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WO2025014842A1 - Bandelettes réactives et procédés de test de bio-analytes à l'aide de nanoparticules de nitrure de titane et de nanoparticules de cuivre revêtues d'or - Google Patents

Bandelettes réactives et procédés de test de bio-analytes à l'aide de nanoparticules de nitrure de titane et de nanoparticules de cuivre revêtues d'or Download PDF

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Publication number
WO2025014842A1
WO2025014842A1 PCT/US2024/036983 US2024036983W WO2025014842A1 WO 2025014842 A1 WO2025014842 A1 WO 2025014842A1 US 2024036983 W US2024036983 W US 2024036983W WO 2025014842 A1 WO2025014842 A1 WO 2025014842A1
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Prior art keywords
nps
nanoparticles
sample
tin
gold
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Inventor
Alexander O. Govorov
Veronica A. BAHAMONDES LORCA
Oscar R. AVALOS OVANDO
Martin E. Kordesch
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Ohio University
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Ohio University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present disclosure incorporates by reference the entirety of United States Provisional Application Ser. No. 63/512,506, filed July 7, 2023, and United States Provisional Application Ser. No. 63/572,981 filed April 2, 2024.
  • TECHNICAL FIELD [0002] The present disclosure relates to detecting analytes using titanium nitride (TiN) nanoparticles (NPs) and gold-coated copper (Au@Cu) nanoparticles.
  • Antibodies and more specifically, antibodies that interact with analytes of interest, are attached to TiN nanoparticles and gold-coated copper nanoparticles. The attached antibodies may then be placed in test strips, and the test strips may be used to for rapid analyte testing.
  • Rapid analyte testing where tests rapidly return results regarding the presence or absences of an analyte, can provide quick results that are easy to interpret in both clinical and residential settings. However, rapid analyte tests that provide inaccurate results, or results that are difficult to interpret are of little use. Even if the rapid analyte tests provide accurate, clear results, the rapid analyte test may require large amounts of materials that place further strain on Earth’s diminishing resources.
  • NPs plasmonic nanoparticles
  • Au Docket No. OHU23015WO NPs Au Docket No. OHU23015WO NPs
  • Example embodiments disclosed herein are directed to a medical test strip comprising detection antibodies comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof, wherein the detection antibodies bind a first epitope or a first binding site on an analyte.
  • the medical test strip further comprises a test line comprising test antibodies, wherein the test antibodies bind a second epitope or second binding site on the analyte.
  • the Docket No. OHU23015WO medical test strip also comprises a control line comprising control antibodies, wherein the control antibodies bind the detection antibodies.
  • Example embodiments disclosed herein are directed to methods of detecting an analyte, comprising obtaining a medical test strip comprising detection antibodies comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof, wherein the detection antibodies bind a first epitope or a first binding site on an analyte.
  • the method further comprises obtaining a sample, applying the sample to the sample pad of the test strip, passing the sample over the test line and the control line, and observing the test line and the control line.
  • FIG. 1A shows a schematic representation of a lateral flow assay (LFA) showing the sample pad containing the marked antibody against the target antigen and the test and control lines. After the sample is applied to the sample pad, the sample plus the antibodies diffuse through the strip. Only if the target antigen is present, the marked antibody will bind to both the test and control lines (+ result). If the target antigen is not present, the target antibody will only bind to the Docket No.
  • LFA lateral flow assay
  • FIG.1B shows characterization (scheme, colloid, and TEM) for the three kinds of NPs used throughout this work: Au in H2O [from 35 ], Cu@Au in Tris EDTA (TE) buffer, and TiN in H2O. Scale bars: 40 nm for Au, 20 nm for Cu@Au, and 100 nm for TiN. [0014] FIG.
  • the shaded areas show three different assessment spectra: i) in light yellow, the day-vision sensitivity of the human eye (photopic vision), S day ⁇ vision ⁇ ⁇ ⁇ ; in light gray, a typical tungsten LED; iii) in light blue, a typical white LED, S white ⁇ LED ⁇ ⁇ ⁇ .
  • FIG. 1D shows the VEC and the MEC efficiencies for each NP as shown, for the three spectral sources from FIG. 1C.
  • FIG.1E shows bulk material trade prices for titanium, 36 copper 37 and gold 38 (left panel) and sale prices for different gold products: trade, bullion, powder, and colloids [from Ref. 13 ] (right panel).
  • FIG. 2A shows a visual characterization of each type of NPs before and after antibody conjugation to demonstrate they do not aggregate nor precipitate.
  • FIG. 2B shows a table and plot comparing the plasmons for each type of NPs before (solid lines) and after (dashed lines) conjugation.
