WO2022058308A1 - Procédé et kit pour mettre en évidence la présence de toxines et d'agents pathogènes par des tests de liaison de ligands au moyen de particules de colloïde déformables - Google Patents

Procédé et kit pour mettre en évidence la présence de toxines et d'agents pathogènes par des tests de liaison de ligands au moyen de particules de colloïde déformables Download PDF

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Publication number
WO2022058308A1
WO2022058308A1 PCT/EP2021/075233 EP2021075233W WO2022058308A1 WO 2022058308 A1 WO2022058308 A1 WO 2022058308A1 EP 2021075233 W EP2021075233 W EP 2021075233W WO 2022058308 A1 WO2022058308 A1 WO 2022058308A1
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WIPO (PCT)
Prior art keywords
analyte
partner
particle
binding partner
analyte binding
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PCT/EP2021/075233
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German (de)
English (en)
Inventor
Lisa Hannusch
Gerhard RÖDEL
Christian Dahmann
Kai Ostermann
Tilo Pompe
David Rettke
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Technische Universität Dresden
Universität Leipzig
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Publication of WO2022058308A1 publication Critical patent/WO2022058308A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G01N33/546—Synthetic resin as water suspendable particles

Definitions

  • the invention relates to a method for detecting analytes by means of an immobilized analyte binding partner and deformable particles and a kit and their use for detecting toxins and pathogens, in particular metal ions, viruses or bacteria in food, animal feed, detergents, dishwashing detergents, personal care products , cosmetics, pharmaceuticals, process water, soil, water, drinking water and/or waste water samples and/or body fluids.
  • toxins and pathogens in particular metal ions, viruses or bacteria in food, animal feed, detergents, dishwashing detergents, personal care products , cosmetics, pharmaceuticals, process water, soil, water, drinking water and/or waste water samples and/or body fluids.
  • EP 2 752 664 A1 or WO 2014/106 665 A1 discloses an alternative method for detecting analytes by immobilizing an analyte on hydrogel particles which subsequently interact with a ligand immobilized on a surface. Depending on the degree of interaction, the hydrogel particles are deformed by attachment to the surface, so that the deformation is detected using reflection interference contrast microscopy. This allows the analyte to be detected or characterized.
  • DE 10 2018 130 134 A1 describes a competitive detection method for the detection of low-molecular analytes, in particular glyphosate, using a surface functionalized with an analyte binding partner and a deformable particle, functionalized with a competitor, a fusion protein with a hydrophobin domain is used. Detection is by means of reflection interference contrast microscopy.
  • US 2014/0255916 A1 discloses a method and a kit for measuring the ability of a test sample to inhibit the binding of a pathogen receptor, preferably a sialic acid receptor, to a host cell ligand of the pathogen.
  • the method comprises an immobilized receptor which is contacted with a test sample and a particle reagent containing the ligand, preferably sialic acid, the particle reagent being a biological reagent selected from erythrocytes, erythrocyte vesicles, erythrocyte ghost cells, membrane fragments, membrane vesicles, proteins and combinations, in particular in form of colloids, beads or combinations thereof; wherein the particle is coated and/or magnetic, electrically conductive and/or semiconductive. The amount of particle reagent bound to the surface is then measured.
  • WO 00/14538 A1 discloses a linker-assisted immunoassay for glyphosate and a method for producing glyphosate antibodies comprising the production of glyphosate conjugates with a carrier molecule, preferably porcine thyroglobulin, bovine serum albumin, human serum albumin, ovalbumin or limpet hemocyanin; and immunizing a host with the conjugate.
  • a carrier molecule preferably porcine thyroglobulin, bovine serum albumin, human serum albumin, ovalbumin or limpet hemocyanin
  • the linker-assisted immunoassay for detecting the analyte in a test sample comprises preparing a linker-analyte conjugate using a test sample, contacting it with a glyphosate antibody, and contacting the test sample with a solid phase having an immobilized second carrier molecule covalently bound to glyphosate, coupled to a glyphosate derivative or a glyphosate salt.
  • the second carrier molecule is selected from porcine thyroglobulin, bovine serum albumin, human serum albumin, ovalbumin or sloth limpet hemocyanin, but differs from the first carrier molecule used to produce the glyphosate antibody.
  • the amount of bound antibody is then determined on the solid phase, which is indirectly proportional to the amount of glyphosate in the test sample.
  • the detection is preferably carried out by means of enzymatic detection, the antibody being conjugated with biotin and a labeled enzyme which binds biotin being added.
  • the enzyme is preferably alkaline phosphatase or horseradish peroxidase.
  • a disadvantage of the competitive detection methods, as disclosed in EP 2 752 664 A1, US 2014/0255916 A1, WO 00/14538 A1, is that the direct immobilization of different analytes due to e.g. B. the small size, availability, toxicity or pathogenicity is not possible.
