Patent Application
20230384301
The presently disclosed subject matter relates generally to the detection of molecules, such as DNA, proteins, drugs, and the like, and more particularly to methods for signal enhancement and limit of detection (LOD) improvement in plasmonic nanoparticle assisted enzyme-linked immunosorbent assay (ELISA) in a fluidics device.
In the field of microfluidics, there is difficulty in producing low-cost, sensitive, and portable optical detection systems, especially with respect to portable point-of-care (POC) diagnostics. For example, polymerase chain reaction (PCR) is standard for viral detection, but it requires dedicated equipment and trained lab personnel, making it unsuitable for POC testing. Traditional POC assays are done using lateral flow immunoassays (LFIAs), but LFIAs have inadequate sensitivity for direct viral detection in saliva and are prone to errors. ELISA is the gold-standard for viral antigen analysis, and commercial kits are available. However, like PCR, lab ELISAs are complex, lengthy, and require specialized equipment and trained operators. In addition, the sensitivity and lower detection limits of conventional fluorescent dyes for optical detection (e.g., 3,3′,5,5′-tetramethylbenzidine (TMB)) is limited.
Enzyme-linked immunosorbent assay (ELISA) based detection systems have been widely used for the detection of various kinds of disease biomarkers. 3,3′,5,5′-tetramethylbenzidine (TMB) is an important substrate that can be oxidized to TMB+ (blue) or TMB2+ (yellow) for colorimetric immunoassays. It was reported that TMB2+ can quantitatively and efficiently etch AuNRs, which converted the color of TMB2+ to the color change of AuNRs to increase the sensitivity of detection. However, the utility of this method is limited by the extreme conditions required for this reaction to occur. Hydrogen peroxide (H2O2) is an important molecule in biology. As a byproduct of many enzymatic reactions, H2O2 is a popular target of many sensors. A classic H2O2 sensor is based on the color change of 3,3′,5,5′-tetramethylbenzidine (TMB). In the presence of peroxidases (e.g., horseradish peroxidase HRP) and H2O2, the chromogenic TMB substrate can be oxidized to colored products TMB+. However, the sensitivities of these colorimetric detectors are limited by the final oxidized product concentrations.
Accordingly, new approaches are needed for addressing these limitations and for improving optical detection capabilities in microfluidics.
It is an object of the present disclosure to detect and quantify proteins (e.g., antibodies) in a sample using plasmonic nanoparticle assisted ELISA.
Plasmonic nanoparticles described herein include nanoparticles of any type, shape, size, and material composition; although, preferably, one or more of the particle dimensions are less than about 100 nm. For nanoparticles consisting of more than one material composition (e.g., core-shell particles) at least one of the particle layers may consist of a plasmonic layer. Aside from the plasmonic layer, the nanoparticles may also include a dielectric, semiconductor, or polymeric material, and/or any other type of material. In plasmonic nanoparticles having a semiconductor layer, the semiconductor layer may be magnetic or superparamagnetic. Nanoparticle types that may be used with this disclosure include (but are not limited to) core-shell particles, rattle-type particles, hollow particles, porous particles, bimetallic particles, and particles consisting of a single metal composition.
It is another object of the present disclosure to quantify protein concentration in samples through colorimetric detection.
Colorimetric change may be observed through the naked human eye and/or with instrumentation. Colorimetric change or color change described herein, includes (but is not limited to) a change in the intensity of a color and/or perceivable color hues.
In one embodiment, the selection of nanoparticles to be integrated in the present inventive device may be optimized for colorimetric detection by the naked human eye and/or with instrumentation (e.g., smartphone camera). Preferably, the initial and/or final readout color from the device has a maximum fluorescence, absorbance, scattering, and or extinction peak between about 380 nm to about 800 nm for colorimetric detection by the naked human eye. The initial and/or final readout color from the present inventive device may have a maximum fluorescence, absorbance, scattering, and or extinction peak in the ultraviolet, visible, and/or infrared wavelengths for colorimetric detection with instrumentation.
Various methods and designs for plasmonic nanoparticle assisted ELISA in a fluidics device are described herein. The present disclosure describes embodiments of the present disclosure whereby methods for signal and/or limit of detection (LOD) enhancement may be applied to the various methods as further described herein. Some non-limiting examples of colorimetric change for protein detection include etching, aggregation, and/or further growth of plasmonic nanoparticles. Colorimetric change may also be observed due to nucleation and growth of plasmonic nanoparticles by reducing metal ion precursors. Colorimetric change may also be observed due to quenching and/or unquenching the fluorescence of fluorescent probes. The various embodiments described herein generally relate to the use of plasmonic nanoparticles to amplify the signal as a result of substrate conversion in ELISA. One embodiment of the present disclosure relates to signal enhancement and/or LOD improvement in plasmonic nanoparticle assisted ELISA and is not limited to any particular type of color change modality (e.g., nanoparticle etching or aggregation). Other embodiments described herein relate to signal enhancement and/or LOD improvement in etching-based plasmonic nanoparticle assisted ELISA. Yet another embodiment relates to signal enhancement and/or LOD improvement in aggregation-based plasmonic nanoparticle assisted ELISA. Other embodiments described herein involve the use of TMB substrate solution, HRP, and gold nanoparticles (e.g., urchin-like gold nanoparticles (AuNUs), also called gold nanourchins) for plasmonic nanoparticle assisted ELISA. HRP may oxidize TMB to TMB+ or TMB2+ which in turn may etch and/or aggregate gold nanoparticles. In such embodiments, a negative control may be used in the assay. The negative control may consist of gold nanoparticles and TMB substrate solution. The TMB need not be oxidized in the negative control solution since HRP may not be present. Positive samples may contain oxidized TMB due to the presence of HRP.
It is another object of the present disclosure to use a black background to maximize observation of scattered light from plasmonic nanoparticles. For example, a black PCB may be used to provide the black background color to maximize observation of scattered light from the plasmonic nanoparticles. The perceived color of solution containing plasmonic nanoparticles may be different on a black background compared to backgrounds of other colors (such as a white background).
In an etching-based plasmonic nanoparticle assisted ELISA assay, an absorber (e.g., dye) may be added to the control(s) and sample(s) to tune the intensity and/or brightness of the perceived color in the control(s) and sample(s) in the event of a positive sample. As a non-limiting example, plasmonic nanoparticles with high scattering efficiency and a LSPR wavelength near the max absorbance wavelength of the selected absorber may be selected for use in an etching-based plasmonic nanoparticle assisted ELISA assay. As another non-limiting example, an etching-based assay involving gold nanourchins with a LSPR near about 650 nm and methylene blue as the selected absorber may be used. As the gold nanourchins are etched (as a result of enzyme-substrate reactions), the LSPR blue-shifts. As the gold nanourchins are etched, the absorbance and/or reflectance profile of the gold nanourchins may not overlap as much with the absorbance profile of methylene blue. This may result in a perceivable color change but also a change in the intensity of the perceived color due to the light scattered by the gold nanourchins. In this specific non-limiting example, a positive sample (where gold nanourchins are etched) may be of a different color and look brighter (and/or more intense) than the negative control (where gold nanourchins remain unetched).
In an etching-based plasmonic nanoparticle assisted ELISA assay, one or more types of surfactants (e.g., CTAB) may be used to promote or catalyze etching of the plasmonic nanoparticles in the event of a positive sample. The rate and/or extent of etching in the positive samples may be significantly greater than in the negative control. One or more of the surfactant types may be a quaternary ammonium surfactant (e.g., CTAB or CTAC). Aside from promoting or catalyzing plasmonic nanoparticle etching in the positive sample, the surfactant(s) may have additional functionalities as well, including, but not limited to stabilizing the negative control (e.g., preventing or reducing aggregation of plasmonic nanoparticles). The surfactant(s) may also prevent or reduce aggregation of plasmonic nanoparticles in the sample as well (positive or negative). The surfactant(s) may be dispersed in the same solution as the plasmonic nanoparticles or be coated, physically adsorbed, or chemically conjugated onto the plasmonic nanoparticles. In such embodiments, CTAB may be used to stabilize plasmonic nanoparticles (e.g., gold nanourchins) in the substrate solution (e.g., prevent nanoparticle aggregation) and to promote etching of plasmonic nanoparticles in solution in which the substrate has been converted by reporter enzymes. CTAB may prevent aggregation of plasmonic nanoparticles in substrate solution (thereby providing a stable negative control) and promote or catalyze etching of plasmonic nanoparticles after substrate conversion (e.g., in a positive sample).
In an etching-based plasmonic nanoparticle assisted ELISA assay, one or more types of halide ions (e.g., chloride, bromide, and iodide) may be added to solution to promote or accelerate etching of plasmonic nanoparticles in the event of a positive sample. The rate of etching in the positive samples may be significantly greater than in the negative control. Halide ions may be added to a solution in which plasmonic nanoparticles are present in the form of a cationic surfactant, as a salt, or in another form. Halide ions may form complexes with byproducts of enzyme-substrate reactions (in a positive sample) that may etch plasmonic nanoparticles faster, more effectively, or in a different manner (e.g., selectively etch specific facets) compared to solely using halide ions or the byproducts of the enzyme-substrate reactions.