  • FIG. 1D shows the VEC and the MEC efficiencies for each NP as shown, for the three spectral sources from FIG. 1C.
  • FIG.1E shows bulk material trade prices for titanium, 36 copper 37 and gold 38 (left panel) and sale prices for different gold products: trade, bullion, powder, and colloids [from Ref. 13 ] (
  • FIG. 2C shows LFA in duplicate showing the identification of FITC at different concentrations (100, 10, 1, 0.1, and 0 nM) using the conjugated NPs.
  • FIG. 2D shows a plot showing the intensity measured via ImageJ of each test band of the LFA. Docket No. OHU23015WO
  • FIG. 3A shows a 0.25 % agarose gel for testing the functionalization of the Cu@Au and TiN NPs. At the bottom of the gel, each solution pre- and post-functionalization.
  • FIG. 3B shows representative schemes of the dot blot (top) and LFA (bottom) used for testing, respectively, the ability of the Cu@Au and TiN NPs to detect the target protein and their specificity.
  • FIG. 3C shows dot blot analysis showing the detection of cardiac troponin (cTnT) at different concentrations ( ⁇ g).
  • the Ponceau S staining corroborates the presence of cTnT at different concentrations in the nitrocellulose membrane.
  • the membrane was cut into 3 sections, which are shown after being incubated for a total of 30 minutes with the conjugated Au, Cu@Au, and TiN NPs.
  • FIG. 3D shows LFA against cTnT diluted in bovine calf serum, showing the specific detection of cTnT when using each conjugated NP.
  • FIG.4A shows cropped test lines and control lines of all strips that tested with different FITC concentrations.
  • FIG. 4B shows results of the machine learning experiments. It is evident that the accuracy of TiN and Cu@Au-based LFAs surpasses that of Au-based LFAs.
  • Figure S Models of TiN-TiO 2 core-shell.
  • A Schematic representation of different aspect ratios between TiN-TiO2 as shown. In all plots, lines are Comsol numerical simulations (left axis) and red symbols are our NPs experimental absorbance (right axis).
  • B Different diameters of TiN, no TiO 2 .
  • the inset shows a visual of the post-transfer solution.
  • Figure S6 TEM images of NPs after functionalization.
  • Figure S7 Normalized absorbance of the Cu@Au and TiN NPs pre and post anti-cTnT conjugation.
  • the medical test strip further comprises a test line comprising test antibodies, wherein the test antibodies bind a second epitope or a second binding site on the analyte.
  • the medical test strip also comprises a control line comprising control antibodies, wherein the control antibodies bind the detection antibodies.
  • antibody or “antibodies” refers to immunoglobulins and immunoglobulin portions, whether natural or partially or wholly synthetic, such as recombinantly, produced, including any portion thereof containing at least a portion of the variable region of the immunoglobulin molecule that is sufficient to form an antigen binding site.
  • an antibody or portion thereof includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding site.
  • an antibody refers to an antibody that contains two heavy chains (which can be denoted H and H′) and two light chains (which can be denoted L and L′), where each heavy chain can be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen binding site (e.g. heavy chains include, but are not limited to, VH, chains VH-CH1 chains and VH-CH1-CH2-CH3 chains), and each light chain can be a full-length light chain or a portion thereof sufficient to form an antigen binding site (e.g.
  • light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H′) pairs with one light chain (L and L′, respectively).
  • antibodies minimally include all or at least a portion of the variable heavy (VH) chain and/or the variable light (VL) chain.
  • the antibody also can include all or a portion of the constant region.
  • the term antibody includes full-length antibodies and portions thereof including antibody fragments.
  • a “detection antibody” is, for example, a monoclonal antibody that is conjugated to a detection label and that is specific for a target analyte of interest.
  • a “test antibody” or a “control antibody” should be understood as an antibody, such as a monoclonal antibody, attached directly or indirectly at the test line or control line, respectively, of the medical test strip of the invention and that is capable of detecting and binding the detection Docket No. OHU23015WO antibody.
  • the detection antibody may be bound to, or immobilized on, the assay strip using a variety of techniques known to those in the art.
  • the term “bound” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antibody and functional groups on the support or may be a linkage by way of a cross-linking agent).
  • detection antibodies are reversibly associated with the conjugation pad such that they may migrate to the test line as a complex with their target analyte for detection by the test antibody present at the test line and the control antibody present at the control line.
  • analyte or “analytes” refers to a compound or composition to be detected or measured and which has at least one epitope or binding site.
  • the analyte can be any substance for which there exists a naturally occurring analyte specific binding member or for which an analyte-specific binding member can be prepared. e.g., carbohydrate and lectin, hormone and receptor, complementary nucleic acids, and the like.
