drives, random access memory, analog-to-digital (A/D) converters, and/or communication modules for communicating with other parts of the system, either wirelessly, or wired. The module may include a controller, memory, a communications module, an input component, and an output component. It is understood that all or part of the controller system may be a separate, wired structure, or integrated into the substrate. The controller system may comprise a BIOS, one or more signal amplifiers, one or more analog-to-digital converters, memory, storage, processor(s), additional electronic hardware components, and/or computer-readable instructions for controlling the module and/or for communication within the system or with a separate device, for example, as are known in the computing arts. The controller system may be contained within a single housing or distributed among two or more devices.
An external module having a communication module for communicating wirelessly or by a wired connection may be connected to the controller system. The external module may include one or more computers, storage, and a communications module for communicating with other elements and, optionally, additional computers and/or computer networks. The external module also may comprise a BIOS, one or more signal amplifiers, one or more analog-to-digital (A/D) converters, memory, storage, processor(s), additional electronic hardware components, and/or computer-readable instructions for controlling the module and/or for communication within the system or with a separate device, e.g., as are known in the computing arts. The external module may be a computer, such as a personal computer, a laptop, a smartphone, or a dedicated controller device. The external module may communicate with the module via a wireless connection, such as by near-field communication (NFC), Zigbee, or Bluetooth protocols. The external module may be connected to any suitable output device, including displays, printers, or may communicate with one or more additional external devices, such as computers or computer networks via any suitable communication means, such as a computer network or over the Internet.
The controller system may comprise a controller for executing functions related to receipt, analysis, and transmission of sensed electrical data. For example, the controller may be a central processing engine including a baseline processor, memory, and communications capabilities. The controller can be any suitable processor comprising computer readable memory and configured to execute instructions either stored on the memory or received from other sources. Computer-readable memory can be, for example, a disk drive, a solid-state drive, an optical drive, a tape drive, flash memory (e.g., a non-volatile computer storage chip), cartridge drive, and control elements for loading new software.
The controller may include an executable program, code, set of instructions, or some combination thereof, executable by the controller system for independently or collectively instructing the controller system to interact and operate as programmed, referred to herein as “programming instructions”. In some examples, the controller is configured to issue instructions to initiate data collection from the electrode assembly and to select types of measurement information that should be recorded. In other instances, the electrode assembly may comprise suitable electronics and instructions configured to automatically transmit electrical signals to the module either in real time or at periodic intervals without first receiving initiation instructions from the controller system to initiate sensing and data transmission.
Processing can include applying filters and other techniques, hardware and/or software based, for removing signal artifacts, noise, baseline waveforms, or other items from captured signals to improve readability. Processing information includes data analysis techniques, such as quantifying various electrical signals based on received data, corroborating or calibrating data from multiple sources, and/or analyzing generated electrical signals to draw conclusions.
The controller system may further comprise an input component and an output component in communication with the controller, which allow a user to interact with and receive feedback from the module. The input component may include one or more of a keyword, touchpad, computer mouse, trackball, or other data entry accessory, for example as are known in the art. The input component can be used to enter information about the test sample which can be used to analyze the measurement data and/or to assist in analysis and training regimens. The input components can also be used to interact with a user interface by, for example, being able to toggle through instruction screens for configuring the electrical signals provided to or received from the sensing electrode. User interface screens that can be shown on a visual display and used for entering information and guiding a user in collecting information about a sample.
The components of the electrode assembly, controller system, and external modules can be combined in various manners with various analog and digital circuitry, including controllers, filters, ADCs (analog-digital chips), memory, communication devices and/or adaptors. As devices become smaller and processors become more powerful and use less energy, it is possible to integrate many more electrical and electronic components into the module. Technologies such as package on package (PoP) and system on a chip (SoC) integrated circuit packages allow manufacture of very small devices with significant capacities. For example, smart phones use PoP technologies to stack memory and processors in a very small volume. One example of a SoC is a microcontroller (MCU), which is a small computer on a single integrated circuit typically containing a processor core, memory, and programmable input/output peripherals. MCUs also may include timer module(s) and analog-to-digital converter(s) for, e.g., converting analog sensor output to a digital signal.