  • an indirect detection is chosen for analytes that are no longer sterically accessible during the interaction with their analyte binding partner.
  • the most common indirect test is an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • An ELISA is understood to mean an antibody-based detection method (assay) in which quantification is carried out by means of an enzymatic color reaction.
  • the disadvantage of an ELISA is that it depends on the availability of suitable antibodies.
  • the object of the present invention is therefore to provide an easy-to-handle, cost-effective and rapid method for the quantitative detection of analytes which cannot be immobilized.
  • the object is solved by the features of the independent claims. Advantageous configurations are specified in the dependent claims.
  • a first aspect of the invention relates to a method for detecting an analyte selected from toxins and pathogens, comprising the steps: a) providing a surface with an immobilized analyte binding partner, the analyte binding partner being able to interact with the analyte, b) providing an interaction partner , wherein the interaction partner is bound to a deformable particle via a functional group, wherein the interaction partner is able to interact with the analyte, c) contacting the surface with an aqueous solution containing the analyte, wherein the analyte interacts with the analyte binding partner, wherein a complex of analyte binding partner and analyte is formed, d) contacting the surface with the interaction partner, the interaction partner interacting with the complex of analyte binding partner and analyte, with a deformation of the deformable particle taking place, and e) detection of the deformation of the d deformable particle
  • the method according to the invention takes place with a sequence of steps a) and b), c), d) and e), steps a) and b) being carried out simultaneously or in the sequence a) and then b) or b) and then a) take place.
  • the surface with the immobilized analyte binding partner comes into contact with the analyte (step c) and the interaction partner (step d) at the same time.
  • analytes are detected which are capable of interacting with at least one analyte binding partner and one interaction partner.
  • interaction is understood to mean a reversible interaction, in particular due to coordinate bonds, ionic bonds, hydrogen bonds, van der Waals forces and hydrophobic effects.
  • the analyte binding partner and the interaction partner can be identical or different.
  • an interaction between the deformable particle and the surface is only possible when the analyte is present in the aqueous solution. If such an interaction between the particle and the surface takes place, the particle deforms.
  • the particle deformation can thus be used for qualitative analysis or for quantification of the analyte.
  • a detection of analytes is advantageously possible with the method according to the invention compared to a competitive detection method, the direct immobilization due to z.
  • the analyte is a metal particle or a metal ion, preferably Cu(I), Cu(II), Co(II), or Zn(II); a virus, preferably bacteriophage or Circoviridae; a bacterium or a biomarker.
  • the analyte binding partner and the interaction partner are each independently selected from a metal or metal ion binding peptide or protein, a chelating agent, an enzyme, a membrane protein, a lipopolysaccharide, a nucleic acid or derivatives thereof.
  • analyte binding partners and interaction partners selected from metal or metal ion-binding peptides or proteins, chelating agents, enzymes, membrane proteins, lipopolysaccharides, nucleic acids or their derivatives, enable the simultaneous interaction of the analyte with the analyte binding partner and the interaction partner.
  • metal or metal ion-binding peptide or protein is understood to mean peptides or proteins which enter into attractive interactions (electrostatic interactions, van der Waals interactions, hydrophobic interactions) with metals or metal ions. Metal ions are positively charged ions of metals.
  • the metal ion binding protein is a metal ion binding transport protein.
  • chelating agent is understood to mean chemical compounds which form at least two coordinate bonds with a metal ion or metal atom.
  • the chelating agent is diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), mercaptoacetyltriglycine (MAG3), 6-hydrazinopridine-3-carboxylic acid (Hynic), Hydroxybenzylethylenediamine (HBED), N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N-diacetic acid (HBED-CC) or 2-(3-(1-carboxy- 5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentanedicarboxylic acid (DCFPyL).
  • DTPA diethylenetriaminepentaacetic acid
  • DOTA 1,4,7,10-tetraazacyclododecane-N,
  • membrane protein is understood to mean proteins which are associated with the cell membrane or a biomembrane of cell compartments or organelles of a cell.
  • membrane proteins are peripheral membrane proteins (also surface proteins) which are bound to the membrane surface, or integral membrane proteins which are integrated with a hydrophobic component in the double lipid layer of the membrane or span the membrane as a transmembrane protein.
  • lipopolysaccharides refers to molecules that consist of different regions.
  • the region that serves to anchor the LPS to the hydrophobic region of the bacterial outer membrane is lipid A, consisting mostly of N-acetylglucosamine with attached fatty acids.
  • the polysaccharide moiety contains a "core" polysaccharide and a region of repeating sugar moieties.
  • nucleic acid is understood to mean macromolecules made up of nucleotides with a sugar-phosphate backbone.
  • the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA).