  • analytes include virtually any compound, composition, aggregation, or other substance that may be immunologically detected. That is, the analyte, or portion thereof, will be antigenic or haptenic having at least one determinant site, or will be a member of a naturally occurring binding pair.
  • one or more analyte detected is an antibody (e.g., IgG, IgM) in a sample (e.g., urine, oral fluid, blood, plasma or serum sample) where the antibody is specific for a virus or virus component, bacteria or bacteria component, cancer cell or tumor antigen.
  • allergy detection testing comprises detecting the presence of specific IgG, IgM and/or IgE Ab in a subjects oral fluid, whole blood, urine, plasma or serum to specific allergens.
  • Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, bacteria, viruses, amino acids, nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes as well as those administered for illicit purposes), pollutants, pesticides, and metabolites of or antibodies to any of the above substances.
  • analyte also includes any antigenic substances, haptens, antibodies, macromolecules, and combinations thereof.
  • a non-exhaustive list of exemplary analytes is set forth in U.S. Pat. No. 4,366,241, at column 19, line 7 through column 26, line 42; Docket No. OHU23015WO U.S. Pat. Nos. 4,299,916; 4,275,149; and 4,806,311, all of which are hereby incorporated herein by reference. Therefore, in various embodiments, one or more analyte is detected from a sample obtained from a subject. [0039] A sample is any material to be tested for the presence and/or concentration of an analyte.
  • a biological sample can be any sample taken from a subject, e.g., non-human animal or human and utilized in the test devices.
  • a biological sample can be a sample of any body fluid, cells, or tissue samples from a biopsy.
  • Body fluid samples can include without any limitation blood, urine, sputum, semen, feces, saliva, bile, cerebral fluid, nasal swab, urogenital swab, nasal aspirate, spinal fluid, etc.
  • Biological samples can also include any sample derived from a sample taken directly from a subject, e.g., human.
  • a biological sample can be the plasma fraction of a blood sample, serum, protein or nucleic acid extraction of the collected cells or tissues or from a specimen that has been treated in a way to improve the detectability of the specimen, for example, a lysis buffer containing a mucolytic agent that breaks down the mucens in a nasal specimen significantly reducing the viscosity of the specimen and a detergent to lyse the virus thereby releasing antigens and making them available for detection by the assay.
  • a sample can be from any subject animal, including but not limited to, human, bird, porcine, equine, bovine, murine, cat, dog, or sheep.
  • a sample can be derived from any source, such as a physiological fluid, including blood, serum, plasma, saliva or oral fluid, sputum, ocular lens fluid, nasal fluid, nasopharyngeal or nasal pharyngeal swab or aspirate, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, transdermal exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid, semen, cervical mucus, vaginal or urethral secretions, amniotic fluid, and the like.
  • a physiological fluid including blood, serum, plasma, saliva or oral fluid, sputum, ocular lens fluid, nasal fluid, nasopharyngeal or nasal pharyngeal swab or aspirate, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid,
  • test sample can be used such as water, food products, soil extracts, and the like for the performance of industrial, environmental, or food production assays as well as diagnostic assays.
  • a solid material suspected of containing the analyte can be used as the test sample once it is modified to form a liquid medium or to release the analyte.
  • Other fields of interest include the diagnosis of veterinary diseases, analysis of meat, poultry, fish for bacterial contamination, inspection of food plants, food grains, fruit, dairy products (processed or unprocessed), restaurants, hospitals and other public facilities, analysis of Docket No. OHU23015WO environmental samples including water for beach, ocean, lakes or swimming pool contamination.
  • Analytes detected by these tests include viral and bacterial antigens as well as chemicals including, for example, lead, pesticides, hormones, drugs and their metabolites, hydrocarbons and all kinds of organic or inorganic compounds.
  • spherical copper nanoparticles covered with a gold shell (Cu@Au) and spherical titanium nitride (TiN) NPs as they have been proved to be stable, bio-friendly and capable of being manufactured at large scale. 17-20
  • the choice of these NPs would be especially profitable when they are produced by cost-efficient and scalable methods such as laser ablation, which is much cheaper than colloidal chemistry under high production rates (> 500 mg/hour).
  • TiN NPs show a plasmonic peak in the range of 650-800 nm and high photothermal conversion efficiency.
  • 16,21 TiN biomedical application has been limited by the difficulty of fabricating large-scale spherical-shaped in water.
  • laser-synthesized TiN NPs present a safe choice for future biomedical applications.