The microfluidic test device includes a microfluidic portion that includes a microfluidic channel, an inlet flowing into the microfluidic channel, and an outlet flowing out of the microfluidic channel. The microfluidic portion of the microfluidic test device may be fabricated using a soft-replica molding method (see, e.g.,
The microfluidic portion of the microfluidic test device may comprise polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass, sapphire, or a combination thereof. For example, the microfluidic portion of the microfluidic test device may be PDMS, where the microfluidic portion of the microfluidic test device is formed using a soft-replica molding method.
The microfluidic channel may be less than or equal to 5 mm in depth, such as less than or equal to 2 mm in depth, such as less than or equal to 1 mm in depth, such as 0.5 mm in depth. The width of the microfluidic channel may vary from 2 mm to 0.5 mm. For example, one part of the microfluidic channel may be 2 mm wide, and another part of the microfluidic channel may be 1 mm wide.
The inlet delivers a fluid to be analyzed into the microfluidic channel (
The fluid may be a biological fluid, such whole blood, serum, plasma, urine, or saliva. The fluid may contain an analyte that is the binding partner of the binding reagent that is coated on the 3D electrode array. The fluid may not contain an analyte that is the binding partner of the binding reagent that is coated on the 3D electrode array.
For example, the 3D electrode array may be coated with an antigen or epitope of a protein of a coronavirus, an ebola virus, a human immunodeficiency virus (HIV), an influenza virus, a herpes virus or a zika virus and the fluid to be analyzed may have an antibody of a coronavirus, an ebola virus, a human immunodeficiency virus (HIV), an influenza virus, herpes virus, or a zika virus. The 3D electrode array may be coated with an antibody of a coronavirus, an ebola virus, a human immunodeficiency virus (HIV), an influenza virus, a herpes virus or a zika virus and the fluid to be analyzed may have an antigen or epitope of a protein of a coronavirus, an ebola virus, a human immunodeficiency virus (HIV), an influenza virus, a herpes virus or a zika virus. The 3D electrode array may be coated with an antigen or epitope of a protein of a bacteria, such as E. coli or M. tuberculosis and the fluid to be analyzed may have an antibody of a bacteria, such as E. coli or M. tuberculosis. The 3D electrode array may be coated with an antibody of a bacteria, such as E. coli or M. tuberculosis, and the fluid to be analyzed may have an antigen or epitope of a protein of a bacteria, such as E. coli or M. tuberculosis. The 3D electrode array may be coated with a spike S1 protein of SARS-CoV-2 and the fluid to be analyzed may be plasma collected from an individual with COVID-19 or suspected as having COVID-19. The 3D electrode array may be coated with a spike RBD protein of SARS-CoV-2 and the fluid to be analyzed may be plasma collected from an individual with COVID-19 or suspected as having COVID-19. The 3D electrode array may be coated with a spike S1 antibody of SARS-CoV-2 and the fluid to be analyzed may be plasma collected from an individual with COVID-19 or suspected as having COVID-19. The 3D electrode array may be coated with a spike RBD antibody of SARS-CoV-2 and the fluid to be analyzed may be plasma collected from an individual with COVID-19 or suspected as having COVID-19.
The microfluidic test device may also include a plasma separator, which is a microfluidic channel that can separate plasma from red blood cells. The plasma separator reduces pre-processing requirements by allowing the direct use of blood samples, e.g., heparin-treated blood samples, in the microfluidic test device. The plasma separator may include a single channel that splits into two separate channels, where the split forms a Y-shape. According to the Zweifach-Fung bifurication law, a channel bifurcates into two outlets, forming a Y-shape junction, with a minimum flow ratio of 2.5:1, separates blood cells from the plasma due to the variation of fluidic pressure at the Y-shaped junction. The plasma may then be directed to the microfluidic channel in direct contact with the sensing electrode for analysis and the blood cells may be discarded through an outlet.