In an etching-based plasmonic nanoparticle assisted ELISA assay, one or more types of thiosulfates (e.g., ammonium thiosulfate or sodium thiosulfate) and/or thiosulfonates may be added to solution to promote or accelerate etching of plasmonic nanoparticles in the event of a positive sample. The rate and/or extent of etching in the positive sample may be significantly greater than in the negative control. Thiosulfates and/or thiosulfonates may form complexes with byproducts of enzyme-substrate reactions (in a positive sample) that may etch plasmonic nanoparticles faster, more effectively, or in a different manner (e.g., selectively etch specific facets of the nanoparticles) compared to solely using thiosulfates/thiosulfonates or the byproducts of the enzyme-substrate reactions. As a non-limiting example, sodium thiosulfate may etch gold nanorods slowly in dissolved oxygen but much faster in the presence of oxidized TMB (TMB+ and/or TMB2+).
In an etching-based plasmonic nanoparticle assisted ELISA assay, one or more types of metal ions (e.g., Cu2+ or Fe3+) may be added to solution to promote or accelerate etching of plasmonic nanoparticles in the event of a positive sample. The rate of etching in the positive samples may be significantly greater than in the negative control. Metal ions may provide a synergetic effect to other etchants produced as byproducts of enzyme-substrate reactions (in a positive sample) thereby etching plasmonic nanoparticles faster, more effectively, or in a different manner (e.g., selectively etch specific facets of the nanoparticles) compared to solely using metal ions or the byproducts of the enzyme-substrate reactions.
It is yet another object of the present disclosure to use a combination of surfactants, halide ions, thiosulfates/thiosulfonates, and metal ions to promote or accelerate etching of plasmonic nanoparticles in the event of a positive sample. The rate of etching in the positive samples may be significantly greater than in the negative control.
In an aggregation-based plasmonic nanoparticle assisted ELISA assay, one or more types of polymers may be added to solution to promote or accelerate aggregation, flocculation, and/or reduce interparticle distance of plasmonic nanoparticles in the event of a positive sample. Polymers may be ionic or non-ionic. Polymers may promote or accelerate nanoparticle aggregation and/or flocculation through one or more mechanisms, including but not limited to the mechanisms described below. Polymers may provide a synergistic effect to other aggregants, flocculants, and/or coagulants produced as byproducts of enzyme-substrate reactions (in a positive sample) to reduce interparticle distance between plasmonic nanoparticles and thereby providing a greater colorimetric signal enhancement and/or LOD improvement. A non-ionic polymer (e.g., TWEEN 20) may provide a synergistic effect to oxidized TMB (which is converted from TMB substrate by reporter enzymes such as HRP) in aggregating plasmonic nanoparticles (e.g., citrate coated gold nanourchins). A non-ionic polymer (e.g., Polysorbate 20) may flocculate plasmonic nanoparticles through polymer bridging in tandem with charge neutralization of the plasmonic nanoparticles byproducts in enzyme-substrate reactions in positive samples. One or more of the byproducts produced in the enzyme-substrate reactions may be of an opposite charge to the initial surface charge of the plasmonic nanoparticles to induce charge neutralization.
In one embodiment, a plasmonic particle assisted method of detecting a target analyte, is provided, the method including:
In the method, the surface can be a magnetic bead. The capture biomolecule can be immobilized on the surface or on the magnetic bead and the plasmonic particle can be introduced in a fluidic suspension subsequent to introduction of the substrate.
In the method, the plasmonic particles can be introduced as a fluidic suspension in a droplet in conjunction with the substrate or the plasmonic particles can be introduced as a fluidic suspension in a droplet separately from the substrate.
In one embodiment, the capture biomolecule and the plasmonic particle are both immobilized on the surface. In another embodiment, the capture biomolecule is immobilized on the surface and the plasmonic particle is immobilized on a separate surface.
In the method, the measuring of the optically detectable change can be performed by one or both the naked eye, with an instrument, or with a camera.
In the method, the sample fluid can be a bodily fluid from a human or an animal. The target analyte can be a protein, an antigen, an antibody, an IgG antibody, an IgM antibody, a virus, a molecule or molecular structure from a virus, a bacteria, or any other pathogen, or a molecule or molecular structure bound to the outer surface of a virus, a bacteria, or any other pathogen. An internal target molecule or target molecular structure can be exposed by disrupting the integrity of the virus, the bacteria, or any other pathogen prior to introducing the sample to the microfluidic device.
In one embodiment of the method, the target analyte is a target antibody and the capture biomolecule is an antigen that the target antibody binds to. In another embodiment, the target analyte is a virus and the capture biomolecule is an antibody that binds to the virus.
In the method, the optically detectable change can be a colorimetric change.
In one embodiment of the method, the capture biomolecule is immobilized on the surface, the surface is a magnetic bead, and the antibody is conjugated indirectly to the enzyme as a result of both the antibody and the enzyme being conjugated to a second surface that can be, for example, a non-magnetic bead.
In another embodiment, the capture molecule is immobilized on the surface which is a magnetic bead, and the antibody is immobilized on a second surface.
In one embodiment of the method, the enzyme is horseradish peroxidase (HRP), the substrate is TMB, and the optically detectable change is a result of etching of the plasmonic particles by the oxidized TMB substrate.
In another embodiment, the enzyme is alkaline phosphatase (AP).
In the method, one or more dimensions of the plasmonic particles is less than about 100 nm. In various embodiments, the plasmonic particles have more than one layer, more than one material, or combinations thereof. The plasmonic particles can have one or a combination of a plasmonic layer, a dielectric layer, a semiconductor layer, or a polymeric layer. The semiconductor layer can be a magnetic, a paramagnetic, or a superparamagnetic semiconductor layer. The plasmonic particle can be a core-shell particle, a rattle-type particle, a hollow particle, a porous particle, a bimetallic particle, a single metal particle, a gold nanorod, or a gold nanourchin. The core-shell particle can include a gold core and an outer porous (or mesoporous) metal oxide layer, thereby facilitating enzyme catalyzed etching of the particle core for optical detection of the target analyte. The core-shell particle can include a silver core and an outer porous (or mesoporous) metal oxide layer, thereby facilitating enzyme catalyzed etching of the particle core for optical detection of the target analyte. The metal oxide layer can be silicon dioxide (i.e., SiO2@Ag or SiO2@Au).
In one embodiment, the method further includes introducing a fluorescent probe. The fluorescent probe can be adhered or chemically bound to the plasmonic particle. The fluorescent probe can be a quantum dot. The optically detectable change can be caused by etching of the plasmonic particles which allows the fluorescent probe or quantum dot to be released or detached from the plasmonic particles. In the method including introducing a fluorescent probe, the method can further include introducing a fluorescence quencher/acceptor moiety. The fluorescence quencher/acceptor moiety can be a black hole quencher. The fluorescence quencher/acceptor moiety can be a short oligomer dual-labeled with a FRET pair. The short oligomer can be a peptide, an aptamer, or a carbohydrate-based molecule.
In the method, the plasmonic particle can include two or more types of plasmonic particles, thereby increasing the sensitivity and/or the range of detection for the target analyte. The plasmonic particles can be a mixture of one or more aggregation states of the same type of plasmonic particle, thereby increasing the sensitivity and/or range of detection for the target analyte. The plasmonic particles can be a mixture of one or more aggregation states of one or more different types of plasmonic particles, thereby increasing the sensitivity and/or range of detection for the target analyte.
In the method, the optically detectable change can be a colorimetric change, and the method can further include using a background color that is not changed in response to the enzyme-substrate reactions, thereby providing a constant measurable color. The background color can be provided by a dye. The background color be provided by a second plasmonic particle that is not changed in response to the enzyme-substrate reactions. The second plasmonic particle can have a protective surface modification, thereby providing resistance to a colorimetric change in response to the enzyme-substrate reactions.
In the method, the reduced interparticle distance can be mediated by an addition or a presence of ionic molecules containing one or more functional groups that can covalently bind to the particles, where the addition or the presence of the ionic molecules is a result of the enzyme-substrate reactions. The one or more functional groups can include one or a combination of a carboxyl group, a thiol group, or an amine group. In one example, the one or more functional groups are present on the plasmonic particles, and in response to the enzyme-substrate reactions a chemical bond is formed between the plasmonic particles, thereby aggregating or reducing the interparticle distance of the plasmonic particles. The chemical bonding of the plasmonic particles can be provided by one or more different functional groups used in a click chemistry reaction. The click chemistry reaction can be a thiol-ene reaction or a copper(I)-catalyzed azide-alkyne cycloaddition.
In one embodiment of the method, the plasmonic particle is a core-shell particle and the method further includes introducing a metal ion precursor. The optically detectable change is caused by growth of the plasmonic particle in response to reduction of the metal ion precursor mediated by the enzyme-substrate reactions. In this case, the core-shell particle includes a thin shell formed in response to the enzyme-substrate reactions on top of a plasmonic core. The plasmonic core and the thin shell can be the same metallic material.
In one embodiment, a method is provided for detecting a target analyte, where the method includes:
In another embodiment, a system is provided for performing plasmonic particle assisted detection of a target analyte, the system including:
Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:
“Absorber” means a dye, chromophore, nanoparticle, or other species which absorbs wavelengths of light.