  • ultrastable Cu@Au NPs have been recently synthetized via a seed-mediated galvanic replacement approach, which unlike CuAu alloys, the thin Au shell here provides enhanced stability. Furthermore, they show a superior photothermal efficiency in solar-induced water evaporation as compared to Au NPs. 17
  • TiN and Cu- Au NPs are promising to be useful in several fields such as catalysis, light harvesting, optoelectronics, and biotechnologies. 25-30 [0043] Examples described herein include medical test strips comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof.
  • Examples described herein further include a medical test strip comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof having a detection means such as a detection antibody attached thereto.
  • the titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof provide a detectable change in the visible absorption spectrum in response to the binding of an analyte. This change may be observed by instrument or by the naked eye.
  • Docket No. OHU23015WO Many metal nanoparticles are small enough to interact intimately with biological or chemical species. Such interaction may be facilitated by their comparable size and by the large surface area to volume ratio of the nanoparticles. Molecular species can be readily attached to the nanoparticle surface.
  • the attachment which can be by non-specific adsorption or interactions involving covalent or electrostatic bonding, affects the surface plasmon resonance (SPR) of the nanoparticle and alters the spectral response.
  • SPR surface plasmon resonance
  • This alteration of the spectral response can be observed either as a wavelength shift in spectral peak, a diminishment or enhancement of the peak absorbance, or a combination of these.
  • This sensitivity of the surface of these nanoparticles to the molecules in the surrounding environment makes them ideal for sensor applications.
  • Metal nanoparticles that differ in size, shape and composition scatter light of different wavelengths according to their distinct SPR. This is again due to the influence of these factors on the spectral response of the SPR.
  • the most typical metal nanoparticle shape is spherical and these have a characteristic single SPR spectral peak. If a metal nanoparticle has a non-spherical shape, for example ovoid, then the SPR will exhibit more than one peak. This occurs as the nanoparticles are no longer isometric and the SPR electrons have more than one oscillation axis. In the case of ovoid nanoparticles, electronic oscillation about the major and minor axes will result in at least two peaks in the SPR spectrum.
  • An advantage of non-isometric metal nanoparticles is their increased sensitivity, which in part arises from the presence of the additional SPR spectral peaks.
  • FIG. 1A A schematic illustration of a medical test strip is provided in FIG. 1A.
  • a conjugation pad of the medical test strip are nanoparticles to which a detection antibody is attached.
  • a sample containing a carrier fluid is introduced onto the sample pad. If the sample contains any analyte specific to the detection antibody, the analyte binds to the detection antibody. By capillary action, the sample flows from the conjugation pad to the absorbent pad.
  • test line contains a substance to which analyte binds and to which the detection antibody does not bind.
  • the control line contains a substance to which the detection antibody binds but to which the analyte does not bind. If analyte is present, the test line changes color based on the type of nanoparticle bonded to the detection antibody. Whether analyte is Docket No. OHU23015WO present or absent in the sample, the control line changes color based on the type of nanoparticle bonded to the detection antibody.
  • Titanium nitride nanoparticles, zirconium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof have a strong SPR spectrum comparable to that of gold nanoparticles, as shown in FIG. 1C.
  • the methods described herein enable the preparation of titanium nitride nanoparticles, or gold-coated copper nanoparticles that are stable over long time periods and do not aggregate.
  • the titanium nitride nanoparticles may be nanospheres.
  • the titanium nitride nanospheres may have a size distribution of from 1 nanometers (nm) to 100 nm, from 1 nm to 80 nm, from 1 nm to 60 nm, from 1 nm to 40 nm, from 1 nm to 30 nm, from 1 nm to 5 nm, from 1 nm to 10 nm, from 5 nm to 40 nm, from 10 nm to 40 nm, from 20 nm to 40 nm, from 5 nm to 30 nm, from 10 nm to 30 nm, from 20 nm to 30 nm, from 5 nm to 20 nm, from 10 nm to 20 nm, from 5 nm to 20 nm, from 10 nm to 20 nm, from 5 nm to 20 nm, or even from 5 nm to 10 nm.
  • the titanium nitride nanospheres exhibit plasmon adsorption in the visible light spectrum.
  • the titanium nitride nanospheres attached to the detection antibodies may appear blue to the naked eye.
  • Example characterizations of titanium nitride nanoparticles are provided in FIGS. 3, 4, 5, and 6.
  • FIGS. 7A and 7B illustrate medical test strips using TiN nanoparticles compared to medical test strips using gold nanoparticles.
  • the gold-coated copper nanoparticles may be nanospheres.
  • the gold-coated copper nanospheres may have a size distribution of from 4 nm to 14 nm, from 4 nm to 12 nm, from 4 nm to 10 nm, from 4 nm to 8 nm, from 4 nm to 6 nm, 6 nm to 14 nm, from 6 nm to 12 nm, from 6 nm to 10 nm, from 6 nm to 8 nm, 8 nm to 14 nm, from 8 nm to 12 nm, from 8 nm to 10 nm, 10 nm to 14 nm, from 10 nm to 12 nm, or even from 12 nm to 14 nm.