The fluid that enters the microfluidic channel directly contacts the reference electrode, the counter electrode, and the working electrode. A fluid that contains the binding partner to the binding reagent that is coated on the 3D electrode array is sensed or detected by the 3D electrode array on the working electrode. The binding of the binding partner to the binding reagent to form a binding pair may be sensed or detected based on chemical impedance spectroscopy, cyclic voltammetry, chronoamperometry, or differential pulse voltammetry. If the fluid does not contain the binding partner to the binding reagent a signal will not be sensed or detected. Different amounts of the binding partner analyte to the binding reagent in the sample to be tested may result in different signals, permitting calibration and quantification of the analyte.
The outlet of the microfluidic test device permits egress of fluid, such as excess fluid, from the microfluidic channel. Excess fluid includes any fluid that was not analyzed or had been previously analyzed. Fluid can be channeled through the microfluidic channel from the inlet to the outlet to permit analysis of volumes of fluid greater than the volume of the microfluidic channel. For example, fixed volumes of fluid, such as 1, 2, 5, or 10 mL of fluid, can be passed through the microfluidic channel depending on expected analyte concentration, and calibration methods for the device.
The binding partner analyte may be eluted from the binding pair after analysis, thereby regenerating the binding reagent on the 3D electrode array. The sensing electrode may be rinsed with an acidic aqueous solution having a pH that is greater than 2.0 and less than 7.4. For example, the acid may be formic acid having a pH of 2.5. Other liquids, such as aqueous solutions and/or non-aqueous solutions may be used to elute analyte from the electrode assembly, and may be combined for optimal elution and preservation of the electrode assembly. Elution solutions may include solutions with different, e.g., non-physiological, salt concentrations, alcohols, surfactants, emulsifiers, etc. The 3D electrode array may further be rinsed with, for example and without limitation, phosphate buffered saline (PBS) or normal saline after completing an elution step, such as an acid rinse. As such, the electrode assembly can be used to analyze a fluid more than once, such as two times, such as three times, such as four times, such as five times, such as six times, such as seven times, such as eight times, or such as nine times, or more.
The microfluidic test device may include two or more sensing electrodes in direct contact with the microfluidic channel, where each of the sensing electrodes are the same or are different. The microfluidic test device may include a single sensing electrode which may include two or more working electrodes, where each of the working electrodes are the same or different. When different, the two sensing electrodes or two working electrodes may be used to test for at least two different analytes simultaneously, for example two different coronavirus antigens, or antigens from two different variants of a virus (such as two SARS-CoV-2 variants), or two different viruses or other infectious diseases simultaneously. The microfluidic test device may include two, three, four, or more different (multiplex) sensing electrodes or two, three, four, or more working electrodes, for example to detect two or more different infectious diseases. For example, the microfluidic test device may include a sensing electrode or a working electrode specific for the detection of a coronavirus, such as SARS-CoV-2, and a sensing electrode or working electrode specific for the detection of influenza virus.
The sensing electrode having the 3D electrode array may alternatively be open to the air without being in contact with a microfluidic channel, for example within a chamber configured to house the electrodes and having an opening into which a liquid may be deposited to contact the electrodes. For example, a fluid to be analyzed may be applied to the one or more 3D electrode arrays of the sensing electrode(s) as a droplet.
The following examples are presented to demonstrate the general principles of the invention. The invention should not be considered as limited to the specific examples presented.
To construct the 3D array electrode using aerosol jet (AJ) printing, a commercial gold (Au) nanoparticle ink (UTDAu40, UT Dots Inc., Champaign, Ill.) was used. The average Au particle size was 4 nm, the ink viscosity was 3 centipoise (cP), and particle loading in the ink was 40 weight percent (wt %). The Au nanoparticles were dispersed in an organic non-polar solvent, which was aerosolized during AJ printing via ultrasound energy. Polydimethylsiloxane (PDMS) (SYLGARD™ 184 Silicone Elastomer Kit, Dow Corning, Midland, Mich., USA) with a base to hardener ratio of 10:1 was used to create the microfluidic channel of the 3D Printed COVID-19 Test Chip (3DcC) device.
