WO2020098851A1 - Dispositif et procédé pour déterminer la fluorescence et le nombre d'anticorps sur des exosomes - Google Patents

Dispositif et procédé pour déterminer la fluorescence et le nombre d'anticorps sur des exosomes Download PDF

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Publication number
WO2020098851A1
WO2020098851A1 PCT/DE2019/000294 DE2019000294W WO2020098851A1 WO 2020098851 A1 WO2020098851 A1 WO 2020098851A1 DE 2019000294 W DE2019000294 W DE 2019000294W WO 2020098851 A1 WO2020098851 A1 WO 2020098851A1
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WO
WIPO (PCT)
Prior art keywords
antibodies
exosomes
fluorescence
filter
lasers
Prior art date
Application number
PCT/DE2019/000294
Other languages
German (de)
English (en)
Inventor
Hanno Wachernig
Clemens Hambrecht
Sacha Raschke
Jens Schiffmann
Original Assignee
Particle Metrix Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Particle Metrix Gmbh filed Critical Particle Metrix Gmbh
Publication of WO2020098851A1 publication Critical patent/WO2020098851A1/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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N15/1433—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N2015/0038—Investigating nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1486—Counting the particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1488—Methods for deciding

Definitions

  • the application relates to a device and a method for determining the fluorescence and the number of antibodies on exosomes.
  • Vesicles surrounded by a single or double membrane or a net-like shell made of proteins.
  • the vesicles form their own
  • Extracellular vesicles are membrane particles that are secreted by almost every cell and can be taken up again by a large number of cells. They transfer a complex set of information from one cell to another.
  • Exosomes are small vesicles with a size of approx. 50 - 150 nm
  • Microvesicles have a size of approx. 100 - 1000 nm
  • Apoptotic bodies are the largest group of extracellular vesicles. Generally, extracellular vesicle proteins. RNA or mico-RNA, and in the case of apopotic vesicles also contain DNA and other cellular components.
  • the possible areas of application are wide-ranging and include:
  • Exosomes are extracellular microvesicles that are released into biological fluids by living cells, particularly large numbers of tumor cells and platelets. They have gained considerable importance because they have biological functions and because they can also serve as biomarkers for diseases, in particular tumors.
  • Exosomes are extracellular microvesicles that are released by living cells through exocytosis, contain a large number of proteins and RNA and perform biological functions.
  • micro-RNA and m-RNA in exosomes from tumors serve to prevent tumor growth and the settlement of metastases and their
  • Exosomes originate from intracellular multivesicular bodies in which vesicles of 30 nm to 200 nm in size bud, which exocytose from a cell into the surrounding interstitial fluid, blood and urine.
  • Exosome microvesicles contain a large number of proteins, THEN and RNA, especially mRNA and micro-RNA. They perform various functions in cell-cell communication by influencing the function of neighboring cells or even distant cells of the body. It is believed that new therapeutic concepts can be developed by influencing such interactions.
  • Microvesicles from endothelial progenitor cells promote new vessel formation by transferring m-RNA.
  • Endothelial progenitor cells produce microvesicles that protect cardiac muscle cells from hypertrophy and apoptosis induced by angiotensin II. Endothelial cell microvesicles protect other enothelial cells from damage and malfunction caused by reoxygenation after hypoxia.
  • micro-RNA detectable in exosomes
  • miR-12 is found particularly high in serum when there is an HCC. As more precise studies show, these micro-RNAs are not dissolved in the serum, but are located in microvesicles (exosomes).
  • miR e.g. miR-122
  • miR-122 Different miR (e.g. miR-122) are used in one
  • NAFLD nonalcoholic fatty liver disease
  • miR - 122 decreases in liver fibrosis depending on the severity.
  • exosome analysis can do the familial one to be distinguished from the sporadic form, and to identify those at risk of relapse at an early stage.
  • Exosome-associated microRNA is found in the urine of certain organs
  • Kidney disease increased.
  • Exosomes are biological nanoparticles (BNP)
  • BNP biological nanoparticles
  • Exosomes come from the cells and represent very small vesicles (bubbles) that are covered with a bio-membrane. They can be removed from living cells and migrate outside the cells to other cells and be reintroduced there. Since they can escape from the cells, they are also called