  • the gold-coated copper nanospheres also exhibit plasmon adsorption in the visible light spectrum.
  • the gold-coated copper nanospheres attached to the detection antibodies may appear blue to the naked eye.
  • Molecular species, such as antibodies may be readily attached to the surface of the titanium nitride nanoparticles, or gold-coated copper nanoparticles.
  • Example characterizations of gold-coated copper nanoparticles are provided in FIGS. 8, 9, and 10. Docket No. OHU23015WO FIGS. 11A and 11B illustrate medical test strips using gold-coated copper nanoparticles compared to medical test strips using gold nanoparticles.
  • the medical test strip may be made of, for example, a porous matrix, such as a flat piece of nitrocellulose or other support structure that may be coated with or impregnated or otherwise including nitrocellulose or any other polymer suitable for a chromatographic process.
  • the medical test strip may be in a form of a plain narrow piece, or it may, for example, be coil- shaped in order to increase its length in the same volume, and hence, improve separation of the components of the liquid sample.
  • the medical test strip is small and portable such that it requires only a small amount of sample for testing but is large enough to provide a separate test area for detection. For example the dimensions may be about 2 cm long and 3 mm wide.
  • the medical test strip may preferably include a backing to provide rigidity to the medical test strip (e.g., plastic) and/or a housing (e.g., plastic) that can optionally cover portions of the porous matrix to protect the matrix and any antibodies bound thereto during storage and transport prior to use and during use.
  • a housing e.g., plastic
  • Such housing may be removable or remain in place so long as the sample pad portion of the matrix is accessible to sample and results at the single test area can be easily read. Lateral flow assay devices that are capable of being handheld are known in the art.
  • Methods of detecting an analyte include obtaining a medical test strip as described herein. A sample that has been obtained is then applied to the medical test strip. In some examples, the sample is applied to a sample pad of the medical test strip.
  • the sample is applied directly to the sample pad of the medical test strip.
  • the sample is mixed with or suspended in a sample solution before applying the sample to the sample pad of the medical test strip.
  • the sample pad may be soaked in a sample solution before the sample is applied to the sample pad.
  • a sample solution may be added to the sample pad after the sample has been applied to the sample pad.
  • the sample may be obtained before obtaining the medical test strip.
  • the medical test strip may be obtained before obtaining the sample.
  • the detection antibodies comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof may be present on the sample pad.
  • the detection antibodies may be present on the sample pad before the sample is Docket No. OHU23015WO applied to the sample pad. In some examples, the detection antibodies may be added to the sample pad after the sample is applied to the sample pad. [0054] After the sample has been applied to the sample pad, detection antibodies comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof may bind a first epitope or a first binding site on an analyte if the analyte is present in the sample. The sample may then be passed away from the sample pad and passed over the test line and the control line.
  • test antibodies bound to a test line will bind a second epitope or a second binding site on the analyte.
  • Some of the analyte will be bound by both detection antibodies comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, and combinations thereof, and test antibodies.
  • detection antibodies comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, and combinations thereof will gather at the test line.
  • detection antibodies comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof may bind control antibodies that are bound to the medical tests strip on a control line.
  • a visible line on the control line will appear because the detection antibodies will be bound to the control antibodies.
  • the control line will help to ensure that the medical test strip is functioning properly. If a visible line does not appear at the control line, this may indicate that sample or the detection antibody was not properly passed over the control line or the test line.
  • the detection antibodies comprising titanium nitride nanoparticles, gold-coated copper nanoparticles, or combinations thereof will only be bound by the control line.
  • Nanoparticles can be conjugated with diverse biomolecules and employed in biosensing to detect target analytes in biological samples. This proven concept was primarily used during the COVID-19 pandemic with gold NPs-based lateral flow assays (LFAs).
  • NPs plasmonic nanoparticles
  • TiN titanium nitride
  • Cu@Au gold shell
  • our study represents the 1st application of laser-ablation-fabricated nanoparticles (TiN) in the LFA and dot- blot biotesting.
  • OHU23015WO inexpensive NPs in bio-applications in where the NPs’ plasmonic features in combination with their successful functionalization capabilities, may be useful in photothermal therapies, delivery, imaging and sensing technologies.
  • Method and Materials [0062] Synthesis of Cu@Au NPs. Typically, 33.2 mg of copper(II) acetylacetonate (Cu(acac)2, ⁇ 99.9%, Sigma-Aldrich) was dissolved in 10 mL of Oleylamine (OLA, 70%, Sigma- Aldrich) under nitrogen atmosphere. Then, the solution was kept at 230 °C for 3 h to produce Cu NPs.