Human recombinant SARS-CoV-2 spike S1-His protein (50 micrograms per milliliter (μg/mL)) and SARS-CoV-2 spike RBD-His protein (50 μg/mL) expressed in HEK293 cells, were the antigens purchased from the Sino Biological US Inc., Wayne, Pa. Before immobilizing on the 3D electrode surface, both the antigens were diluted to 5 μg/mL using a carbonate buffer solution having a pH of approximately 9.6. Two rabbit IgG antibodies, SARS-CoV-2 spike S1 antibody (10 microliters (μL)), and SARS-CoV-2 spike RBD antibody (10 μL) were also obtained from Sino Biological US Inc., Wayne, Pa. Both the antibodies were diluted in phosphate buffer saline solution (pH 7.4) containing a 5 millimolar (mM) ferro/ferricyanide before their introduction into the microfluidic channel for measurement. These solutions were stored at −20 degrees Celsius (° C.) before their use. Mouse monoclonal antibody (MAb) of human recombinant SARS-CoV-2 nucleoprotein (Cat. No. 40143-MM05) was purchased from Sino Biological Inc., Wayne, Pa. E. coli derived human recombinant interleukin-6 (IL-6; Cat. No., 206-IL) antigen was purchased from R&D Systems, Inc., Minneapolis, Minn. Bovine serum albumin (BSA), sodium bicarbonate, sodium carbonate, formic acid, phosphate buffered saline (PBS) powder, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) were acquired from Sigma Aldrich, St. Louis, Mo. A room temperature curable silver/graphene conductive epoxy (type G6E-RTSG, Graphene Supermarket, Inc., Ronkonkoma, N.Y.) was used to connect wires to the pads of the working electrode (WE), counter electrode (CE), and reference electrode (RE) (
Fabrication of Electrodes for 3DcC Device:
The schematic of the AJ 3D printer (Model AJ-300, Optomec, Inc., Albuquerque, N. Mex. USA) is shown in
The process of the functionalization of the WE is shown in
Fabrication of PDMS housing and Assembly of the 3DcC Device: The PDMS housing of the 3DcC device was fabricated by soft-replica molding method (
Electrode Characterization: An electrochemical workstation with Zview software (VersaSTAT 3 Potentiostat Galvanostat, Princeton Applied Research, Oak Ridge, Tenn.) was used to record the electrochemical signals and analyze the impedance spectra. In addition, a smartphone-based reading platform was enabled, where the sensor was interfaced with an Android mobile phone using a portable device (Sensit Smart Device, PalmSens, Inc., Randhoeve 221, GA Houten, The Netherlands). The Sensit Smart is a microcontroller-based system having a 30.5 mm×18 mm×2.6 mm system-on-module (SOM) potentiostat. The potentiostat device could be directly connected to a smart phone via a USB-C interface. Further, a mobile app ‘PStouch’ was used to record the data. The Au micropillar electrodes were imaged by a scanning electron microscope (FEI Sirion SEM, Hillsboro, Oreg.). The elemental analysis was carried out using Energy Dispersive X-ray spectroscopy (EDX) in the same instrument as the SEM imaging. The Raman spectra were collected using the NT-MDT AFM/Raman (NT-MDT America, Tempe, Ariz.) that used a 532 nm green laser with 12 milliwatt (mW) laser power for excitation.
Electrochemical Simulations: The modeling and 3D simulation of the different electrode structures was conducted using finite element software, COMSOL Multiphysics® (Version 5.5, COMSOL Inc., Burlington, Mass.). This study was carried out to investigate the diffusion profiles for different geometries of the electrodes and their corresponding electrochemical currents which were generated due to an electrochemical reaction at the surface of the electrode. In this electroanalysis scheme, a redox species B was considered to be oxidized to form a product (A) by losing an electron (B↔A+e−). At the boundary, the product concentration was zero, but the bulk concentration of oxidative species was taken to be 1 mole per cubic meter (mol/m3) and uniform. In this study, Fick's second law of diffusion was utilized as the domain equation which is given as:
wherein the ci=1 mol/m3. The diffusivities were taken for the 2D and 3D electrodes as obtained from the cyclic voltammograms at different scan rates, along with the Randles—Sevcik equation (Elgrishi, N. et al., A Practical Beginner's Guide to Cyclic Voltammetry, J. Chem. Edu. 2018, 95, 197).
