WO2025042890A1 - Diagnostic basé sur une nanostructure de la tuberculose - Google Patents

Diagnostic basé sur une nanostructure de la tuberculose Download PDF

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Publication number
WO2025042890A1
WO2025042890A1 PCT/US2024/043037 US2024043037W WO2025042890A1 WO 2025042890 A1 WO2025042890 A1 WO 2025042890A1 US 2024043037 W US2024043037 W US 2024043037W WO 2025042890 A1 WO2025042890 A1 WO 2025042890A1
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WO
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Prior art keywords
sensor device
sensor
liquid
antigen
nanostructures
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PCT/US2024/043037
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English (en)
Inventor
Alexander Star
Jieyu WANG
Michael R. SHURIN
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University Of Pittsburgh - Of The Commonwealth System Of Higher Education
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Publication of WO2025042890A1 publication Critical patent/WO2025042890A1/fr

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Classifications

    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4146—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS involving nanosized elements, e.g. nanotubes, nanowires
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/492—Determining multiple analytes
    • 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
    • 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

Definitions

  • Tuberculosis caused by rnwhcrctenw tubera/faw (MTB), is a highly contagious and airborne disease spread through the air when a patient coughs, speaks, or sneezes.
  • MTB is one of infectious agents that kill the most people in the world, and TB remains a leading cause of morbidity and mortality in many developing countries.
  • TB is clinically dichotomized into active TB and latent. TB forms.
  • Latent TB is the case where the concentration of MTB is too low to show symptoms.
  • TB diagnosis focuses on screening tools (that is, chest X-ray), the detection of bacilli by microscopic techniques (i.e., smear microscopy) and bacterial growth cultures, detection of host immune response to the pathogen (that is, Mantoux test), and bacterial nucleic acid amplification methods.
  • the interferon-v release assays like QuantiFERON-TB Gold Plus, widely used in the USA and Europe, can also diagnose TB by detecting IFN-y secretion from the collected blood lymphocytes after their stimulation with ESAT-6 and CFP-10 antigens that are quite specific for MTB, A chest X-ray showw low sensitivity to latent TB.
  • the monoclonal antibody against recombinant Ag85B protein was immobilized onto the biosensor for antigen detection and the LOD was determined as 0.12 pgZmL.
  • a silicon nanowire-based field-effect transistor (SiNW- FET) biosensing platform was developed to detect the MTB, Based on the binding between Ag85B and anti ⁇ Ag85B antibody, that biosensor demonstrated sensitivity and obtained responses from sputum samples of TB patients.
  • SiNW- FET silicon nanowire-based field-effect transistor
  • the plurality of nanostructures comprise a plurality of semiconductor enriched single-walled carbon nanotubes, and a plurality of rabbit polyclonal Ag85B antibodies are covalently attached to the plurality of semiconductor enriched singlewalled carbon nanotubes.
  • the liquid may include phosphate buffered saline in a concentration range of approximately O. lx to 0.001 x.
  • a blocking buffer may be applied to the sensor before the liquid is deposited thereon, wherein the blocking buffer comprising bovine serum albumin, a non-ionic surfactant and polyethylene glycol.
  • the sensor device further includes at least one measurement system in operative connection with the sensor.
  • the sensor device may be otherwise characterized as described above and elsewhere herein.
  • the body fluid sample may, for example, be sputum or a blood fluid.
  • the body fluid sample may, for example, be a blood fluid.
  • the ionic strength and mass loading of the one or more recognition entities immobilized on the plurality of nanostructures are selected so that an average height of immobilized recognition entities is within a determined range of a maximum mass loading at which the average height remains within a Debye screening length of the liquid (for example, to achieve a determined limit of detection).
  • the sensor device further includes electronic circuitry including at least one measurement system in operative connection with the sensor to measure a variable providing a measure of change in at least one property of the sensor medium which is dependent upon the presence of the antigen.
  • the sensor device is a field-effect transistor device and the liquid functions as a gating liquid.
  • a blocking buffer which is interactive with non-selective binding sites may be applied to the sensor before the liquid is deposited thereon.
  • the protein may be covalently attached to the nanostructures.
  • the protein is crosslinked via a crosslinking agent.
  • the protein may, for example, be bovine serum albumin.
  • the protein is bovine serum albumin and the crosslinking agent is glutaraldehyde.
  • the average height of the one or more immobilized recognition entities may, for example, be within 0.2 to 2.0 times of the maximum mass loading at which the average height remains within a Debye screening length of the liquid, within 0.5 to 1.5 times of the maximum mass loading at which the average height remains within a Debye screening length of the liquid, or within 0.75 to 1 .25 times of the maximum mass loading at which the average height remains within a Debye screening length of the liquid.