  • Oleylamine Oleylamine
  • the solution was alkalinized by adding 140 ⁇ L of NH4OH (Sigma). Then, dropwise, 90 ⁇ L of 3-Mercaptopropionic acid (3-MPA, Sigma) was added. The solution was heated in a hot plate at 75°C for 1h with manual sporadic agitation to yield the 3-MPA capped Cu@Au NPs. The NPs were then precipitated by adding 10 mL of distilled water, 2.5 mL of isopropanol, and 1% Triton x100. The Cu@Au NPs were collected by centrifugation at 7000 rpm for 10 min, rinsed with water, and resuspended in 1X Tris-EDTA (TE), pH 8.0 buffer.
  • TE 1X Tris-EDTA
  • TiN NPs synthesis TiN NPs were synthesized by the technique of femtosecond laser ablation in liquid ambient.
  • a TiN target (MaTeck, Germany, 99+%) was fixed in a vertical position inside a glass cuvette (Hellma, Germany, optical glass, 88 mL, 2.5 mm wall thickness) filled with 80 mL of acetone (Acros Organics, 99.5+%).
  • the thickness of the liquid layer between the target and a cuvette wall was 3 mm.
  • a femtosecond laser beam (s-Pulse HP, Amplitude Systems, France, Yb:KGW, 490 fs, 10 kHz) was focused through the cuvette wall on the surface of the target using a 75 mm convex lens. The energy was attenuated down to 150 ⁇ J per pulse using a half-wave plate and Brewster polarizer. To prevent ablation from the same area, the target was constantly moved at a speed of 2.5 mm s-1 by a translation stage (scanned area was 5 x 5 mm2). Docket No. OHU23015WO [0065] TiN NPs water dispersion.
  • TEM Transmission Electron Microscope
  • NPs were centrifuged and dispersed in their corresponding buffer (Au: TE pH 8.0, Cu@Au: Borate pH 7.0, and TiN: 1X TE pH 8.48).2 ⁇ g of anti-FITC (200-032-037, Jackson ImmunoResearch Laboratories, Inc.) per 40 ⁇ L of NPs solution was added and incubated in a rotor at 4°C for 30 min. 10% BSA was added to the NPs to have a BSA final concentration equal to 1 %. The samples were incubated for an extra hour at 4°C with constant rotation. The NPs were then rinsed twice and centrifuged at 13,000 rpm for 10 min each.
  • NPs were resuspended in their corresponding buffers.
  • Functionalization of NPs with anti-cTnT Au and TiN NPs were centrifuged and then dispersed via sonication in their corresponding buffer (Au: 1X TE pH 8.0 and TiN: 1X TE pH 8.48).10 ⁇ L/mL of Anti-Cardiac Troponin T antibody [1F11] (cTnT, ab10214, Abcam) was added to each sample and incubated in a rotor at room temperature for 30 min. 10% BSA was added to the NPs to obtain a BSA final concentration equal to 1 %. The samples were incubated for an extra hour at room temperature with constant rotation.
  • NPs were then rinsed twice and centrifuged at 4 °C and 12,000 rpm for 10 min each. Finally, the NPs were resuspended in their corresponding buffers. For the Cu@Au NPs, after centrifugation, NPs were resuspended in 100 mM MES buffer pH 6.0 containing excess of freshly prepared 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, 1 mg/mL, # 22980, Thermo ScientificTM).
  • EDC 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
  • sulpho-SANPAH Excess of sulpho- sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (sulpho-SANPAH, 1 mg/mL, # 22589, Thermo ScientificTM) was added immediately to the NPs solution and samples were incubated in dark, at room temperature and under constant rotation for 20 min.2-Mercaptoethanol (10 ⁇ L/mL) was added to the NPs to quench EDC, samples were incubated under the same condition for an additional 20 min.
  • 0.25 % agarose gel was prepared in 0.5 X Tris-borate-EDTA (TBE) buffer (40 mM Tris-base, 40 mM Boric acid, and 1 mM EDTA). The samples were loaded with sucrose 50 % and run at 100 Volts for 5 to 10 min.
  • Lateral flow assay (LFA) For FITC-LFA, 20 ⁇ L of anti-FITC functionalized NPs were mixed with 0.2 ⁇ L of the oligo FITC-T10-Biotin (Metabion International AG, Germany) diluted at the specified concentrations. Commercial LFA strips (MGHD1, Milenia GenLineHybridetect, Germany) were used, which were produced at request without the gold NPs pad.