Calculation of Limit-of-Detection for the 3DcC Device: The limit-of-detection (LoD) was calculated using a method described by Armbruster et al. (Armbruster, D. A. et al., Limit of blank, limit of detection, and limit of quantification, The Clin. Biochem. Rev. 2008, 29, S49). The calculation involved evaluating limit-of-blank (LoB), the limit of detection of the signal (YLOD), and the LoD in terms of concentration (Lavín, Á. et al. On the Determination of Uncertainty and Limit of Detection in Label-Free Biosensors, Sensors 2018, 18, 2038) using:
LoB=Mean of signal (blank sample)+1.645×(Standard deviation of blank sample) Equation 2
Y
LoD=LoB+1.645×(Standard deviation of target at low concentration) Equation 3
LoD=(YLoD−c)/(slope of the sensor calibration) Equation 4
where, ‘c’ is the intercept of the calibration curve of the sensor (
Design and Construction of COVID-19 Test Chip using Nanomaterials: The schematic of the 3DcC device along with AJ nanoprinting of the three-dimensional electrodes is shown in
For detection of antibodies from the fluid introduced in the electrochemical cell, the 3D printed microelectrode was functionalized by viral antigens using rGO nanoflakes. This process is depicted in
The 3DcC device works on the principle of electrochemical transduction. When an AC potential is applied between WE and RE, an electrical double layer is formed at the WE-electrolyte interface (
Physical Characterization of 3DcC Device:
The top surface of a solid pillar is pointed and had a 15 μm deep and a 20 μm diameter dip which was a result of the printing process depicted in the schematic of
Raman spectroscopic measurements were conducted to investigate the defect (D) and graphitic (G) bands present in the rGO-Au micropillars (
Electrochemical Characterization of 3DcC Device: Cyclic voltammetry (CV) studies were carried out to investigate the electrochemical properties of the AJ printed 3D Au electrode (i.e. with micropillar geometry shown in
This is due to larger-surface area and porous feature of the AJ printed 3D Au electrode. The 3D Au electrode provided both radial and linear diffusion of electrons which enhanced the electrochemical current, while planar 2D Au electrode provided a linear diffusion of electrons. This was confirmed by COMSOL simulations. This difference is partly responsible for the high redox current of the 3D electrode. Further, the simulation shows that the diffusion of redox species reached equilibrium in 50s for electrodes with 2D geometry, while it took only 0.5s to reach equilibrium for the 3D micropillar geometry. The simulation indicates a higher concentration of redox species for the 3D micropillar electrodes when compared to 2D planar electrodes. After applying the rGO coating, the electrochemical signal was decreased (
The electrodes were also characterized by the EIS method (
In order to understand the electrochemical processes in the 3DcC device, simulations of the CV experiments using COMSOL Multiphysics Software for the 2D and 3D electrodes were completed. The bulk concentration was assumed to be 1 mol/m3, while the diffusion coefficient was taken from that obtained in the experiments from the Randles-Sevcik equation.
COMSOL simulations were also conducted to investigate the effect of the AJ printed electrode geometry on the current of the electrochemical cell. The simulation results are given in
Sensing of COVID-19 Antibodies: SARS-CoV-2 S and N proteins have been used for serologic assays for detecting antibodies of SARS-CoV-2 infection. The S protein binds to viral entry receptor angiotensin-converting enzyme-2 (ACE2) and mediates viral entry. It is present as a trimer with three receptor-binding S1 heads sitting on top of a trimeric membrane fusion S2 stalk. The receptor-binding subunit S1 contains the N-terminal domain (NTD) and the RBD while the membrane fusion subunit S2 contains the fusion peptide (FP), two heptad repeats (HR1 and 2), a transmembrane anchor (TM) and the intracellular tail (IC). Serologic assays using both recombinant S1 and RBD proteins have been shown to detect specific antibodies in COVID-19 patients. For antibody tests in this work, we thus chose the spike S1 and RBD antigens of SARS-CoV-2 to develop the 3DcC testing platform.