  • the maximum mass loading at the se l ected ionic strength may, for example, be determined by experimentally determining a maximum response for a determined Ag85 antigen concentration over varying mass loading.
  • the selected ionic strength may, for example, be determined experimentally by determining response to the antigen at each of a plurality of ionic strengths.
  • the maximum mass loading at the selected ionic strength may be determined by experimentally determining a maximum response for a determined antigen concentration over varying mass loading.
  • the selected ionic strength may be determined experimentally by determining response to the antigen at each of a plurality of ionic strengths.
  • the nanostructures are carbon nanotubes.
  • the carbon nanotubes may, for example, be single walled carbon nanotubes.
  • the single-walled carbon nanotubes are semiconductor enriched single-walled carbon nanotubes.
  • the semiconducting content of the semiconductor enriched single-walled carbon nanotubes may be at least 90%, at least 95%, at least 99%, or at least 99.9%.
  • the ionic strength of the liquid may experimentally optimized based on the limit of detection for a given sensor configuration.
  • the mass loading of the recognition entities may be experimentally optimized based upon limit of detection for a given sensor configuration.
  • the one or more recognition entities may be immobilized upon a metal nanoparticles immobilized upon the plurality of nanostructures.
  • the metal nanoparticles include or are gold nanoparticles.
  • the plurality of nanostructures includes a plurality of semiconductor enriched single- walled carbon nanotubes, and a plurality of the antibodies or antibody fragments are covalently attached to the plurality of semiconductor enriched singlewalled carbon nanotubes.
  • a method of detecting an antigen includes applying a body fluid sample to a sensor device and measuring a variable which provides a measure of change in at least one property of a sensor medium which is dependent upon the presence of the antigen.
  • the sensor device includes a sensor including a substrate, a first electrode, a second electrode spaced from the first electrode, and a sensor medium on the substrate between the first electrode and the second electrode.
  • the sensor medium includes a plurality of nanostructures. At least one of one or more recognition entities selected from the group consisting of antibodies and an tibody fragments is immobilized on the plurality of nanostructures. Each of the one or more recognition entities includes at least one acti ve binding site for the antigen.
  • a liquid is deposited over the sensor medium.
  • An ionic strength of the liquid is selected at which binding of the antigen to the one or more recognition entities occurs.
  • the ionic strength and mass loading of the one or more recognition en tities immobilized on the plurality of nanostructures are selected so that an average height of immobilized recognition entities is within a determined of a maximum mass loading at which the average height remains within a Debye screening length of the liquid (for example, to achieve a determined limit of detection).
  • the sensor device further includes at least one measurement system in operative connection with the sensor.
  • the sensor device may be otherwise characterized as described above and elsewhere herein.
  • the body fluid sample may, for example, be sputum or a blood fluid.
  • the body fluid sample may, for example, be a blood fluid.
  • the method includes determining an ionic strength of the liquid at which binding of the antigen to the one or more recognition entities occurs and determining a mass loading of the one or more recognition entities immobilized on the plurality of nanostructures so that an average height of immobilized recognition entities is within a determined range of a maximum mass loading at which the average height remains within a Debye screening length of the liquid (for example, to achieve a determined limit of detection).
  • FIG. 1 A illustrates a schematic illustration of a Ab85B ⁇ SWCNT FET for detection of MTB antigen Ag85B including interdigitatedgold electrodes (illustrated as blocks), contacting a network of SWCNTs (illustrated as transparent), which are configured as the source (S) and drain (D) electrodes, wherein in a number of embodiments, the source-drain voltage (Vsa) was 50 mV, and the gate voltage ( V g ) was applied through an Ag/AgCl reference electrode inserted into the gating electrolyte.
  • interdigitatedgold electrodes illustrated as blocks
  • SWCNTs illustrated as transparent
  • Vsa source-drain voltage
  • V g gate voltage
  • FIG. IB illustrates a scanning electron microscopy (S.EM) image of a bare-SWCNT FET device.
  • FIG. 1C illustrates an SEM image of SWCNT network deposited between channels.
  • FIG. I D illustrates an AFM image of an Ab85B-SWCNT FET device.
  • FIG. IE illustrates schematic representation of an embodiment of an FET sensor device hereof
  • FIG. 1 F illustrates schematic representation of an embodiment of a chemiresistor sensor device.
  • FIG. 3B illustrates calibration plot for Ag85B detection, effect from solvent and non-specific binding detection, wherein all data points plotted in the calibration plots are mean ⁇ standard error of the mean and the number of devices (n) used is indicated in. the parenthesis.
  • FIG, 4F illustrates the relati ve responses after adding 10 pl. TB clinical blood samples onto the SWCNT FET devices, wherein all data plotted are mean x standard error of the mean and the number of devices is six for the positive sample and seven for the negative sample.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Ecology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Nanotechnology (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