  • sample mix 50 ⁇ L of running buffer, 25 ⁇ L 1:1 bovine calf serum/PBS containing 0.156 ⁇ g of recombinant human cTnT (ab86685, abcam), and 1 ⁇ L of biotinylated anti- cTnT antibody [bs-10648R-Biotin, Bioss] was loaded in the LFA strips.
  • the sample mix for the negative controls only contains running buffer, biotinylated anti-cTnT antibody and bovine calf serum in PBS (1:1). [0071] Dot blot.
  • Recombinant human cTnT (ab86685, abcam) was diluted in 1X PBS buffer. 50 ⁇ L of the protein at different concentration were blotted onto a nitrocellulose membrane (0.2 ⁇ m, #88024, Thermo ScientificTM). After aspiration was completed, the membranes were stained with Ponceau S to check the presence of the blotted protein on them. The membranes were rinsed with TBS-T buffer until completely removed the stain and then blocked in 5 % milk for 30 minutes. The membranes were rinsed once with each of the corresponding NPs-buffers followed by the incubation for a maximum of 30 min at 4°C with the conjugated NPs.
  • the scattering cross-section is calculated by integrating the scattered intensity over a fictitious sphere around the NP, whereas the formalism for the absorption cross- section is based on the following equations: Q ⁇ ⁇ abs abs [0076] I 0 , (S2) where Q abs is the absorbed power by the system, is the dielectric constant of the ⁇ ur metal nanoparticle, is the angular frequency of the incident light, E ⁇ is the complex electric field inside the metal, and I 0 is the photon flux magnitude (intensity for simplicity), given by [0079] where ⁇ ⁇ is the dielectric constant of the medium, c 0 is the speed of light in vacuum, ur is the vacuum permittivity, and E 0 is the electric field magnitude of the incident electromagnetic wave.
  • NPs are simulated with sizes estimated from experimental characterization, as described in Fig.1.
  • a thin shell of TiO2 is needed in the accurate simulation of the extinction, as previously described elsewhere 1 .
  • For gold and copper we use the permittivity ⁇ NP from Johnson and Christy 2 , for TiN from Guler 3 , and for TiO 2 from Siefke 4 . The sum of ⁇ ⁇ and ⁇ ⁇ allows to obtain ⁇ ⁇ , related to the experimental measurement of absorbance. Docket No.
  • Contrastive learning uses negative sampling and contrastive loss functions to effectively Docket No. OHU23015WO distinguish between different classes or categories of data.
  • our contrastive learning model employs a Siamese network 5 consisting of two identical subnetworks, each with a simple architecture that includes a single fully connected layer. This Siamese network processes a pair of strips from the training set, generating two corresponding vectors, as shown in Fig. S3B.
  • Figure S6A was obtained as indicated in the main text, in the materials and methods section.
  • Figure S6B the TiN NPs were functionalized using a different approach, to demonstrate the flexibility of conjugation these NPs have.
  • TiN NPs shown in Figure S6B were Docket No. OHU23015WO functionalized with DNA, composed of a thiol group at the 5’ end of 19 consecutive thymine bases, ordered from biomers, that will react with the titanium forming a titanium sulfide bridge between the TiN NPs and the DNA.
  • VEC volume extinction coefficient
  • MEC mass extinction coefficient
  • Cu@Au and TiN NPs preserved their characteristics after being transferred to an aqueous solvent.
  • the TiN NPs were synthesized via laser ablation, allowing the production of large amount of spherical size-specific NPs, which do not aggregate due to electrostatic stabilization. These NPs of ⁇ 30-40 nm were grown in acetone, showing great stability and capability of by polyethylene glycol-coating.
  • the stability analysis in the aqueous medium after measuring the plasmon at day zero and >40 days indicates that the stability of the Cu@Au NPs is preserved (Fig. S4C).
  • the characterization of the TiN NPs indicates these particles have an average size of 35 nm (Weibull fit, max.34.5 nm) when suspended in water (Fig. S4D).
  • the observed plasmon was around 650 nm, in good agreement with the observed plasmon before their transfer to water (Fig. S4E).
  • TiN NPs were also stable through time since their plasmon was preserved after >40 days (Fig. S4F). Together, these results show that after transferring Cu@Au and TiN NPs to aqueous media, the NPs size and optical properties were preserved.
  • Figure 2B shows a plasmon red-shift for all the NPs, suggesting a few nm shell of new dielectric media (in our case, biomolecules), evidencing a successful modification step. Moreover, the efficiency of the functionalization was similar for the three NPs, as demonstrated by their absorbances (Fig. 2B). For testing the ability of the functionalized NPs to detect the antigen of interest, LFA were run.