  • derivative means a molecule, in particular a metal ion-binding peptide, a membrane protein, a lipopolysaccharide or a nucleic acid; having a high degree of structural identity with the molecule, preferably the same backbone, wherein at least one atom, group of atoms, functional group or substructure is replaced by another atom, group of atoms, functional group or substructure, e.g an alkyl group, especially methyl group; a glucuronic acid group, a halogen, a hydroxyl group or a sulfate group.
  • Derivatives advantageously interact with the analyte.
  • Derivatives also expediently include peptides or proteins with a sequence identity of at least 90%, preferably 95%, particularly preferably 99%, with the difference in the amino acid sequence of at most 10%, preferably at most 5%, particularly preferably at most 1%, in particular a shortening of the amino acid sequence , an extension of the amino acid sequence and/or a replacement of individual amino acids.
  • the peptide derivatives or protein derivatives preferably have a sequence identity of at least 90%, preferably 95%, particularly preferably 99%, outside the binding sites of the analyte binding partner or interaction partner for the analyte and a sequence identity of the binding sites of the analyte binding partner or interaction partner for the analyte of 100%.
  • peptide or protein derivatives include D-amino acids, pseudopeptide bonds, amino alcohols, non-proteinogenic amino acids, amino acids with modified side chains, and/or circular peptides.
  • nucleic acid derivatives are selected from peptide nucleic acids, such as ⁇ -peptide nucleic acids.
  • the interaction partner interacts with the complex of analyte binding partner and analyte.
  • the interaction partner interacts with the analyte or with the analyte and with the analyte binding partner.
  • the analyte binding partner and the interaction partner are identical.
  • the analyte binding partner and the interaction partner are different.
  • the selectivity of the detection can be increased by using different analyte binding partners and interaction partners.
  • the analyte binding partner is immobilized on the surface via a protein domain selected from hydrophobins, ECM proteins, S-layer proteins, peptide linkers and protein tags, or via a polymer linker, e.g. B. Polyethylene glycol (PEG). This enables directed immobilization on the surface.
  • immobilized is understood to mean an irreversible or pseudo-irreversible bond comprising covalent, coordinate, metallic, ionic bonds, hydrogen bonds and/or van der Waals interactions.
  • the immobilization takes place by covalent binding via a functional group of the analyte binding partner.
  • functional group means an atomic group in a compound that determines the reaction behavior of the compound.
  • functional groups are functional groups comprising heteroatoms (O, N, S, P or halogens), in particular carboxylic acids, halides, thiocarboxylic acids, sulfonic acids, thiolesters, sulfoxides, carboxylic acid anhydrides, carboxylic acid esters, carboxamides, nitriles, aldehydes, thioaldehydes, ketones, thioketones , oximes, hydrazones, alcohols, thiols, amines, imines, hydrazines, ethers or thioethers; and functional groups without heteroatoms, in particular double bonds, triple bonds or aromatics.
  • heteroatoms O, N, S, P or halogens
  • the analyte binding partner is a fusion protein.
  • the fusion protein has, for example, different protein domains which have different functionalities, in particular an analyte binding site and a protein domain selected from hydrophobins, ECM proteins, S-layer proteins, peptide linkers and protein tags.
  • the analyte binding site serves to bind the analyte to the analyte binding partner.
  • the analyte binding partner is preferably a fusion protein with a protein tag or hydrophobin, particularly preferably a fusion protein with a His tag.
  • the analyte binding partner is immobilized on the surface from a solution, in particular an aqueous solution.
  • the analyte is a metal particle, in particular a metal nanoparticle, or a metal ion, in particular a heavy metal particle or heavy metal ion.
  • the term "heavy metal” means a metal with a density of at least 4.5 g/ cm3 , in particular antimony, arsenic, lead, cadmium, chromium, iron, germanium, gold, cobalt, copper, manganese, molybdenum, nickel, Platinum, polonium, mercury, rhodium, selenium, silver, tantalum, thallium, titanium, vanadium, bismuth (bismuth), zinc, tin or zircon.
  • a disadvantage is that many heavy metals are harmful or toxic to the human organism even in slight overconcentrations.
  • the analyte is a metal ion, particularly a monovalent or divalent metal ion.
  • the analyte is Cu(I), Cu(II), Co(II), or Zn(II).
  • a concentration of 100 pM Cu(I) could advantageously be detected with the method according to the invention.
  • the limit of detection (LOD) below a concentration of 100 pM Cu(I) is also advantageous.
  • detection limit is understood to mean the lowest possible concentration at which the method according to the invention can detect an analyte within the aqueous solution with a certain degree of confidence.
  • the analyte binding partner and the interaction partner are each independently selected from a metal or metal ion-binding peptide or protein, in particular a metal ion-binding transport protein, and a chelating agent.