“Byproducts” mean any one or more products or side products produced by an enzyme reaction that is not the primary product.
“Capture biomolecules” means biomolecules (e.g., antigens, antibodies etc.) to which target analytes bind. Thus, in some instances, the terms “capture biomolecule” and “antigen” and “antibody” are used interchangeably herein. For the purposes of the claims, the term “capture biomolecule” refers to the first capture biomolecule (which in some examples is an antibody or an antigen) that is introduced to the target analyte to form a target-capture biomolecule complex. For the purposes of the claims, the term “antibody” refers to an antibody that binds directly or indirectly to the target analyte at a different site or epitope than the capture biomolecule and is introduced after introduction of the capture biomolecule. Thus, the term “target antibody” refers to a target analyte that is an antibody and is not the same as the term “antibody” or the term “detection antibody” or the term “secondary antibody”. The terms “antibody”, “secondary antibody” and “detection antibody” are herein used interchangeably for the purposes of the specification and claims. “Controls” means positive and negative controls.
“Core-shell nanoparticles” means nanoparticles that consist of a core particle encapsulated by a shell.
“Infrared wavelengths” means wavelengths of from about 700 nm to about 1000 nm.
“Localized surface plasmon resonance” means the collective oscillation of electrons at the interface of metallic structures.
“Nanoparticles” means particles with one or more dimensions less than 100 nm.
“Nanourchins” means nanoparticles with a spiky uneven surface.
“Negative control” means a solution containing nanoparticles and other components which should not undergo a colorimetric change.
“Optical density” means a measure of the amount of light that is transmitted through a solution. The solution may or may not contain plasmonic nanoparticles. It is the negative logarithm of the percentage of light transmitted through a solution.
“Oxidized TMB” refers to TMB+ or TMB2+.
“Particles” means particles with one or more dimensions greater than 100 nm.
“Plasmonic nanoparticles” or “plasmonic particles” mean particles whose electron density can couple with electromagnetic radiation of wavelengths larger than the particle. Plasmonic nanoparticles exhibit intense light absorbance, scattering, and/or extinction properties. Plasmonic nanoparticles typically consist of at least one layer or component of noble metals (e.g., gold, silver, palladium, platinum, etc.).
“Positive control” means a solution containing nanoparticles and other components which should undergo a colorimetric change.
“Positive sample” means samples which contain detectable levels of the target analyte.
“Rattle-type nanoparticles” mean nanoparticles that consist of a shell encapsulating one or more freely moving core particles in a solvent.
“Reporter enzymes” mean enzymes that perform a chemical reaction (e.g., substrate conversion) which leads directly or indirectly to a detectable optical change such as, for example, a color change. For the purposes of the specification and claims, the terms “reporter enzyme” and “enzyme” are herein used interchangeably.
“Sample” means fluid that is tested for detection and, optionally, quantification of one or more target analytes. The fluid may be artificially spiked with target analytes, including, for example, but not limited to antibodies, antigens, proteins, viruses, bacteria and other pathogens, and/or other constituents. The fluid may also be collected from humans or animals, such as sweat, saliva, blood, urine, mucous, tear fluid, etc.
“Substrate” means a molecule which gets acted upon by enzymes (e.g., reporter enzymes). In some examples, reporter enzymes catalyze chemical reactions involving the substrate.
“Ultraviolet wavelengths” means wavelengths of from about 10 nm to about 400 nm.
“Visible Wavelengths” means wavelengths of from about 400 nm to about 800 nm.
The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the presently disclosed subject matter are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
In some embodiments, the presently disclosed subject matter provides methods for signal enhancement and limit of detection (LOD) improvement in plasmonic nanoparticle assisted enzyme-linked immunosorbent assay (ELISA) in a fluidics device.
In some embodiments, a digital microfluidics (DMF) system that includes a DMF cartridge is provided for performing the presently disclosed methods of plasmonic particle assisted ELISA for the detection and quantification of, for example, proteins (e.g., antibodies), viruses, bacteria, and/or any other pathogens.
In some embodiments, using the presently disclosed methods of plasmonic particle assisted ELISA in the DMF system and/or DMF cartridge provides increased sensitivity and lower detection limits as compared with conventional dyes (e.g., 3,3′,5,5′-tetramethylbenzidine (TMB)).
In some embodiments, the presently disclosed methods of plasmonic particle assisted ELISA in the DMF system and/or DMF cartridge may be portable and may be used in, for example, point-of-care (POC) applications.
In some embodiments, using the presently disclosed methods of plasmonic particle assisted ELISA in the DMF system and/or DMF cartridge provides selective detection of multiple proteins (e.g., antibodies IgG and IgM) using, for example, multi-modality plasmonic sensors.
In some embodiments, using the presently disclosed methods of plasmonic particle assisted ELISA in the DMF system and/or DMF cartridge provides detection and quantification of, for example, proteins (e.g., antibodies), viruses, bacteria, and/or any other pathogens. For example, using plasmonic particle assisted ELISA, plasmonic particles of various shapes, sizes, and compositions, which display tunable localized surface plasmon resonance peaks and/or fluorescence quenching through Forster resonance energy transfer (FRET), may be processed (e.g., etched, grown, cleaved and/or aggregated in response to selective detection of proteins (e.g., antibodies)).
In some embodiments, using the presently disclosed methods of plasmonic particle assisted ELISA in the DMF system and/or DMF cartridge, colorimetric change may be observed due to (1) etching of plasmonic particles; (2) aggregation of plasmonic particles; (3) growth of plasmonic particles; (4) nucleation and growth of plasmonic particles by reducing metal ion precursors, and (5) quenching and/or unquenching the fluorescence of fluorescent probes (e.g., quantum dots).
In some embodiments, the presently disclosed methods of plasmonic particle assisted ELISA in the DMF system and/or DMF cartridge may include, but are not limited to, an etching-based plasmonic ELISA process in which colorimetric particles are in suspension, an etching-based plasmonic ELISA process in which colorimetric particles are immobilized, an etching-based plasmonic ELISA process in which colorimetric particles are immobilized and antigens are bound to the plasmonic particles, an aggregation-based plasmonic ELISA process, a FRET-based plasmonic ELISA process, a colorimetric detection of multiple proteins process, a signal amplification process, and a colorimetric signal amplification process.
In some embodiments, the presently disclosed methods of plasmonic particle assisted ELISA in the DMF system and/or DMF cartridge may include enzyme catalyzed cascade reaction ELISA for DMF.
Referring now to
DMF cartridge 110 may facilitate DMF capabilities generally for fluidic actuation including droplet merging, splitting, dispensing, diluting, and the like. One application of these DMF capabilities may be sample preparation. However, the DMF capabilities may be used for other processes, such as waste removal. DMF cartridge 110 of DMF system 100 may be provided, for example, as a disposable and/or reusable cartridge.
DMF system 100 may further include a controller 150, a DMF interface 152, an illumination source 154, an optical measurement device 156, and thermal control mechanisms 158. Controller 150 may be electrically coupled to the various hardware components of DMF system 100, such as to DMF cartridge 110, illumination source 154, and optical measurement device 156. In particular, controller 150 may be electrically coupled to DMF cartridge 110 via DMF interface 152, wherein DMF interface 152 may be, for example, a pluggable interface for connecting mechanically and electrically to DMF cartridge 110. Together, DMF cartridge 110, controller 150, DMF interface 152, illumination source 154, and optical measurement device 156 may comprise a DMF instrument 105.
Controller 150 may, for example, be a general-purpose computer, special purpose computer, personal computer, tablet device, smartphone, smart watch, microprocessor, or other programmable data processing apparatus. Controller 150 may provide processing capabilities, such as storing, interpreting, and/or executing software instructions, as well as controlling the overall operations of DMF system 100. The software instructions may comprise machine readable code stored in non-transitory memory that is accessible by the controller 150 for the execution of the instructions. Controller 150 may be configured and programmed to control data and/or power aspects of these devices. For example, with respect to DMF cartridge 110, controller 150 may control droplet manipulation by activating/deactivating electrodes. Generally, controller 150 may be used for any functions of the DMF system 100. For example, controller 150 may be used to authenticate the DMF cartridge 110 in a fashion similar to how printer manufacturers check for their branded ink cartridges, controller 150 may be used to verify that the DMF cartridge 110 is not expired, controller 150 may be used to confirm the cleanliness of the DMF cartridge 110 by running a certain protocol for that purpose, and so on.
Additionally, in some embodiments, DMF cartridge 110 may include capacitive feedback sensing. For example, a signal may be generated or detected by a capacitive sensor that can detect droplet position, velocity, and size. Further, in other embodiments, instead of capacitive feedback sensing, DMF cartridge 110 may include a camera or other optical device to provide an optical measurement of the droplet position, velocity, and size, which can trigger controller 150 to re-route the droplets at appropriate positions. The feedback may be used to create a closed-loop control system to optimize droplet actuation rate and verify droplet operations are completed successfully.