Un dispositif de capteur pour le diagnostic de la tuberculose comprend un capteur comprenant un substrat, une première électrode, une seconde électrode espacée de la première électrode, et un milieu capteur sur le substrat entre la première électrode et la seconde électrode. Le milieu capteur comprend des nanostructures et au moins une entité parmi une ou plusieurs entités de reconnaissance comprenant au moins un site de liaison actif pour l'antigène Ag85. Un liquide est déposé sur le milieu capteur. Une force ionique du liquide est sélectionnée au niveau de laquelle se produit la liaison de l'antigène Ag85 à la ou aux entités de reconnaissance. La force ionique et le charge de masse de la ou des entités de reconnaissance immobilisées sur la pluralité de nanostructures sont sélectionnés de telle sorte qu'une hauteur moyenne d'entités de reconnaissance immobilisées se situe dans une plage déterminée d'une charge de masse maximale à laquelle la hauteur moyenne reste dans une longueur de Debye de criblage du liquide.
PCT/US2024/043037 2023-08-21 2024-08-20 Diagnostic basé sur une nanostructure de la tuberculose WO2025042890A1 (fr)

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US202363520728P 2023-08-21 2023-08-21
US63/520,728 2023-08-21

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060029926A1 (en) * 1998-05-06 2006-02-09 Metrika, Inc. Blocking compositions for immunoassays
US20210072235A1 (en) * 2017-12-22 2021-03-11 Sugentech, Inc. Method for diagnosing tuberculosis
US20220365078A1 (en) * 2021-05-07 2022-11-17 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Nanostructure-based sensors and methods for detecting antigens and antibodies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060029926A1 (en) * 1998-05-06 2006-02-09 Metrika, Inc. Blocking compositions for immunoassays
US20210072235A1 (en) * 2017-12-22 2021-03-11 Sugentech, Inc. Method for diagnosing tuberculosis
US20220365078A1 (en) * 2021-05-07 2022-11-17 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Nanostructure-based sensors and methods for detecting antigens and antibodies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN ZHE, ZHOU JIAN, TANG HAO, LIU YI, SHEN YIPING, YIN XIAOBO, ZHENG JIANGPO, ZHANG HONGSHUAI, WU JIANHUI, SHI XIANGLONG, CHEN YI: "Ultrahigh-Frequency Surface Acoustic Wave Sensors with Giant Mass-Loading Effects on Electrodes", ACS SENSORS, AMERICAN CHEMICAL SOCIETY, US, vol. 5, no. 6, 26 June 2020 (2020-06-26), US, pages 1657 - 1664, XP093286228, ISSN: 2379-3694, DOI: 10.1021/acssensors.0c00259 *

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