  • Extracellular vesicles (EV 's ).
  • Extracellular vesicles can contain short pieces of cell genetic material; they circulate in the fluid outside the cells when they have emerged from cells, do not mutate and cannot reproduce themselves. They presumably serve the communication between different cells and can contain natural substances inside, but also artificially introduced active substances which cause other cells to behave in a certain way if the vesicles are taken up by another cell. They are therefore of the greatest interest for research, both for diagnostic and therapeutic applications.
  • Antibodies are very specific and recognize and bind only to a certain protein.
  • the entire complex consisting of exosome antibody X fluorescent dye can be detected by the Zetaview device.
  • Colocalization means that two or more different proteins are represented side by side on the surface of the exosomes. In our example, this would be Protein X and Protein Y. So we want to know if these two proteins coexist on the exosome surface.
  • Antibody X can be labeled with a red fluorescent dye, for example, and antibody Y with a blue fluorescent dye.
  • both labeled antibodies recognize and bind or represent the corresponding proteins on the exosome surface (colocalized)
  • signals are recorded in both fluorescence channels of the ZetaView device. If only protein X is represented, a signal is only detected in the red fluorescence channel (because only antibody X can bind), but not in the blue (and vice versa).
  • Antibodies on exosomes with the following characteristics:
  • the beams from a plurality of different lasers (1, 2, 3, 4) are each directed separately onto a beam path (21) into a measuring cell (22) with a sample (9) containing particles by means of a separate collecting prism (14), wherein the focusing of the laser steel (21) in interaction with the sample (9) forms the center of a convergent beam consisting of light from the fluorescence plane (5) and the scattered light plane (8), which after passing through a liquid lens with an optical control ( 18) is registered in a video camera (15),
  • the convergent beam path passes through a color filter (16) which is moved by means of a change wheel (17) and a controller (26).
  • a display (19) with a touch screen (19) and an overall control (20) with a particle tracking program are used to operate a video camera (15) and that instead of the color filter (16) a diffusive optical element ( 24) and a gray filter 38 with a changing device (40) is used and instead of the video camera CCP (15) a graph based
  • Antibodies on exosomes with the following characteristics:
  • fluorescent antibodies and particles for example exosomes, filled as a sample (9) into a measuring cell (10),
  • the measuring cell (10) is irradiated in vitro in rapid change and imaged by means of an optical device (15), the change in lasers and filters being seen in a fraction of a second because of the Brown ' molecular movement have to be done
  • a Particle Metrix own PTA measuring arrangement serves as the overall control of the measuring arrangement and the unbounded antibody and possibly free fluorescent dye which is annoying when preparing a measurement by means of size exclusion chromatography is eliminated and a computer program with a program code for carrying out the Method steps when the program is executed in a computer and a machine-readable medium with the program code
  • the solution with the fluorescent antibodies is applied to the exosomes given, the antibodies bind to the corresponding proteins on the surface of the exosomes. Duration 2 hours. The antibodies that do not do this are separated.
  • FIG. 2 a special design of the NTA from FIG. 1
  • Fiq.4 an analysis - course of the position of the color filter and laser
  • Fig. 5 Schematic representation of the diffractive deflection by the DOE 24
  • Fig. 1 shows a Particle Metrix PTA nanoparticle tracking measuring arrangement which enables the detection of several antibodies on the same exosome. For this purpose, several combinations of lasers and filters have to follow each other quickly. Since the nanoparticles see a Brown 'Zappelzi perform, they may after a few
  • Seconds disappear from the video image so that you only have a few seconds to measure several fluorescences on the same particle at the same time, but in vitro. .That means having to change the laser / filter combination in a fraction of a second. Since several lasers can already be guided into one fiber and can be quickly switched from one to the next wavelength by electronic switching, this is no longer a problem. It remains only have to deal with the quick filter change.