Further, in some embodiments, controller 150 may be external to DMF instrument 105 (not shown in
Optionally, DMF instrument 105 may be connected to a network. For example, controller 150 may be in communication with a networked computer 160 via a network 162. Networked computer 160 may be, for example, any centralized server or cloud server. The servers may be, for example, virtual servers, with logical drives distributed across geographically diverse physical drives. Network 162 may be, for example, a local area network (LAN) or wide area network (WAN) for connecting to the internet. Though
In DMF system 100, illumination source 154 and optical measurement device 156 may be arranged with respect to the portion of DMF cartridge 110 at which plasmonic particle assisted ELISA 112 occurs. The illumination source 154 may be, for example, a light source for the visible range (400-800 nm), such as, but not limited to, a white light-emitting diode (LED), a halogen bulb, an arc lamp, an incandescent lamp, lasers, and the like. Illumination source 154, in some embodiments, may be a “smart” bulb, capable of being operated and controlled via a mobile device. In addition to different wave lengths (i.e. different colors), the brightness may also be adjusted via the mobile device. Illumination source 154 is not limited to a white light source. Illumination source 154 may be any color light that is useful in DMF system 100. Optical measurement device 156 may be used to obtain light intensity readings. Optical measurement device 156 may be, for example, a charge coupled device, a photodetector, a spectrometer, a photodiode array, a smartphone camera, or any combinations thereof. Further, DMF system 100 is not limited to one illumination source 154 and one optical measurement device 156 only. DMF system 100 may include multiple illumination sources 154 and/or multiple optical measurement devices 156 to support multiple sensing elements. Thermal control mechanisms 158 may be any mechanisms for controlling the operating temperature of DMF cartridge 110. Examples of thermal control mechanisms 158 may include Peltier elements and resistive heaters.
Referring still to DMF system 100 of
DMF system 100 may be used for the detection and quantification of, for example, proteins (e.g., antibodies), viruses, bacteria, and/or any other pathogens proteins in DMF cartridge 110 using plasmonic particle assisted ELISA 112. Further, proteins may refer to both unbound proteins (e.g., after cell lysis) and proteins on the outer surface of cells and pathogens (e.g., viruses and bacteria). Using plasmonic particle assisted ELISA 112, plasmonic particles of various shapes, sizes, and compositions, which display tunable localized surface plasmon resonance peaks and/or fluorescence quenching through FRET may be processed. The plasmonic particles may be integrated into a digital microfluidic ELISA scenario in which the particles may undergo etching, growth, and/or aggregation in response to selective detection of proteins (e.g., antibodies). Etching, growth, and aggregation of the particles results in changes in the light absorbance, scattering, extinction and/or fluorescence quenching properties of the particles that can be detected with the naked eye and/or with instrumentation (e.g., a smartphone camera, which is one example of optical measurement device 156).
Generally, in DMF system 100 and/or DMF cartridge 110, plasmonic particle assisted ELISA 112 may be used to detect and quantify proteins (e.g., antibodies) in a sample. The plasmonic particles described herein may include nanoparticles of any type, shape, size, and material composition. In one example, the plasmonic particles are nanoparticles wherein one or more of the particle dimensions should be less than about 100 nm. In another example, for particles (e.g., nanoparticles) that include more than one material composition (e.g., core-shell particles) at least one of the particle layers must consist of a plasmonic layer. In yet another example, aside from the plasmonic layer, the particles (e.g., nanoparticles) may also include a dielectric, semiconductor, or polymeric material, and/or any other type of material. For example, in plasmonic particles having a semiconductor layer, the semiconductor layer may be magnetic, paramagnetic or superparamagnetic. In still another example, particle types (e.g., nanoparticles) that can be used with this disclosure may include, but are not limited to, core-shell particles, rattle-type particles, hollow particles, porous particles, bimetallic particles, and particles consisting of a single metal composition.
In DMF system 100 and/or DMF cartridge 110 and using plasmonic particle assisted ELISA 112, analyte concentration in samples may be quantified through colorimetric detection. Further, colorimetric change may be observed through the naked human eye and/or with instrumentation. For example, the colorimetric change or color change described herein, may include, but is not limited to, a change in the intensity of a color and/or perceivable color hues.
In DMF system 100 and/or DMF cartridge 110 and using plasmonic particle assisted ELISA 112, the selection of particles to be integrated in the device may be optimized for colorimetric detection using the naked human eye and/or using instrumentation (e.g., a smartphone camera, which is one example of optical measurement device 156). The initial and/or final readout color from the device should have a maximum fluorescence, absorbance, scattering, and or extinction peak of from about 380 nm to about 800 nm for colorimetric detection by the naked human eye. Further, the initial and/or final readout color from the device should have a maximum fluorescence, absorbance, scattering, and or extinction peak in the ultraviolet, visible, and/or infrared wavelengths for colorimetric detection with instrumentation.
In DMF system 100 and/or DMF cartridge 110 and using plasmonic particle assisted ELISA 112, colorimetric change may be observed due to one of several scenarios.
In colorimetric change scenario #1 and using plasmonic particle assisted ELISA 112 of DMF system 100, colorimetric change may be observed due to etching of plasmonic particles by one or more of the modalities shown in Table 200 of
For example, Table 200 shows that etching of plasmonic particles changes the shape of the particles which shifts the LSPR peak of the particles. Table 200 also shows that etching of plasmonic particles changes the particle feature sharpness which shifts the LSPR peak of the particles. For example, rounding out sharp vertices is an example of changing particle feature sharpness. Table 200 also shows that etching of plasmonic particles changes the material composition ratio of a particle that includes more than one layer and/or material (e.g., silver coated gold nanospheres) which shifts the LSPR peak. Table 200 also shows that etching of plasmonic particles changes the size of the particles which shifts the LSPR peak. Table 200 also shows that etching of plasmonic particles reduces the surface area of the particles which allows fluorescent probes (e.g., quantum dots) that were adhered or chemically bound to the particles to be released or detached from the plasmonic particles. For example, the fluorescence of the fluorescent probes is quenched when they are bound to the plasmonic particles (or within a short distance from the surface of the plasmonic particles) through a mechanism known as FRET.
Continuing colorimetric change scenario #1 and using plasmonic particle assisted ELISA 112 of DMF system 100, the etching of plasmonic particles due to ELISA occurs as a result of oxidizing byproducts produced during enzyme-substrate reactions within the device. For example, if the enzyme is directly or indirectly linked to the target antibody and/or capture biomolecule, the amount of enzyme-substrate reactions is proportional to the number of target antibodies bound to capture biomolecules (e.g., antigens) in the device.
Examples 1-4 provided herein describe signal enhancement and limit of detection improvement in plasmonic nanoparticle assisted ELISA in a fluidics device. More specifically, Example 1 describes a high sensitivity sensor for H2O2 using TMB+-mediated etching of gold nanomaterials. Example 2 describes camera detection of gold nanoparticle etching by oxidized TMB on a DMF. Example 3 shows that biotin-HRP conjugated magnetic beads can oxidize TMB substrate on a DMF cartridge, and the oxidization of TMB can be detected by gold nanourchin etching, which results in a colorimetric change that can be measured by tracking the maximum LSPR peak position of the gold nanourchins. Example 4 describes a plasmonics assisted ELISA for detecting spike protein that includes capturing the spike protein (target analyte”) with anti-spike antibody conjugated magnetic beads (capture biomolecule), labelling the bead-spike protein complex with secondary anti-spike HRP (antibody having conjugated enzyme), incubating the beads with TMB (substrate), followed by etching of gold nanourchins by the oxidized TMB, which results in a colorimetric change that can be measured by tracking the maximum LSPR peak position of the gold nanourchins.
Continuing colorimetric change scenario #1 and using plasmonic particle assisted ELISA 112 of DMF system 100, the colorimetric change due to etching may be observed by one or more types of particles. An example of colorimetric detection due to etching multiple particles types is colorimetric plasmonic ELISA that is based on etching a dual particle system.
For example, a combination of two or more particle types may be used to increase the sensitivity and/or range of detection for a specific target antibody. Further, the number of perceivable color hues (by the naked human eye and/or with instrumentation) over the target antibody detection range using multiple particle types may be greater than the number of perceivable color hues using one of the particle types. Further to the example, a Table 300 shown in
Additionally, two different hues may be achieved by combining different aggregation states of the same particle. For example, combining an NP population as single particles and a second population of a controlled aggregation state (e.g., a grouping of 6× nanoparticles) may result in two distinct LSPR peaks, one being red and another being blue.
Continuing colorimetric change scenario #1 and using plasmonic particle assisted ELISA 112 of DMF system 100, the colorimetric change due to etching may be observed by one or more aggregation states of one or more types of particles. For example, a combination of aggregation states of one or more particle types may be used to increase the sensitivity and/or range of detection for a specific target antibody. Further, the number of perceivable color hues (by the naked human eye and/or with instrumentation) over the target antibody detection range using multiple particle aggregation states may be greater than the number of perceivable color hues using one particle aggregation state.