  • metal-binding or metal ion-binding peptides or protein are provided using phage display.
  • the analyte binding partner and the interaction partner are selected from Cox17 and Sco1, depending on one another.
  • the analyte binding partner comprises Cox17 and the interaction partner comprises Sco1, preferably a truncated mutant of Sco1 lacking a transmembrane domain.
  • the analyte binding partner comprises Sco1 and the interaction partner comprises Cox17.
  • the term "Sco1" refers to a chaperone of cytochrome c oxidase in the mitochondrial respiratory chain.
  • the truncated mutant of Sco1 lacking a transmembrane domain has the amino acid sequence SEQ ID no. 1 on.
  • Cox17 means a copper transport protein.
  • Cox17 has the amino acid sequence SEQ ID no. 2 on.
  • the interaction partner Cox17 is attached to the deformable particle by means of hydrophobin functionalization, preferably with the amino acid sequence SEQ ID no. 3 or SEQ ID no. 4 tied.
  • the analyte binding partner Sco1 is immobilized by means of a His tag on the functionalized surface, preferably with the amino acid sequence SEQ ID no. 5.
  • the analyte is a virus, preferably a bacteriophage, such as M13 and ⁇ t>X174; or Circoviridae.
  • virus is understood to mean infectious organic structures comprising a nucleic acid, in particular DNA or RNA.
  • the virus is present as a virion, preferably outside cells (extracellular), or as a virus in the form of a nucleic acid, preferably inside a suitable host cell (intracellular).
  • the virion comprises a nucleic acid, in particular DNA or RNA, and an enclosing protein capsule (capsid) or a ribonucleoprotein.
  • the virion comprises a lipid bilayer containing viral membrane proteins.
  • bacteria refers to viruses that specialize in bacteria as host cells.
  • M13 refers to a filamentous bacteriophage consisting of single-stranded, circular DNA (ssDNA) of positive polarity, which comprises 6407 nucleotides and has approximately 2700 copies of the major coat protein P8 and 5 copies of two different minor coat proteins (P9, P6 , P3) is encapsulated at the ends. M13 infects Escherichia coli (E. coli).
  • ⁇ t>X174 means a bacteriophage consisting of a single-stranded, circular DNA (ssDNA) which comprises 5386 nucleotides. X174 infects Escherichia coli (E. coli).
  • Circoviridae is understood to mean a virus family which has single-stranded, circular DNA with negative or ambisense polarity as a genome and non-enveloped capsids.
  • the analyte binding partner and the interaction partner are each independently composed of a membrane protein, a lipopolysaccharide, a nucleic acid, in particular a single-stranded DNA (ssDNA) or microRNA (miRNA); or their derivatives selected.
  • ssDNA single-stranded DNA
  • miRNA microRNA
  • the analyte binding partner and the interaction partner are each independently composed of a protein according to Letarov and Kulikov, in particular a receptor from Table 1; Silva et al., in particular a receptor from Tables 1 to 3; Stone et al., in particular a receptor from Table 1; selected (Letarov and Kulikov 2017, Silva et al. 2016, Stone et al. 2019).
  • the analyte is a biomarker, in particular a protein- and/or carbohydrate-based tumor marker or an endotoxin; or a bacterium.
  • biomarker is understood to mean molecules which indicate toxins, pathogens or diseases, in particular components of pathogens, cells, genes or gene products, preferably tumor markers or endotoxins.
  • endotoxins is understood to mean lipopolysaccharides which are decomposition products of bacteria, in particular of the outer cell membrane of gram-negative bacteria or cyanobacteria. Endotoxins can trigger health or life-threatening physiological reactions in humans and some animal species.
  • bacteria is understood to mean prokaryotes which have a cell membrane enclosing the DNA, the cytoplasm and the ribosomes, the DNA being freely present in the cytoplasm as a densely packed, self-contained molecule. Furthermore, bacteria have an RNA polymerase consisting of 5 subunits (a (2x), ß, ß' and w).
  • the analyte binding partner and the interaction partner are each independently selected from a peptide, a protein, an enzyme, a nucleic acid or derivatives thereof.
  • the analyte binding partner and the interaction partner comprises the active site or substrate binding domain of an enzyme, e.g. a sushi peptide.
  • ushi peptide refers to an endotoxin binding site of factor C, a lipopolysaccharide-sensitive serine protease of the horseshoe crab.
  • the analyte binding partner and/or the interaction partner is sushi peptide S1 and/or S3.