  • the diameter of the filter in the narrowed beam of the focus can be much smaller (less mass faster switching speed of the filter) than if the filter were placed in the parallel beam.
  • the imaging optics consist of a lens with an optional additional liquid lens 6, which is aimed at the particulate sample 9 in the measuring cell 10, and a video camera 15, or a detector, which records the movement of the particles.
  • the lasers 1 to 4 shown here serve as a stimulating light source for scattered light and fluorescent light and mostly radiate into the laser passage window 22 of the measuring cell 10 at an angle of 90 degrees.
  • a change wheel 17 for color filters is arranged on a filter wheel axis 1 1 and a counterbearing 7 via a change clutch 12 and a filter wheel drive 13, the change wheel being equipped with a color filter 16, and the control of the
  • Color filter wheel by means of a device 26.
  • the light passing through the color filter 16 is registered in the optics through window 23 of the measuring cell 10.
  • a diffusive optical element (DOE) 24 and a gray filter 38 are installed.
  • DOE diffusive optical element
  • a graphene-based light sensor 25 is used.
  • the DOE 24 can consist of a combination of several differently arranged gratings, depending on the optical deflection properties
  • Wavelength have exist. This creates different deflection angle ranges (locations) depending on the wavelength of the light.
  • the fluorescent light emissions of the particles generated by the different lasers then strike in different areas (locations) 33-36 of the light sensor surface 37 of the G light sensor 25.
  • the penetrating light signal is somewhat weakened by the DOE 24, which impairs the quality of a detection on a CCD light sensor.
  • the graph-based light sensor 25 compensates for this deficiency.
  • the graphene-based light sensor 25 has a light sensitivity that is 1000 times higher and allows the analysis to be concentrated on a narrow spectrum around the central wavelengths of fluorescence. This has the advantage of relating the fluorescent light emanating from a particle in two different directions precisely to that one particle. The intensity of light is highest in the center of fluorescence and localization is most precise.
  • the distance 44, see FIG. 5, from the maximum of the fluorescence in the region 33 to the maximum of the fluorescence in the region 34 is to be regarded as a constant.
  • the antibodies of the fluorescence in the region 33 from particles (exsosome) 41 to the fluorescence in the region 34 of the particle 41 can be correlated to the same particle.
  • the area of the central fluorescence wavelength and the associated exciting laser show the area (location) 33 of the wavelength 430 nm, for example on the sensor surface 37.
  • the laser change time is less than 0.002 sec.
  • fluorescent particles can be determined.
  • the filter 27 in the form of an insert made of quartz glass is to be regarded as neutral, while the filter with the number 28 opens at a wavelength from 420 nm.
  • the filter with the number 29 opens at a wavelength from 500 nm, the filter 30 opens at a wavelength from 540 nm and the filter 31 opens at 660 nm.
  • Fig. 4 shows an analysis of the course of the position of the respective color filter of the fluorescence emission measurement and of the respective laser.
  • the rectangular box shown on the right shows which of the seven antibodies shown by way of example for each
  • the filter wheel is set to filter 27, all lasers 1 to 4 are active.
  • the number of particles in the sample 9 is measured here
  • the time for the filter change is approx. 20 ms, the time for the fluorescence measurement is approx. 50 ms.
  • Fig.4c is the filter 29 (500nm) and the laser 2 (color blue and
  • Wavelength 488 nm switched on.
  • the particles b), c) and e) light up.
  • the filter change is approx. 20 ms, the fluorescence measurement approx. 50 ms)
  • the filter 30 (540 nm) and the laser 3 (color green and wavelength 520 nm) are switched on.
  • the particles shine at a) and d).
  • the filter change is approx. 20 ms, the fluorescence measurement approx. 50 ms).
  • Fiq 4e is the filter 31 (660 nm) and the laser 4 (color red and
  • Wavelength 640 nm switched on.
  • the particles d) and g) light up.
  • the filter change is approx. 20 ms, the fluorescence measurement approx. 50 ms).
  • Particle a (exosome) is populated with two different antibodies (4b and 4d.)
  • Particle b (exosome) is populated with two different antibodies (4b and 4c)
  • Color filter edge filter or bandpass filter
  • DOE Diffractive optical element
  • Filter opens from 420 nm
  • Filter opens from 500 nm