Continuing colorimetric change scenario #1 and using plasmonic particle assisted ELISA 112 of DMF system 100, the plasmonic particles may be suspended in solution during etching. Plasmonic particles may be transported in a fluidic suspension (or in a droplet) with the substrate to the site (in the device) where target antibodies are present. In one example,
Continuing colorimetric change scenario #1 and using plasmonic particle assisted ELISA 112 of DMF system 100, the plasmonic particles may be immobilized on a surface during etching as a result of enzyme-substrate reactions during ELISA. Capture biomolecules that selectively bind the target antibodies may be immobilized on the same surface or on a separate surface as shown in
Continuing colorimetric change scenario #1 and using plasmonic particle assisted ELISA 112 of DMF system 100, one or more layers, features, and/or components of the particles may be etched as a result of enzyme-substrate reactions during ELISA. A Table 800 of
In colorimetric change scenario #2 and using plasmonic particle assisted ELISA 112 of DMF system 100, colorimetric change may be observed due to the aggregation of plasmonic particles or by reducing the interparticle distance. Particle aggregation and changes in the interparticle distance is due to modulating the attractive or repulsive forces among particles which is catalyzed by enzyme-substrate reactions during ELISA in the device. Further, if the enzyme is directly or indirectly linked to the target antibody and/or capture biomolecule, the amount of enzyme-substrate reactions is proportional to the number of target antibodies bound to capture biomolecules (e.g., antigens) in the device.
Additionally, particle aggregation (and also etching) may be provided in the presence of another background color, created by a dye or another population of particles which does not aggregate or etch (etching or lack of aggregation can perhaps be prevented by a protective surface modification). Oftentimes, mass aggregation results in more of a loss of color compared with a change in color. So here, as aggregation/etching of the particle increases, it's particular color absorbance peak may disappear, changing the hue to the constant color present (that which doesn't change).
Continuing colorimetric change scenario #2 and using plasmonic particle assisted ELISA 112 of DMF system 100, changes in the interparticle distance may be stimulated by one or more mechanisms. These mechanisms may include, but are not limited to, hydrogen bonding between particles, decreasing the surface charge of particles, conjugating particles together through chemical reactions (e.g., cycloaddition), and by bridging particles together through bioconjugation.
Continuing colorimetric change scenario #2 and using plasmonic particle assisted ELISA 112 of DMF system 100, the surface charge associated with plasmonic particles may be modified by ionic molecules containing one or more functional groups that can covalently bind to plasmonic particles (e.g., carboxyl, thiol, and/or amine groups). The addition or presence of these functional groups is catalyzed by enzyme-substrate reactions during ELISA.
Continuing colorimetric change scenario #2 and using plasmonic particle assisted ELISA 112 of DMF system 100, the interparticle distance between plasmonic particles may be tuned by enzyme-catalyzed chemical bonding between plasmonic particles with different functional groups. This may include, but is not limited to, click reactions, such as thiol-ene and copper(I)-catalyzed azide-alkyne cycloaddition. In one example,
In colorimetric change scenario #3 and using plasmonic particle assisted ELISA 112 of DMF system 100, colorimetric change may be observed due to the growth of plasmonic particles catalyzed by the detection of target antibodies using ELISA in the device. Growth of plasmonic particles due to ELISA occurs as a result of reducing metal ion precursors catalyzed by enzyme-substrate reactions within the device. The amount of enzyme-substrate reactions is proportional to the number of target antibodies bound to capture biomolecules (e.g., antigens) in the device.
Continuing colorimetric change scenario #3 and using plasmonic particle assisted ELISA 112 of DMF system 100, colorimetric change due to growth of plasmonic particles may be observed by one or more types of particles.
Continuing colorimetric change scenario #3 and using plasmonic particle assisted ELISA 112 of DMF system 100, core-shell particles may be formed due to reduction of metal ion precursors, followed by growth of a thin shell on top of a plasmonic (or non-plasmonic) core.
Continuing colorimetric change scenario #3 and using plasmonic particle assisted ELISA 112 of DMF system 100, metal ion precursors may be reduced and deposited on plasmonic cores or shells containing the same metallic material.
Continuing colorimetric change scenario #3 and using plasmonic particle assisted ELISA 112 of DMF system 100, particle growth may result in uniform growth of all crystal facets, directional growth, and/or anisotropic growth.
Continuing colorimetric change scenario #3 and using plasmonic particle assisted ELISA 112 of DMF system 100, particles may be suspended in solution during enzyme-catalyzed growth. Particles may be transported in a fluidic suspension (or in a droplet) with the substrate, metal ion precursors, and other reagents to the site (in the device) where target antibodies are bound. In another example, the substrate, particles, metal ion precursors, and other reagents may be transported as separate fluidic suspensions (or droplets) to the site where target antibodies are bound.
Continuing colorimetric change scenario #3 and using plasmonic particle assisted ELISA 112 of DMF system 100, enzyme-catalyzed growth can occur on particles that are immobilized on a surface. For example, particles and capture biomolecules that selectively bind the target antibodies may be immobilized on the same surface or on a separate surface.
In colorimetric change scenario #4 and using plasmonic particle assisted ELISA 112 of DMF system 100, colorimetric change may occur due to the nucleation and growth of plasmonic particles catalyzed by the detection of target antibodies using ELISA in the device. Nucleation and growth of plasmonic particles due to ELISA occurs as a result of reducing metal ion precursors catalyzed by enzyme-substrate reactions within the device. The amount of enzyme-substrate reactions is proportional to the number of target antibodies bound to capture biomolecules (e.g., antigens) in the device. This differs from colorimetric change scenario #3 that also describes colorimetric change due to the growth of plasmonic particles catalyzed by the detection of target antibodies using ELISA in that here the nucleation of new particles occurs within the device after enzyme-substrate reactions, rather than growth on pre-formed particles.
Continuing colorimetric change scenario #4 and using plasmonic particle assisted ELISA 112 of DMF system 100, nucleation and growth of plasmonic particles may also take place within the device at zero or low concentrations of bound target antibodies. However, the colorimetric response at zero or low concentrations of bound target antibodies may be clearly distinguished from the colorimetric response at higher concentrations of bound target antibodies by the naked eye and/or with instrumentation.
In colorimetric change scenario #5 and using plasmonic particle assisted ELISA 112 of DMF system 100, colorimetric change may occur due to a change in the fluorescence from fluorescent probes (e.g., quantum dots) catalyzed by the detection of target antibodies using ELISA in the device. Fluorescence modulation by ELISA may be achieved by tuning the distance between fluorescent probes and plasmonic particles, which is mediated by enzyme-substrate reactions within the device. The amount of enzyme-substrate reactions is proportional to the number of target antibodies bound to capture biomolecules (e.g., antigens) in the device. Further, the fluorescence from fluorescent probes is quenched when they are within a short distance from the surface of plasmonic particles through a mechanism known as FRET. Further, the fluorescence intensity from fluorescent probes is higher when the fluorescent probes are not within a short distance from the surface of plasmonic particles or from the surface of another fluorescence quencher/acceptor moiety (e.g., a “black hole quencher”). An example of this type of actuation may be short oligomer (e.g., peptide or aptamer or carbohydrate-based molecules) that are dual-labeled by a FRET pair for enzymatically amplified cascade assay.
Continuing colorimetric change scenario #5 and using plasmonic particle assisted ELISA 112 of DMF system 100, changes in the distance between plasmonic particles and fluorescent probes may be stimulated by one or more mechanisms. These mechanisms may include, but are not limited to, hydrogen bonding between particles and/or fluorescent probes, decreasing the surface charge of particles and/or fluorescent probes, conjugating particles and/or fluorescent probes together through chemical reactions (e.g., cycloaddition), and by bridging particles and/or fluorescent probes together through bioconjugation.
Continuing colorimetric change scenario #5 and using plasmonic particle assisted ELISA 112 of DMF system 100, the surface charge associated with plasmonic particles and/or fluorescent probes may be modified by ionic molecules containing one or more functional groups that can covalently bind to plasmonic particles and/or fluorescent probes (e.g., carboxyl, thiol, and/or amine groups). The addition or presence of these functional groups is catalyzed by enzyme-substrate reactions during ELISA.
Continuing colorimetric change scenario #5 and using plasmonic particle assisted ELISA 112 of DMF system 100, the distance between plasmonic particles and fluorescent probes may be tuned by enzyme-catalyzed chemical bonding. This may include, but is not limited to, click reactions such as thiol-ene and copper(I)-catalyzed azide-alkyne cycloaddition. For example,
Referring now again to
In DMF system 100 and/or DMF cartridge 110 and using plasmonic particle assisted ELISA 112, the assay detection limit may be decreased through the use of multi-functionalized particles (e.g., nanoparticles or microparticles) containing capture biomolecules to selectively detect target proteins and multiple reporter enzymes to amplify the colorimetric response. The multi-functionalized particles may contain a magnetic core (or layer), thereby allowing their movement within the fluidic device to be controlled by an external magnetic field. The multi-functionalized particles may contain a plasmonic layer. The ratio of reporter enzymes to capture biomolecules on the multi-functionalized particles may be greater than 1. The multi-functionalized particles may contain functional groups other than reporter enzymes and capture biomolecules.