  • Another aspect of the invention relates to a combinatorial method for detecting an analyte selected from toxins and pathogens, comprising the steps: a) providing at least two different surfaces with at least two different immobilized analyte binding partners, one analyte binding partner being immobilized on a surface, wherein the at least two different analyte binding partners are capable of interacting with the analyte, b) providing an interaction partner, wherein the interaction partner is bound to a deformable particle via a functional group, wherein the interaction partner is capable of interacting with the analyte, c) contacting the at least two surfaces each with an aqueous solution containing an analyte selected from toxins and pathogens, wherein the analyte interacts with the at least two analyte binding partners, with a complex of analyte binding partner and Analyte is formed, d) contacting the at least two surfaces with the interaction partner, with the interaction partner interacting with the complex
  • the deformable particle is a functionalized particle, with the surface of the deformable particle having a functionalization, in particular functional groups such as carboxyl or amino groups.
  • the functionalization serves to immobilize the interaction partner.
  • the deformable particle is a hydrogel particle, preferably a polyethylene glycol hydrogel particle.
  • the deformable particle has a diameter of 10 ⁇ m to 100 ⁇ m, particularly preferably a diameter of about 25 ⁇ m.
  • the diameter of the deformable particles can be determined by bright field optical microscopy.
  • the deformable particle has a modulus of elasticity in the range from 10 kPa to 100 kPa, particularly preferably in the range from 15 kPa to 50 kPa; on.
  • a high sensitivity is advantageously ensured by the modulus of elasticity in the range from 15 kPa to 50 kPa.
  • an uneven deformation of the particles is advantageously ruled out.
  • the modulus of elasticity of the deformable particles can be determined from atomic force spectroscopy (SFM) force-distance curves.
  • the deformable particle has carboxy functionalization.
  • the synthesis and carboxy functionalization of the deformable particles, in particular the hydrogel microparticles takes place according to the Pussak et al. (Pussak et al. 2012) via emulsion and radical precipitation polymerization of polyethylene glycol diacrylamide or polyethylene glycol diacrylate with subsequent radical grafting of acrylic acid monomers, crotonic acid monomers or other alkene derivatives with functional groups such as amines to introduce the carboxyl, amino or other functional groups.
  • the synthesis and carboxy functionalization of the particles takes place microfluidically by means of photoinitiated radical crosslinking of polyethylene glycol diacrylamide or polyethylene glycol diacrylate (preferred molecular weight in the range from 500 Da to 8,000 Da, particularly preferably about 4,000 Da) with subsequent photoinitiated radical Grafting of acrylic acid monomers to introduce the carboxyl groups, producing monodisperse particles with a diameter in the range from 10 ⁇ m to 100 ⁇ m, particularly preferably around 25 ⁇ m.
  • the interaction partner is bound to a deformable particle via a functional group.
  • the interaction partner is bound to the deformable particle via a linker, the linker having a contour length in the range from 5 ⁇ to 200 ⁇ , preferably in the range from 10 ⁇ to 65 ⁇ ; and/or a degree of polymerization in the range from 1 to 70, preferably in the range from 3 to 20; having.
  • the linker is attached to the functionalized particle surface.
  • a linker molecule advantageously enables the interaction partner to be immobilized appropriately via a functional group, with the coupling group influencing the functionality and sensitivity of the method according to the invention. Furthermore, the resulting affinity of the immobilized interaction partner can advantageously be varied via the length or the degree of polymerization of the linker, and the working range of the method according to the invention can thus be adjusted.
  • the linker is selected from the groups of homo- and heterobifunctional linkers and includes ethylene diamine, oligo- and polyethylene glycol diamines, peptides such as pentaglycine and amino acids or a bifunctional linker with other groups such as thiols or azides.
  • the linker is a polyethylene glycol diamine linker.
  • the linkers contain protecting groups.
  • the protecting group is selected from fluorenylmethoxycarbonyl, tert-butyloxycarbonyl or tert-butyl protecting groups.
  • the protective groups advantageously ensure that no undesired polymerization of the linker molecules or cross-linking of the deformable particles occurs.
  • the detection is carried out using a quartz crystal microbalance (QCM), surface plasmon spectroscopy (SPR), atomic force spectroscopy (SFM), impedance spectroscopy or reflection interference contrast microscopy.
  • QCM quartz crystal microbalance
  • SPR surface plasmon spectroscopy
  • SFM atomic force spectroscopy
  • impedance spectroscopy or reflection interference contrast microscopy.
  • the detection preferably takes place by means of reflection interference contrast microscopy (RICM).
  • RCM reflection interference contrast microscopy
  • Reflectimming interference contrast microscopy is a light microscopic method for determining layer thicknesses using interference patterns, which are created by light reflected at the upper and lower boundary surface of the layer.
  • the contact radii a of the particle and surface as well as the particle radii R HGS are automatically determined using software specially developed for this purpose. According to the Johnson-Kendall-Roberts model (Johnson et al. 1971), these quantities are related to the adhesion energy W adh in the following way:
  • the determined adhesion energy corresponds to the binding density of the particle-bound interaction partner to the analyte and allows the amount of analyte in the sample to be determined.