Abstract

L'invention concerne un procédé pour déterminer la fluorescence et le nombre d'anticorps sur des exosomes, le procédé comprenant les caractéristiques suivantes : pour la préparation d'une mesure, une solution constituée d'anticorps fluorescents et de particules, par exemple d'exosomes, est versée en tant qu'échantillon (9) dans une cellule de mesure (10), la cellule de mesure (10) est irradiée in vitro en alternance rapide au moyen d'une combinaison de lasers et de filtres et représentée au moyen d'un dispositif optique (15), l'alternance de lasers et de filtres devant s'effectuer en fractions de seconde en raison du déplacement moléculaire de Brown. Un ensemble de mesure de suivi individuel des particules propre à Particle Metrix sert de commande globale de l'ensemble de mesure.
PCT/DE2019/000294 2018-11-14 2019-11-12 Dispositif et procédé pour déterminer la fluorescence et le nombre d'anticorps sur des exosomes WO2020098851A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102018008980.1A DE102018008980B4 (de) 2018-11-14 2018-11-14 Vorrichtung und Verfahren zur Bestimmung der Fluoreszenz und der Anzahl von Antikörpern auf Exosomen.
DE102018008980.1 2018-11-14

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WO2020098851A1 true WO2020098851A1 (fr) 2020-05-22

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2340936A (en) * 1998-08-22 2000-03-01 Malvern Instr Ltd Particle size distribution measurement using dual sources of different wavelengths
EP3056892A1 (fr) * 2015-02-13 2016-08-17 Artium Technologies, Inc. Détection améliorée par l'intermédiaire des enregistrements d'analyse en composantes de signal
DE102016013236A1 (de) 2016-11-07 2018-05-09 Particle Metrix Gmbh Vorrichtung und Verfahren zum Messen der Konzentration und der Größe von Nanopartikeln in Flüssigkeiten im Streulichtmodus und im Fluoreszenzmodus

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6995841B2 (en) 2001-08-28 2006-02-07 Rice University Pulsed-multiline excitation for color-blind fluorescence detection

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2340936A (en) * 1998-08-22 2000-03-01 Malvern Instr Ltd Particle size distribution measurement using dual sources of different wavelengths
EP3056892A1 (fr) * 2015-02-13 2016-08-17 Artium Technologies, Inc. Détection améliorée par l'intermédiaire des enregistrements d'analyse en composantes de signal
DE102016013236A1 (de) 2016-11-07 2018-05-09 Particle Metrix Gmbh Vorrichtung und Verfahren zum Messen der Konzentration und der Größe von Nanopartikeln in Flüssigkeiten im Streulichtmodus und im Fluoreszenzmodus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Next Generation Twin Laser NTA System to Improve the Study of Extracellular Vesicles and other Nanoparticles Announced", 19 December 2019 (2019-12-19), XP002797683, Retrieved from the Internet <URL:https://www.labmate-online.com/news/laboratory-products/3/analytik-ltd/next-generation-twin-laser-nta-system-to-improve-the-study-of-extracellular-vesicles-and-other-nanoparticles-announced/47925> [retrieved on 20200213] *
ANONYMOUS: "PMX 220 ZetaView Twin Laser - Standard technical data", April 2018 (2018-04-01), XP002797682, Retrieved from the Internet <URL:https://www.particle-metrix.de/fileadmin/pdf/PMX_ZetaView_Twin_Laser_Specifications_EN.pdf> [retrieved on 20200213] *
MIRANDA JEZID ET AL: "Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth", PLACENTA, W.B. SAUNDERS, GB, vol. 64, 2 March 2018 (2018-03-02), pages 34 - 43, XP085375902, ISSN: 0143-4004, DOI: 10.1016/J.PLACENTA.2018.02.006 *

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DE102018008980A1 (de) 2020-05-14

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