In DMF system 100 and/or DMF cartridge 110 and using plasmonic particle assisted ELISA 112, target proteins on the outer surface of pathogenic particles (e.g., virus particles) can bind to multiple multi-functionalized particles (described above) and in some examples to another surface functionalized with second capture biomolecules (e.g., monoclonal antibodies), wherein the second capture biomolecule is also referred to herein for the purposes of the specification and claims as an antibody that binds to a site on the target analyte that is different than the capture biomolecule. The second capture biomolecule or antibody may be immobilized on another surface referred to herein for the purposes of the specification and claims as a second surface such as, for example, a non-magnetic bead or another non-magnetic surface. The ratio of multi-functionalized particles bound per pathogenic particle may be equal to or greater than 1. Colorimetric detection and quantification of pathogenic particles (and/or target proteins) may be achieved using the processes described hereinabove. Multi-functionalized particles that are not bound to pathogenic particles may be removed prior to introducing specific substrates that bind with the reporter enzymes on the multi-functionalized particles. For example,
In
In
In
In DMF system 100 and/or DMF cartridge 110 and using plasmonic particle assisted ELISA 112, target proteins can bind to capture biomolecules (e.g., monoclonal antibodies) on multi-functionalized particles (described above) and on an additional surface to form a structure similar to conventional sandwich ELISA. The capture biomolecules on the multi-functionalized particles and additional surface may bind different epitopes on the target protein or different target proteins on the viral particle. Further, pathogenic particles may be lysed so that sandwich structures consisting of capture biomolecules and the target protein contain a singular target protein per multifunctional particle. Lysing pathogenic particles may allow for detection of multiple target proteins per pathogenic particle, thereby amplifying the colorimetric response. For example,
Referring now to
When there is virus present, the non-magnetic NP is anchored via the MNP and remains during a wash operation. When TMB is introduced, the HRP present on the non-magnetic NP will convert it to oxidized TMB (e.g., TMB2+) which then etches the gold shell on the MNP, changing the color. However, when virus is not present, no oxidized TMB is produced and therefore no etching will occur. Again, in this process, the MNP (i.e., the core) may be, for example, a 1.5-2 μg avidin/mg particle (50-60 molecules avidin/particle).
In another example of process 2000, the non-magnetic NP may be replaced with an AuNP and the presence of virus can be detected using the accumulation/depletion of color in a process similar to a lateral flow assay (e.g., as described above with reference to
In yet another example of process 2000, any of the aforementioned processes 1400, 1500, 1600, 1700, 1800, 1900 may be applied to process 2000 as the read out for detecting the presence (or absence) of virus. In this example, instead of two particles (e.g., NPs) becoming tethered together in the presence of virus, the two particles are separated in the presence of viral nucleic acid sequences by CRISPR enzyme mediated cleavage of a tether.
In another example of process 2100, the non-magnetic NP may be replaced with an AuNP and the presence of virus can be detected using the accumulation/depletion of color in a process similar to a lateral flow assay (e.g., as described above with reference to
In yet another example of process 2100, any of the aforementioned processes 1400, 1500, 1600, 1700, 1800, 1900 may be applied to process 2100 as the read out for detecting the presence (or absence) of virus.
As shown and described hereinbelow,
Hydrogen peroxide (H2O2) is an important molecule in biology. As a byproduct of many enzymatic reactions, H2O2 is a popular target of many sensors. A classic H2O2 sensor is based on the color change of 3,3′,5,5′-tetramethylbenzidine (TMB). In the presence of peroxidases (e.g., horseradish peroxidase HRP) and H2O2, the chromogenic TMB substrate can be oxidized to colored products TMB+. However, the sensitivities of these colorimetric detectors observed by naked eyes were limited by the final oxidized product concentrations. Gold nanoparticle-etching-based colorimetric sensors are powerful and highly sensitive. In previous work, the dye TMB2+ was found to etch gold nanorods (AuNRs) quantitatively and efficiently. Since the preparation of TMB2+ requires the extra addition of strong acid solution, AuNR-etching-based sensors always need multiple steps and acid conditions. As is described herein below, we developed a new colorimetric biosensing platform for H2O2 detection with urchin-like gold nanoparticles (AuNUs). Compared with AuNRs, the etching of AuNUs can happen under mild conditions in the presence of TMB+ at pH 6. Such mild reaction conditions also ensured the peroxidase activity of HRP during the AuNUs etching process. Therefore, the colorimetric detection of H2O2 by AuNUs can be realized by one-step mixing. In addition, the AuNUs-etching-based sensors were also fast (in 30 min) and sensitive. This system can sensitively detect H2O2 with a limit of 80 nm (2.7 parts-per-billion).
Gold nanoparticles (AuNPs) possess much higher extinction coefficients than organic dyes due to their localized surface plasmon resonance (LSPR), allowing visual observation at low nanomolar and even picomolar concentrations. The positions of LSPR peaks are dependent on the sizes and morphologies of AuNPs. Therefore, colorimetric detection can be made based on morphology changes of AuNPs, especially anisotropic AuNPs. Over the past decades, anisotropic AuNPs etching-based sensors are very prevalent. For example, the etching of AuNRs can happen along the longitudinal direction, leading to continuous color changes. However, a high concentration of cetrimonium bromide (CTAB) appeared essential for etching the AuNRs. In the presence of CTAB, the redox potential of AuBr2−/Au0 (0.93 V vs NHE) can be dramatically decreased by the AuBr2−-(CTA)2+/Au complex (<0.2 V vs NHE). The addition of hydrogen peroxide (H2O2), acids, and O2 can also help the oxidation process of AuNRs. However, a high CTAB concentration (usually over 50 mm) and extreme conditions (acid or heating) limited the applications of AuNRs. Therefore, this example describes the exploration of gold nanostructures other than AuNRs that might be etched more easily under mild conditions.
Urchin-like AuNPs (AuNUs) are important anisotropic AuNPs in a broad range of applications from biosensors to cancer therapy. AuNUs can grow on AuNP seeds when additional Au3+ was reduced by sodium citrate and hydroquinone. The tip areas on the AuNUs are known to be highly reactive because of their high surface energy, and the sharp tips exhibit larger electric fields at their concavo-convex sites compared to neutral curvature areas. With these sharp tips, morphological changes of AuNUs can be triggered easily. For example, upon laser irradiation, the heat generated from the photothermal effect can melt the AuNUs into spherical AuNPs.
Enzyme-linked immunosorbent assay (ELISA) based detection systems have been widely used for the detection of various kinds of disease biomarkers. 3,3′,5,5′-tetramethylbenzidine (TMB) is an important substrate that can be oxidized to TMB+ (blue) or TMB2+ (yellow) for colorimetric immunoassays. It was reported that TMB2+ can quantitatively and efficiently etch AuNRs, which converted the color of TMB2+ to the color change of AuNRs to increase the sensitivity of detection. However, as mentioned above, acids were needed for this reaction to occur.
In this work, TMB+ induced etching of AuNUs was studied, and comparisons were made with AuNRs. In particular, the role of CTAB was investigated and its effect on the surfactant part was separated from its effect on the halide part. In the presence of a low concentration of CTA+ and Br ions, TMB+ can efficiently etch the branches of AuNUs. As a result, the morphology change of AuNUs was accompanied by a vivid color variation. Based on these understandings, we used the TMB+-induced etching of AuNUs and designed a colorimetric biosensing platform for H2O2 detection without any complex steps.
Materials and Methods
Chemicals. Gold (III) chloride trihydrate (HAuCl4·3H2O), H2O2 (30 wt %), 3,3′,5,5′-tetramethylbenzidine (TMB), sodium oleate (NaOL), Triton X-100, Tween 80, and Tween 20, and acetic acid were from Sigma-Aldrich (St Louis, MO). Cetrimonium chloride (CTAC), cetrimonium bromide (CTAB), sodium chloride, sodium fluoride, sodium bromide, sodium hydroxide, sodium citrate, sodium phosphate were from Mandel Scientific (Guelph, Ontario, Canada). Milli-Q water was used for preparing buffers and solutions.
Preparation of spherical Au seeds. Citrate-capped Au seeds were synthesized according to early reported literature. Briefly, the 100 mL 1 mm HAuCl4 was heated and boiled for 30 s. Then, 38.8 mm sodium citrate was added quickly. The mixture solution will change from light yellow to wine red in 2 min. 13 nm AuNPs were obtained after refluxing for another 20 min. The same protocol was used for preparing 38 nm Au seeds. The concentration of HAuCl4 was doubled to obtain 38 nm Au seeds.
Preparation of AuNUs. First, 30 mm hydroquinone solution was freshly prepared with Milli-Q water and used in one day. For a typical synthesis process, 1 mL HAuCl4 was diluted with 180 mL H2O under vigorous stirring. Subsequently, 600 μL Au seeds, 3 mL 38.8 mm sodium citrate, and 10 mL 30 mm hydroquinone was added one by one. 13 nm and 38 nm spherical Au seeds were, respectively, used for generating AuNUs-13 and AuNUs-38 NPs. The solutions were incubated at room temperature for 30 min under stirring. In the end, the AuNUs were washed with 5 mm pH 6 phosphate buffer at 4000 rpm for 8 min and stored at 4° C. for further use. The morphologies of AuNUs were characterized by TEM (Phillips CM10 100 kV) and UV-vis spectra.