  • the surface is transparent, semitransparent or opaque. In further embodiments, the surface is transparent at least in the range from 400 nm to 600 nm.
  • the surface is planar.
  • the surface can be a glass or plastic molding, e.g. B. a slide, coverslip, silicon wafer or the like.
  • the surface is designed as a well plate, in particular a 16-well or 96-well plate.
  • the sample is an aqueous solution.
  • the aqueous solution has a salt concentration in the range from 0 mmol/l to 300 mmol/l, preferably in the range from 90 mmol/l to 180 mmol/l, particularly preferably in the range from 98 mmol/l to 154 mmol/l; on.
  • the aqueous solution is isotonic saline.
  • isotonic saline solution means a saline solution that has the same osmotic pressure as human blood.
  • the aqueous solution has a pH in the range from pH 2.5 to pH 9, preferably in the range from pH 4 to pH 8; on.
  • body fluids such as saliva samples, blood samples, blood plasma samples, blood serum samples, urine samples or mucus samples
  • body fluids such as saliva samples, blood samples, blood plasma samples, blood serum samples, urine samples or mucus samples
  • Water samples drinking water samples, waste water samples, samples from chemical production processes, extracted soil samples, food samples, animal feed samples, samples from biological and biochemical production processes, fermentation solutions, detergent samples, dishwashing liquid samples, body care product samples, cosmetic samples or pharmaceutical samples.
  • the aqueous solution is expediently free from suspended matter.
  • the aqueous solution is filtered before the method according to the invention, in particular before step c) of the method according to the invention.
  • the method can be carried out at a temperature that avoids freezing and denaturing of the analyte, the analyte binding partner and the interaction partner.
  • the method according to the invention is carried out at a temperature in the range from 0° C. to 40° C., preferably at room temperature (20° C.).
  • the method according to the invention has at least one further step, the at least one further step being selected from a calibration with the analyte and the determination of a reference value.
  • calibration is understood to mean a detection of different standard solutions of the analyte with different concentrations using the method according to the invention and the determination of a calibration line. Instrumental errors and matrix effects are advantageously excluded by a calibration. In embodiments, the calibration is done with an internal standard or an external standard.
  • reference value means a sample without an analyte comprising the same matrix.
  • kits comprising: i. at least one surface with an immobilized analyte binding partner, wherein the analyte binding partner is capable of interacting with an analyte selected from toxins and pathogens, and ii. at least one deformable particle having an immobilized interaction partner, the interaction partner being bound to the deformable particle via a functional group, the interaction partner being able to interact with the analyte.
  • the analyte binding partner and the interaction partner are identical or different.
  • the analyte binding partner and the interaction partner are each independently selected from a metal or metal ion-binding peptide or protein, a chelating agent, an enzyme, a membrane protein, a lipopolysaccharide or a nucleic acid.
  • the deformable particle is a hydrogel particle.
  • the surface is transparent, semi-transparent or opaque. In further embodiments, the surface is transparent at least in the range from 400 nm to 600 nm.
  • the surface is designed to be planar.
  • the surface can be a glass or plastic molding, e.g. B. a slide, coverslip, silicon wafer or the like.
  • the surface is designed as a well plate, in particular a 16-well or 96-well plate.
  • the kit according to the invention comprises at least two different surfaces with at least two different immobilized analyte binding partners, one analyte binding partner being immobilized on a surface, the at least two different analyte binding partners being able to interact with the analyte selected from toxins and pathogens, and at least one deformable particle having an immobilized interaction partner, the interaction partner being bound to the deformable particle via a functional group, the interaction partner being able to interact with the analyte.
  • the subject matter of the invention is also the use of the method according to the invention and/or the kit according to the invention for the detection of toxins and pathogens in food, animal feed, detergents, dishwashing detergents, body care products, cosmetics, pharmaceuticals, process water, soil, Water, drinking water and/or waste water samples and/or body fluids such as blood plasma, blood serum or urine samples.
  • toxins and pathogens are detected in aqueous solutions.
  • process water means an aqueous solution in technical production processes. Expediently, individual parameters of the process water in the circuit are examined.
  • the method according to the invention and/or the kit according to the invention is used for the detection of toxins and pathogens in food, animal feed, detergents, dishwashing detergents, body care products, cosmetics, pharmaceuticals, process water, soil, water , drinking water and/or waste water samples and/or body fluids such as blood plasma, blood serum or urine samples; by means of the steps a) providing at least one surface with an immobilized analyte binding partner, the analyte binding partner being able to interact with the analyte, b) providing an interaction partner, wherein the interaction partner is bound to a deformable particle via a functional group, the interaction partner being able is to interact with the analyte, c) contacting the surface with an aqueous food, feed, detergent, dishwashing liquid, personal care product, cosmetic, pharmaceutical, process water, soil, body of water, drinking water and/or or waste water sample and/or body fluid, d) contacting the surface with the interaction partner, with deformation of the deformation of the
  • the deformable particle 1 in particular a hydrogel particle, is functionalized with the interaction partner 4 and the surface 2, in particular a glass chip, with the analyte binding partner 5, in particular via a self-assembling protein 6.