To elute antibodies from the sensor surface, we exposed the sensor electrodes to a solution of formic acid (1.0
For the S1 sensor, the mean of the blank signal and the standard deviation were 3499.4 Ohms (Ω) and 551Ω (n, replicate=5), respectively. From Equation 2, the LoB was 4406Ω. The mean signal at the lowest concentration (0.01 fM of S1 antibody) was 3481.1Ω, with a standard deviation of 16.36Ω [n=4]. The YLoD was 4433Ω, as calculated per Equation 3. The sensor calibration equation (
Two additional spike S1 sensors were also tested with serial dilution and results are shown in
For the RBD sensor, the mean of the blank signal and standard deviation were 3781Ω and 75.99Ω (n, replicate=5), respectively. From Equation 2, this gave a LoB of 3781Ω. The mean signal at the lowest concentration (1 fM of RBD antibody) was 3422.6Ω, with a standard deviation of 201Ω for [n=4]. This gave a YLoD of 4236Ω, as calculated per Equation 3. The sensor calibration equation (
Further, sensing data for two additional sensors of spike RBD antibodies are shown in
To validate the sensor regeneration phenomenon, 3DcC sensors for the detection of spike S1 antibody and RBD antibody were regenerated 9× each as shown in
The sensitivities of the 3DcC device for spike S1 and RBD sensors are shown in
The sensor reproducibility is shown in
The real-time tracking of binding kinetics of antigens and antibodies at the sensor surface were also investigated.
The AJ nanoprinted platform (i.e. 3DcC device) developed can detect antibodies for SARS-CoV-2 within seconds. The 3DcC device of
The surface features (
The 3DcC device has the ability to detect SARS-CoV-2 antibodies non-destructively within seconds at low analytical sensitivities with label-free probing of antibodies and an ability for regeneration within one minute. A signal was obtained in all the 3DcC devices used every time an antibody solution was introduced (
A gold micropillar array for a working electrode was prepared using AJ printing, as described above in Example 1. The gold micropillars were functionalized with rGO as described above in Example 1. The Au micropillar-rGO surface was further functionalized using SARS-CoV-2 antibodies to the Spike S1 antigens. This was achieved by using a coupling reagent consisting of a mixture of EDC (0.2 M) and NHS (0.05 M) in a ratio of 1:1 by volume. A 20 μL solution of the EDC:NHS mixture was spread over the rGO-Au surface to activate the —COOH groups of the rGO sheets. The electrode on the glass substrate was kept in a humid chamber (at approximately 100% of humidity) for four hours and washed with PBS solution. Next, a 20 μL of SARS-CoV-2 spike S1 antibody solution (5 μg/mL) was spread on the surface of the rGO-Au array electrode via drop-casting using a pipette (10-50 μL) and kept for 4 hours in a humid chamber and then washed with PBS. The activation achieved by EDC:NHS chemistry enabled the primary amine (—NH2) groups of the antibody of SARS-CoV-2 spike to form C—N bonds with the —COOH groups of rGO sheets via an amidation reaction.
SAR-CoV-2 spike S1 antigens were detected using the methods as described above in Example 1.
Upon introduction of a formic acid solution for 60 seconds into the sensor, the Rct values dropped (
The gold micropillar array prepared in Example 1 was used to detect SARS-CoV-2 RBD antibodies in human serum samples. The human serum samples tested were collected from individuals that tested positive (17 samples) and negative (3 samples) for COVID-19. Antibodies were detected in the COVID-positive human serum samples as shown in
Having described this invention above, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof.
This application is the United States national phase of International Application No. PCT/US2021/040302 filed Jul. 2, 2021, and claims priority to U.S. Provisional Application No. 63/047,368 filed Jul. 2, 2020, U.S. Provisional Application No. 63/050,182 filed Jul. 10, 2020, and U.S. Provisional Application No. 63/076,174 filed Sep. 9, 2020, the disclosures of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/040302 | 7/2/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63047368 | Jul 2020 | US | |
| 63050182 | Jul 2020 | US | |
| 63076174 | Sep 2020 | US |