Preparation of AuNRs. AuNRs were prepared by a seed-mediated method using a binary surfactant system as reported by C. B. Murray. For seed preparation, first, 5 mL 0.5 mm HAuCl4 was added into 5 mL 0.2 M CTAB solution in a 20 mL scintillation vial. Second, 0.6 mL of ice-cold fresh 0.01 M NaBH4 was diluted to 1 mL with water and was injected into the HAuCl4-CTAB mixture under rapid stirring (1200 rpm). After stirring for 2 mins, the color of the solution changed from yellow to brown, and the seed solution was used after standing for min at room temperature. To prepare a growth solution, 7.0 g CTAB and 1.234 g NaOL were dissolved in 250 mL of warm water (50° C.) in an Erlenmeyer flask. After the solution was cooled to 30° C., 18 mL 4 mm AgNO3 was added to the solution under stirring. The mixture was kept undisturbed at 30° C. for 15 min, then the 250 mL of 1 mm HAuCl4 solution was added and stirred for 90 min. 1.5 mL HCl (37 wt % in water) was further added into the solution and stirred for 15 min. 1.25 mL of 0.064 M ascorbic acid (ΔA) was added into the solution under vigorously stirred for 30 s. Finally, 0.4 mL seed solution was injected into the growth solution with stirring for 30 s. The growth solution was left undisturbed for 12 hrs. Finally, the AuNRs suspension was centrifuged at 7000 rpm for 30 min to remove excess emulsifier, and washed once with water. The final AuNRs was stored in 250 mL 5 mm CTAB solution at 4° C.
Preparation of TMB+ and TMB2+. 2 mL 0.5 mm TMB substrate in 5 mm pH 4 buffer solution was irradiated under UV light (˜370 nm) for 30 min to get blue TMB+. The final concentration of TMB+ was determined by the absorbance of TMB+ at 652 nm with the extinction coefficient (ε=3.9 104 M−1cm−1). TMB2+ was prepared by mixing TMB+ stock solution and 250 mm H2SO4 with a 1:1 volumetric ratio. The final concentration of TMB2+ was determined by the absorbance of TMB2+ at 450 nm with the extinction coefficient (ε=5.9 104 M−1cm−1).
Etching of AuNUs by TMB+. In a typical etching experiment, 80.5 μL H2O, 7.5 μL 100 mm CTAC, 30 μL 100 mm pH 6 phosphate buffer, 20 μL AuNUs, 3 μL 500 mm NaBr were added into microtubes in sequence. Then, 9 μL TMB+ with various concentrations was pipetted into microtubes respectively. The final volumes of samples were 150 μL. After vigorous stirring for 30 s, the samples were incubated at room temperature for 30 min before UV-vis absorbance measurements.
Colorimetric protocol for H2O2 detection. First, a mixture solution was prepared with TMB substrate, pH 6 phosphate buffer, and CTAC. Subsequently, 1 μL 0.1 mg/mL HRP was added into the mixture solution in the 96-well plates, followed by the addition of 20 μL AuNUs-13 NPs, 3 μL 500 mm NaBr, and various amounts of H2O2. The total volume in each well is 150 μL. The final concentrations of TMB substrate, phosphate buffer, and CTAC were respectively 100 μm, 20 mm, and 5 mm. Then, the mixture solution at room temperature for 30 min. Finally, the UV-vis absorbances were monitored by a plate reader, or the colors were distinguished by naked eyes.
TMB+-mediated etching of AuNUs. Referring now to
The AuNUs were synthesized by a seed-mediated growth method. Spherical AuNPs, which were prepared by citrate reduction, were used as seeds. Because the size of the AuNP seeds was around 38 nm, the resulting urchin-like products were called AuNUs-38. AuNUs grown from 13 nm seeds were also made and named AuNUs-13. From the TEM image shown in
These sharp edges have higher surface energy, and they might be more easily etched. TMB is a common chromogenic substrate and upon one-electron oxidation, the TMB+ product has a blue color. An experiment was preformed to use TMB+ to etch the AuNUs to amplify the color change. To avoid potential effects of other molecules, TMB+ was produced by using UV irradiation (so no H2O2 or HPR was added). It needs to be noted that this method only yielded around 11% of TMB+, while the rest 89% was still the unreacted TMB substrate (see
Then TMB+-mediated etching of the AuNUs was studied. In the presence of 5 mm CTAB, the sharp tips of AuNUs-38 were etched and rounded by the added TMB+ (see
AuNUs are more easily etched than AuNRs. Referring now to
Since previous work mainly used AuNRs, the etching of AuNRs and AuNUs was compared. We expected that AuNUs could be more sensitive for TMB+ since the AuNRs lack the branches with high surface energy (see
We also studied AuNUs-13 which are much smaller than AuNUs-38 in size, but with the same morphologies. All the three AuNPs were incubated with various TMB+ concentrations at room temperature. Interestingly, the AuNRs were quite stable under the etching conditions for AuNUs (see
For AuNUs, after one-hour incubation, color changes happened with both AuNUs-38 and AuNUs-13 as described above for
Effects of halides and surfactants. Referring now to
After demonstrating the feasibility, to acquire the best etching performance, the effects from the reaction conditions, such as pH, etching time, surfactant, and halide ion were investigated in detail. The difference in SPR peak positions (Δλ) was chosen as the indicator of the morphological change of the AuNUs.
The experiment was started by optimizing surfactants. The as-synthesized AuNUs were mainly covered by weakly adsorbed citrate groups, which can be easily displaced by other stronger capping agents. During the initial experiments, cetrimonium bromide (CTAB) was used as the capping agent since CTA+ has been well studied on AuNRs etching. Herein, a few common surfactants were also evaluated, and interestingly, TMB+ only etched the AuNUs in the presence of CTAB (see
CTAB contained Br as its counterion, and Br has a strong affinity to the gold surface. Halides counterions of the surfactant have been proved to be critical in the oxidation of CTAB-capped AuNRs. Halides adsorption was widely studied for shape-controlled AuNP synthesis. It is known the adsorbed concentrations of Br− and I− are much higher than that of Cl− ions on Au surface, and the interaction strength of halides with gold has the trend of I−>Br>Cl−. Thus, it was expected that Br might be important during the etching process. Interestingly, without CTA+, only Br failed to induce the etching process (see
To confirm the roles of halide counterions for deeper mechanistic understanding, more etching experiments were conducted with the mixture of cetrimonium chloride (CTAC) and three halides (F−, Cl−, and Br−). I− was omitted here because a low concentration of iodide can cause serious etching of AuNUs without the help of CTA+ groups (see
To get fast etching speeds or large SPR peak shifts, higher CTAB concentrations are better. However, a too high CTAB concentration may produce many bubbles. These bubbles can cause problems for quantitative absorbance measurements. Also, CTAB has poor solubility at room temperature. Therefore, it was necessary to achieve fast etching with the lowest possible surfactant concentration. This goal is possible by using a mixture of CTAC and NaBr.
The CTAC concentration was fixed at 5 mm (10-folder lower than the typical 50 mm CTAB concentration), and larger SPR peak shifts happened with more NaBr added (see
Optimization of etching time and pH. Referring now to
After understanding the effects of surfactants and Br, the etching time and pH conditions were further investigated.
Then the effects of pH on the etching were studied. An acidic environment was shown to facilitate Au nanomaterial etching in the presence of dissolved oxygen. However, many proteins are only stable in a narrow pH range near neutral. For example, horseradish peroxidase (HRP) loses its structural and conformational stability at pH<4. Thus, the etching of AuNUs-38 was studied between pH 4 and 8. From
Visual detection of H2O2. Referring now to
H2O2 is an important by-product of many enzymatic reactions and has been used as a target molecule of many biosensors. For example, the oxidation of glucose by glucose oxidase (GOx) can produce H2O2. It is well known that H2O2 can oxidize AuNRs in the presence of Br under acid conditions at high temperatures. However, the concentration used for AuNRs oxidation is much higher than that used in the experiments described herein. Over 1 mm H2O2 is required to cause slight etching of AuNUs (see
To realize the visual detection, the AuNUs synthesized from smaller Au seeds were used. A more vivid color change was generated with the sharp tips characteristic but smaller as described above for
This is one of the most sensitive colorimetric sensors, which showed significant color change when the concentration of H2O2 is equal to or higher than 400 nm. Under conditions used in this study, the limit of detection for H2O2 is 80.23 nm (3σ/slope, inset) (see
In summary, a new one-step colorimetric biosensing platform for H2O2 detection by TMB+-mediated etching of AuNUs has been successfully developed. All CTA+, TMB+, and Br molecules were important in etching AuNUs. The reaction conditions including time, pH, and surface ligands were also optimized. Although the blue color of TMB+ with low concentrations failed to be discerned with the naked eyes, nanomolar level TMB+ can still cause vivid color changing of AuNUs solution by etching. This work demonstrates a high sensitivity sensor for H2O2 and also expands TMB+-mediated etching of gold nanomaterials.
Reagent Preparation. Reagent 1 was prepared by mixing citrate coated gold nanourchins suspended in deionized water (18 Mohm) with cetrimonium chloride (CTAC). The final concentration of gold nanourchins and CTAC were 10 OD and 4 mM, respectively.