  • the interaction partner 4 and the surface 2 in particular a glass chip
  • the analyte binding partner 5 in particular via a self-assembling protein 6.
  • the analyte 3 there is an interaction between 4 and 5 and consequently a deformation of the deformable particle 1. This can be measured by the radius of the contact surface (a) from the reflection interference contrast microscopy (RICM) image.
  • RCM reflection interference contrast microscopy
  • Rough lipopolysaccharide core oligosaccharide and lipid A
  • EH100 strand the functional group (amino group) for the immobilization of the deformable particles is circled.
  • Fig. 3 Rough lipopolysaccharide 3-deoxy-d-manno-octulonic acid lipid A (KdO2-Lipid A) circled is the functional group (carboxy group) for the immobilization of the deformable particles.
  • the principle of the detection method is shown in FIG.
  • the immobilized analyte binding partners on the surface interact attractively with the analyte and the analyte with the particle-bound interaction partner, resulting in a characteristic contact area between the surface and the particle. Depending on the concentration of the analyte, this increases the contact area.
  • the contact and particle radius to determine the adhesion energy is determined using reflection interference contrast microscopy.
  • Droplet-based templating is used to create microgel particles with a defined size and shape.
  • the experimental setup requires two high-precision syringe pumps (one for the oil phase and one for the aqueous phase), a microfluidic flow cell with flow-focusing geometry (25 pm at the droplet-forming nozzle), a simple light microscope, and a high-speed camera for single-drop observations.
  • the first dispersion phase solution contains 10% by weight PEG-diacrylamide (4-8 kDa) mixed with 1% by weight LAP (lithium phenyl 2,4,6-trimethylbenzoylphosphinate), both dissolved in ultrapure water.
  • the continuous phase contains 2% by weight of a tenside (perfluoropolyether eg polyhexafluoropropylene oxide, alternatively polyacrylonitrile butadiene acrylate) which is dissolved in the fluorinated oil HFE 7500.
  • a tenside perfluoropolyether eg polyhexafluoropropylene oxide, alternatively polyacrylonitrile butadiene acrylate
  • HFE 7500 fluorinated oil
  • the particles were centrifuged again, the supernatant was discarded and a 20% piperidine/water mixture was added. After 15 minutes of reaction, the particles were again centrifuged and rinsed several times with distilled water and HEPES buffer.
  • Exemplary embodiment 1 detection of copper(I) ions
  • the copper-dependent interaction between a copper transport protein, Cox17, and a chaperone of the cytochrome c oxidase in the mitochondrial respiratory chain, Sco1, is used to detect Cu(I) ions.
  • Horng et al describe that Cox17 transfers Cu(l) to Sco1 in the membrane space of the mitochondrion (Horng et al. 2004).
  • the truncated version of Sco1 without a transmembrane domain with a His tag is used as the interaction partner and Cox17 as the analyte binding partner.
  • cover slips (Menzel glasses, 0 32 mm) were placed in a Teflon rack and left in an ultrasonic bath with distilled water for 30 min. After rinsing three times with distilled water, the glasses were cleaned for a further 30 min in denatured ethanol using an ultrasonic bath. The coverslips were then rinsed three times with distilled water. Further purification was performed using a mixture of 50 mL H2O2 (35%), 50 mL 25% aqueous NHs solution and 250 mL distilled water, for a total volume of 350 mL. The solution was heated to 60°C on a hot plate and the Teflon rack with the coverslips was left in the solution for 10 minutes. After rinsing twice with distilled water, the coverslips were dried in a stream of nitrogen.
  • a 20 mM 3-aminopropyltriethoxysilane solution in 9:1 (v/v) isopropanol/distilled water was prepared (225 ml isopropanol, 25 ml distilled water and 1.16 ml APTES).
  • the Teflon rack with the cover glasses was then left in the solution for 120 min, rinsed thoroughly with isopropanol and dried in a stream of nitrogen. Thereafter, the cover slips were placed in a Petri dish and heated at 120 ° C. for 60 min.
  • the coverslips were left in 250 ml of a 240 mM succinic anhydride solution (6 g in 250 ml) in tetrahydrofuran (THF) (6 g in 250 ml) for 1 h (room temperature), then twice with THF and once washed with water and dried in a stream of nitrogen.