  • Figure 2C shows the functionalized TiN and Cu@Au NPs identified the target protein (FITC-T10-Biotin) present at a concentration between 100 and 1 nM.
  • NPs plasmonic characteristics were preserved even when attached to the test and control line of the LFA strip, as demonstrated by their ability to absorb at specific wavelength (see Figure S5).
  • Functionalized Cu@Au and TiN NPs can detect proteins with biological significance.
  • NPs were functionalized for detecting cardiac troponin (cTnT), a protein that increases in the blood of patients suffering a heart attack.
  • the Au (positive control), Cu@Au and TiN NPs were modified with an antibody anti-cTnT.
  • Figure 3A shows a change in the migration pattern in a 0.25% agarose gel, confirming the functionalization of the three NPs. Additionally, the visual analysis (Fig. 3A) and TEM imaging (see Figure S6A) of the NPs solutions confirm that the NPs do not aggregate and remain mono-dispersed. In addition, a red-shift in the Cu@Au and TiN NPs absorbance pattern ( Figure S7) further confirms their successful functionalization (note that in Fig. S7 the post-functionalization absorbances have been normalized to facilitate comparison). Next, different concentrations of recombinant human cTnT were blotted in a nitrocellulose membrane as schematically shown in Figure 3B.
  • the TiN Docket No. OHU23015WO NPs show an advantage in recognition accuracy that speaks to the potential of new materials in biosensing.
  • the terms “substantially” and “about” may be utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. These terms are also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
  • the term “substantially” is used herein also to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
  • first component is described as “comprising” or “including” a second component, it is contemplated that, in some embodiments, the first component “consists” or “consists essentially of” the second component. Additionally, the term “consisting essentially of” is used in this disclosure to refer to quantitative values that do not materially affect the basic and novel characteristic(s) of the disclosure. [00122] It should be understood that any two quantitative values assigned to a property or measurement may constitute a range of that property or measurement, and all combinations of ranges formed from all stated quantitative values of a given property or measurement are contemplated in this disclosure. Docket No.

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Abstract

Des bandelettes réactives médicales contiennent des anticorps de détection comprenant des nanoparticules de nitrure de titane, des nanoparticules de cuivre revêtues d'or, ou des associations de celles-ci, les anticorps de détection se liant à un premier épitope ou à un premier site de liaison sur un analyte ; une ligne de test comprenant des anticorps de test, les anticorps de test se liant à un second épitope ou à un second site de liaison sur l'analyte ; et une ligne de commande comprenant des anticorps de témoin, les anticorps de témoin se liant aux anticorps de détection. Des procédés de détection d'un analyte consistent à appliquer un échantillon sur le tampon à échantillon de la bandelette réactive ; à faire passer l'échantillon et les anticorps de détection comprenant des nanoparticules de nitrure de titane, des nanoparticules de cuivre revêtues d'or, ou des associations de celles-ci sur la ligne de test et la ligne de témoin ; et à observer la ligne de test et de la ligne de témoin.
PCT/US2024/036983 2023-07-07 2024-07-07 Bandelettes réactives et procédés de test de bio-analytes à l'aide de nanoparticules de nitrure de titane et de nanoparticules de cuivre revêtues d'or Pending WO2025014842A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100136566A1 (en) * 2008-12-03 2010-06-03 Abaxis, Inc. Lateral flow strip assay with immobilized conjugate
US20170234817A1 (en) * 2012-01-31 2017-08-17 Regents Of The University Of Minnesota Lateral flow assays with thermal contrast readers
US20170234866A1 (en) * 2016-02-11 2017-08-17 Massachusetts Institute Of Technology Multiplexed lateral flow assay
US20220074934A1 (en) * 2014-08-13 2022-03-10 Zoetis Services Llc Signal amplification in plasmonic specific-binding partner assays
US20220099667A1 (en) * 2019-06-12 2022-03-31 Gmd Biotech, Inc Conjugate for immunodetection based on lateral flow assay, and immunodetection method using same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100136566A1 (en) * 2008-12-03 2010-06-03 Abaxis, Inc. Lateral flow strip assay with immobilized conjugate
US20170234817A1 (en) * 2012-01-31 2017-08-17 Regents Of The University Of Minnesota Lateral flow assays with thermal contrast readers
US20220074934A1 (en) * 2014-08-13 2022-03-10 Zoetis Services Llc Signal amplification in plasmonic specific-binding partner assays
US20170234866A1 (en) * 2016-02-11 2017-08-17 Massachusetts Institute Of Technology Multiplexed lateral flow assay
US20220099667A1 (en) * 2019-06-12 2022-03-31 Gmd Biotech, Inc Conjugate for immunodetection based on lateral flow assay, and immunodetection method using same

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