Commercial 3,3′,5,5′-Tetramethylbenzidine (TMB) was mixed with biotin-HRP to prepare oxidized TMB. The concentration of oxidized TMB was measured with a UV-Vis spectrophotometer.
Gold Nanourchin Etching on Digital Microfluidic (DMF) Cartridge. All reagents listed in Table 1 were loaded independently onto the DMF cartridge. The reagents were dispensed and transported as aqueous droplets through the oil filled DMF cartridge to specific locations on the cartridge, where reagent mixing took place.
Eight samples were prepared on cartridge using various combinations of Reagents 1-3 (Table 1). Each sample consists of two droplets of Reagent 1, one droplet of Reagent 2, and one droplet of Reagent 3. First, Reagent 1 & 2 were mixed and held as a stationary droplet for a few minutes. During this period, images were taken with the Blackfly S USB3 camera (Model bfs-u3-200s6c, Data not shown). Then, Reagent 3 was mixed with the combined Reagent 1&2 droplet. The combined droplet was then held as a stationary droplet. More images were taken with the Blackfly S USB3 camera Data not shown.
Image Analysis. The HSV color scale was used for measuring the colorimetric change of the samples and the average hue values of the samples were measured using GIMP (Table 2). During image analysis, areas of the droplet that were severely impacted by lighting, shadows or bubbles were excluded from hue measurements.
Results. After mixing Reagent 1 and Reagent 2, all samples appeared blue to the eye and had an average hue between 223.5 to 230.0. After mixing with Reagent 3, the samples that received 4 uM oxidized TMB appeared to be more purple to the eye compared to the other samples and had a higher average hue (see Table 3). The greater change in hue among these samples is due to etching of the gold nanourchins by oxidized TMB.
Reagent Preparation. Streptavidin coated magnetic beads were conjugated with biotin-HRP and suspended in buffer. Citrate coated gold nanourchins suspended in deionized water (18 Mohm) were mixed with cetrimonium bromide (CTAB) such that the final concentration of gold nanourchins and CTAB were 7 OD and 30 mM, respectively.
TMB Oxidation & Gold Nanourchin Etching on DMF Cartridge. Streptavidin coated magnetic beads, Biotin-HRP conjugated magnetic beads, gold nanourchins (10 OD) mixed with CTAB (30 mM), and TMB substrate were independently loaded onto the DMF cartridge. Each reagent was loaded into different wells on the cartridge for dispensing. All reagents were transported through the oil filled cartridge as aqueous droplets.
Streptavidin coated magnetic beads & biotin-HRP conjugated magnetic beads were magnetically separated from the original buffer solution on the DMF cartridge, and then resuspended in TMB. The TMB was allowed to incubate with magnetic beads for 15 minutes. After the 15 minute incubation period, the magnetic beads were magnetically separated from the TMB solutions. The TMB solutions mixed with deionized water at a 1:1 volume ratio on the cartridge.
The diluted TMB solutions were then mixed with gold nanourchins (at a 1:1 volume ratio). The maximum LSPR peak position of the gold nanourchins was tracked over time with optical fibers immersed in the gold nanourchins+CTAB+TMB solution (see
Gold nanourchins that received TMB incubated with streptavidin coated magnetic beads are labelled as the negative control (NEG.CTL) in
Results. The maximum LSPR peak position of gold nanourchins mixed with TMB incubated by biotin-HRP conjugated beads blue-shifted significantly more than the NEG.CTL. This result shows that biotin-HRP conjugated magnetic beads can oxidize TMB substrate on a DMF cartridge, and the oxidization of TMB can be detected by gold nanourchin etching, which results in a colorimetric change that can be measured by tracking the maximum LSPR peak position of the gold nanourchins.
Reagent Preparation. Citrate coated gold nanourchins suspended in deionized water (18 Mohm) were mixed with cetrimonium bromide (CTAB) such that the final concentration of gold nanourchins and CTAB were 5.1 OD and 24 mM, respectively. R001-MNPs were prepared by conjugating magnetic beads with anti-spike antibody (otherwise referred to herein as “capture biomolecule”).
Spike Protein (S1 Subunit) Plasmonic ELISA on DMF. The following reagents were independently loaded into separate wells on the DMF cartridge: citrate coated gold nanourchins (7 OD) mixed with CTAB (30 mM), R001-MNPs, anti-spike HRP (D001-HRP; “secondary antibody” or “antibody” having conjugated “enzyme”), TMB substrate, deionized water, wash buffer (phosphate buffered saline+1% bovine serum albumin), spike protein (S1 subunit; “target analyte”) and phosphate buffered saline.
R001-MNPs were incubated with 250 μM spike protein (or phosphate buffered saline in the case of the negative control). The Spike-R001-MNPs (and R001-MNPs) were washed several times with wash buffer (by magnetic separation and resuspension). The magnetic beads were then incubated with secondary antibody, anti-spike HRP (D001-HRP). The magnetic beads were washed several times with wash buffer (by magnetic separation and resuspension). Next, the magnetic beads were suspended in TMB substrate and allowed to incubate for 15 minutes.
After the 15 minute incubation period, the magnetic beads were magnetically separated from the TMB solutions. The TMB solutions mixed with deionized water at a 1:1 volume ratio. The diluted TMB solutions were then mixed with gold nanourchins (at a 1:1 volume ratio). The maximum LSPR peak position of the gold nanourchins was tracked over time with optical fibers immersed in the gold nanourchins+CTAB+TMB solution (see
Gold nanourchins that received TMB incubated with magnetic beads that mixed with buffer instead of 250 pM spike protein are labelled as the negative control (NEG.CTL) in
Results. The maximum LSPR peak position of gold nanourchins mixed with TMB incubated by beads that mixed with 250 pM spike protein blue-shifted significantly more than the NEG.CTL (
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
Throughout this specification and the claims, the terms “comprise,” “includes,” and “including” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments ±100%, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.
Unless specifically stated otherwise, terms such as “receiving,” “routing,” “updating,” “providing,” or the like, refer to actions and processes performed or implemented by computing devices that manipulates and transforms data represented as physical (electronic) quantities within the computing device's registers and memories into other data similarly represented as physical quantities within the computing device memories or registers or other such information storage, transmission or display devices. Also, the terms “first,” “second,” “third,” “fourth,” etc., as used herein are meant as labels to distinguish among different elements and may not necessarily have an ordinal meaning according to their numerical designation.
Examples described herein also relate to an apparatus for performing the operations described herein. This apparatus may be specially constructed for the required purposes, or it may comprise a general purpose computing device selectively programmed by a computer program stored in the computing device. Such a computer program may be stored in a computer-readable non-transitory storage medium.
The methods and illustrative examples described herein are not inherently related to any particular computer or other apparatus. Various general purpose systems may be used in accordance with the teachings described herein, or it may prove convenient to construct more specialized apparatus to perform the required method steps. The required structure for a variety of these systems will appear as set forth in the description above.
It should also be noted that in some alternative implementations, the functions/acts noted may occur out of the order noted in the figures. For example, two figures shown in succession may in fact be executed substantially concurrently or may sometimes be executed in the reverse order, depending upon the functionality/acts involved.
Although the method operations were described in a specific order, it should be understood that other operations may be performed in between described operations, described operations may be adjusted so that they occur at slightly different times or the described operations may be distributed in a system which allows the occurrence of the processing operations at various intervals associated with the processing.
Various units, circuits, or other components may be described or claimed as “configured to” or “configurable to” perform a task or tasks. In such contexts, the phrase “configured to” or “configurable to” is used to connote structure by indicating that the units/circuits/components include structure (e.g., circuitry) that performs the task or tasks during operation. As such, the unit/circuit/component can be said to be configured to perform the task, or configurable to perform the task, even when the specified unit/circuit/component is not currently operational (e.g., is not on). The units/circuits/components used with the “configured to” or “configurable to” language include hardware—for example, circuits, memory storing program instructions executable to implement the operation, etc. Reciting that a unit/circuit/component is “configured to” perform one or more tasks, or is “configurable to” perform one or more tasks, is expressly intended not to invoke 35 U.S.C. 112, sixth paragraph, for that unit/circuit/component. Additionally, “configured to” or “configurable to” can include generic structure (e.g., generic circuitry) that is manipulated by software and/or firmware (e.g., an FPGA or a general-purpose processor executing software) to operate in manner that is capable of performing the task(s) at issue. “Configured to” may also include adapting a manufacturing process (e.g., a semiconductor fabrication facility) to fabricate devices (e.g., integrated circuits) that are adapted to implement or perform one or more tasks. “Configurable to” is expressly intended not to apply to blank media, an unprogrammed processor or unprogrammed generic computer, or an unprogrammed programmable logic device, programmable gate array, or other unprogrammed device, unless accompanied by programmed media that confers the ability to the unprogrammed device to be configured to perform the disclosed function(s).
Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.
This application claims the benefit of priority of U.S. provisional patent application No. 66/104,006 filed on Oct. 22, 2020 and No. 63/228,970 filed on Aug. 3, 2021, the disclosure of which is incorporated herein by this reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051491 | 10/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63104006 | Oct 2020 | US | |
| 63228970 | Aug 2021 | US |