  • THF tetrahydrofuran
  • NTA-NH2 N-hydroxysulfo-succinimide
  • the surfaces were then washed several times with distilled water, covered with 1.5 ml each of a 20 mM NiCh solution in citrate buffer, and after 30 minutes with distilled water rinsed and dried in a stream of nitrogen.
  • the citrate buffer prevents unwanted precipitation of the nickel.
  • the Ni-NTA coating allows the immobilization of a protein with a hexahistidine sequence.
  • the surfaces according to the invention were first glued to a 16-well I-carrier (CS16-CultureWellTM, Grace Biolabs) with a self-adhesive underside and a volume of 400 ⁇ l/well, and the wells were filled twice with 250 ⁇ l loading buffer (150 mM Na2HPO4, 10 mM imidazole, pH 8.0).
  • CS16-CultureWellTM Grace Biolabs
  • 250 ⁇ l loading buffer 150 mM Na2HPO4, 10 mM imidazole, pH 8.0.
  • At least a copper(I) concentration of 100 pM Cu(I) can be detected.
  • Bacteriophage 0X174 interact with the lipopolysaccharides (LPS) on the surface of Escherichia coli host cells. Therefore, to detect bacteriophage 0X174, the LPS are isolated (see Sun et al. 2014, Sun et al. 2017, Schön et al. 1995) and immobilized on a glass slide and on a deformable particle, in particular a hydrogel particle, for coating the surface.
  • LPS lipopolysaccharides
  • LPS can be isolated using various methods (see Davis and Goldberg 2012, Henderson et al. 2013, Westphal and Lüderitz 1954).
  • the deformable particles, in particular the hydrogel probes are coated by COOH activation using EDC/NHS and the coupling of rough lipopolysaccharides (core oligosaccharide and lipid A, EH100 strand) (see FIG. 2).
  • EDC/NHS rough lipopolysaccharides
  • KdO2-Lipid A is coupled (see Fig. 3) (Wang et al. 2015).
  • microRNA in particular miR-335, miR-193b or miR-302, of different pathological and physiological states are detected with the method according to the invention.
  • a y-peptide nucleic acid (y-PNA) of a segment of a complementary sequence of the target miRNA is terminally coupled to the surface via linkers, in particular a PEG linker, and another part of a y-PNA complementary to the target miRNA is terminally coupled by means Linkers, in particular PEG linkers, attached to the deformable particle.
  • linkers in particular PEG linkers
  • endotoxins will be detected in analyte solutions.
  • sushi peptides in particular S1 and S3, are immobilized on the surface and on the deformable particles, which are derived from the factor C sushil and sushi3 domain and specifically bind lipopolysaccharides from Gram-negative bacteria (Ding et al. 2008 ).
  • a terminal coupling of the peptides takes place via an inserted biotin on the streptavidin-modified surface and the particle.
  • the specific interaction of the lipopolysaccharides is then quantitatively detected via the adhesion and deformation of the microparticle on the surface using reflection interference microscopy as a function of the presence of the lipopolysaccharides in the analyte solution.
  • the surface for example a glass surface, and the particles were coated, they could be used for reflection interference contrast microscopy (RICM).
  • RCM reflection interference contrast microscopy
  • the radial intensity profiles of the hydrogel particles on the functionalized surface were recorded using the reflection interference contrast method using an inverted microscope system (Olympus IX 73) with a 60x immersion objective (Olympus UPlanSAPO 60x Oil Microscope Objective). From the recorded profiles, contact radii a of particles and surface as well as the particle radii R HGS could then be automatically determined using software specially developed for this purpose. According to the Johnson-Kendall-Roberts model, these quantities are related to the adhesion energy W adh as follows (Johnson et al. 1971):
  • a deformable particle has a modulus of elasticity E HGS of 15 kPa and a Poisson's ratio v of 0.5, from which the corresponding adhesion energy of the system can be determined if the particle and contact radius are known.

Abstract

L'invention concerne un procédé pour mettre en évidence la présence d'analytes au moyen d'un partenaire de liaison d'analyte immobilisé et de particules déformables, ainsi qu'un kit et leur utilisation pour mettre en évidence la présence de toxines et d'agents pathogènes, notamment d'ions métalliques, de virus ou de bactéries dans des échantillons d'aliments, d'aliments pour animaux, de détergents, de produits de lavage, de produits de soins corporels, de produits cosmétiques, de produits pharmaceutiques, d'eaux de processus, de sol, de masse d'eau, d'eau potable et/ou d'eaux usées, et/ou dans des fluides corporels.
PCT/EP2021/075233 2020-09-17 2021-09-14 Procédé et kit pour mettre en évidence la présence de toxines et d'agents pathogènes par des tests de liaison de ligands au moyen de particules de colloïde déformables WO2022058308A1 (fr)

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