EP2139999A1 - Verfahren zur herstellung eines substrats zur immobilisierung einer zelle, substrat und verwendungen davon - Google Patents

Verfahren zur herstellung eines substrats zur immobilisierung einer zelle, substrat und verwendungen davon

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Publication number
EP2139999A1
EP2139999A1 EP08736151A EP08736151A EP2139999A1 EP 2139999 A1 EP2139999 A1 EP 2139999A1 EP 08736151 A EP08736151 A EP 08736151A EP 08736151 A EP08736151 A EP 08736151A EP 2139999 A1 EP2139999 A1 EP 2139999A1
Authority
EP
European Patent Office
Prior art keywords
cell
solid support
compound
peptide
fusogenic
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP08736151A
Other languages
English (en)
French (fr)
Inventor
Gérard DELERIS
Sandra Rubio Albenque
Bernard Bennetau
Bernard Desbat
Frédéric BUFFIERE
Jean-Luc Chagnaud
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Universite Victor Segalen Bordeaux 2
Original Assignee
Centre National de la Recherche Scientifique CNRS
Universite Victor Segalen Bordeaux 2
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 Centre National de la Recherche Scientifique CNRS, Universite Victor Segalen Bordeaux 2 filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP2139999A1 publication Critical patent/EP2139999A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/096—Polyesters; Polyamides
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica

Definitions

  • the present invention relates to biochemistry, medicine, biology, and in particular analytical biochemistry and immunoassay. More particularly, the present invention relates to the design of an analysis and diagnostic instrument (analysis chip or biosensor) making it possible to examine samples of different types and in particular biological samples.
  • This analysis chip comprises a support, optionally functionalized with an organic matrix, a penetration agent such as a fusogenic compound capable of being inserted into the cell membranes and optionally a cell or part of a cell.
  • the present invention relates to the method of manufacture and the use of such a support for biomedical diagnosis and / or for health surveillance.
  • the present invention makes it possible to solve the technical problems cited above in the example of irregular antibodies but which can be declinable in many other cases by proposing a tool which is more precisely defined as an analytical system (bioreceptor) composed of an associated biological element. to a solid support and a measurement chain
  • the molecular recognition properties of the biological element confer high selectivity and affinity to the biomolecule / target analyte interaction.
  • the latter generates a signal that can be translated by different physico-chemical methods into a quantitatively and / or qualitatively correlatable measurement to the target analyte, which may be a biomolecular or cellular system.
  • the present invention proposes the use of a supramolecular inclusion system that can penetrate the membranes and this, to immobilize cellular elements. This represents an intermediate technique between simple adsorption and covalent coupling that has not been achieved until now.
  • the present invention allows easy, direct and rapid visualization of the binding or non-binding interaction between two biomolecules with high reliability, and the best possible sensitivity.
  • the possibility of miniaturization and automation makes it possible to compile multiple tests in a single analysis.
  • the present invention is remarkable in that it is not only useful for looking for irregular antibodies during blood transfusions but that it finds uses in any biological, medical, agri-food and other fields, likely to a cell chip.
  • the present invention firstly relates to a method for preparing a solid support capable of immobilizing at least one cell and / or at least one cell part, said method comprising a step of attaching to said solid support a fusogenic compound capable of insert into cell membranes.
  • fusogenic compound in the context of the present invention any compound that can anchor in a phospholipid membrane such as a cell membrane and lead to the immobilization of said membrane and therefore to that of a cell.
  • the fusogenic compound may be chosen from the compounds non-peptide fusogenic and peptic fusogenic compounds.
  • non-peptide fusogenic compound any molecule containing neither amino acid nor amino acid analogue and capable of anchoring in a phospholipid membrane such as a cell membrane and lead to the immobilization of said membrane.
  • the glycosyl phosphatidylinositol (GPI) unit or the polyisoprene units such as the farnesyl unit (isoprene unit with 15 carbon atoms) or the geranylgeranyl unit (20 carbon isoprene unit), are especially known.
  • Alkaline phosphatase has, at its C-terminus, such a well-defined anchoring structure (Ronzon, 2001, University Claude Bernard-Lyon Thesis).
  • Isoprenylation is a post-translational modification that adds a farnesyl or geranylgeranyl moiety to a protein having a particular C-terminal pattern (Maurer-Stroh and Eisenhaber, 2005, Genome Biology, Vol 6, R55).
  • peptide compound in the context of the present invention any molecule consisting of amino acids or amino acid analogues such as peptides, glycopeptides, lipopeptides, pseudopeptides or peptidomimetics. These fusogenic peptide compounds may be linear or branched, comprising between 5 and 50 amino acids, in particular between 7 and 40 amino acids and, in particular, between 10 and 30 amino acids. In the peptide sequences of the present invention, the amino acids are represented by their one-letter code but they can also be represented by their three-letter code according to the following nomenclature:
  • the fusogenic peptide compound used is a basic peptide derived from viral proteins, transcription factors or toxins.
  • Penetratin Several basic peptides derived from viral proteins, transcription factors and toxins possess the ability to cross membranes without altering them (Thoren et al., 2000, FEBS Lett., Vol.482, pages 465-8). This led to the development of the first basic 16 amino acid vector called Penetratin
  • the fusogenic peptide compound used is a peptide whose amino acid composition is rich in hydrophobic amino acids ie rich in alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine.
  • the fusogenic peptide compound comprises at least 40%, especially at least 50% and, in particular, at least 60% of hydrophobic amino acids relative to the total number of amino acids in its sequence.
  • Such fusogenic peptides are advantageously peptides derived from signal peptides and, more particularly, from the hydrophobic domain of the latter or peptide fragments of membrane proteins, in particular viruses such as HIV (human immunodeficiency virus), HTLV (human T cell lymphoma virus), MLV (murine leukemia virus) and Herpes virus.
  • viruses such as HIV (human immunodeficiency virus), HTLV (human T cell lymphoma virus), MLV (murine leukemia virus) and Herpes virus.
  • fusogenic peptide derivative is intended peptides which have 60%, 65%, 70%, 75%, 80%, 85%, 90% and / or 95% identity with the sequences of the preferred fusogenic peptides given above.
  • the fusogenic peptide derivatives may also have, relative to the fusogenic peptide sequences given above, at least one additional C-terminal and / or N-terminal amino acid, a post-translational modification and / or a chemical modification, in particular glycosylation, amidation, acylation, acetylation, methylation, as well as peptides which carry a protective group which makes it possible to prevent their degradation.
  • the fusogenic peptide derivatives may also be those in which one or more amino acids are selected from the group consisting of enantiomers, diastereoisomers, natural D-conforming amino acids, beta amino acids, alpha-substituted amino acids, amino acids hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid and synthetic amino acids including ornithine, norleucine, norvaline, cyclohexyl-alanine and omega-amino acids.
  • the derivatives of the fusogenic peptides cover, according to the invention, also the retropeptides and the retroinversopeptides, as well as the peptides whose side chain of one or more amino acids is substituted by groups which do not modify the fusogenic activity of said fusogenic peptides.
  • the fragments of the preferred fusogenic peptides advantageously have more than 5 amino acids, especially more than 10 amino acids or more than 15 amino acids.
  • the fusogenic peptides, their derivatives and their fragments may be natural products, recombinant products obtained according to techniques of molecular biology and genetic engineering well known to those skilled in the art or be synthesized chemically according to techniques such as phase synthesis solid or liquid also well known to those skilled in the art.
  • the solid support on which the fusogenic compound is attached is in particular an inorganic solid support.
  • the solid support according to the present invention is chosen from the group consisting of glasses, quartz, silicas, ceramics (for example of oxide type), metals (for example, aluminum, chromium, copper, zinc, silver, nickel, tin or gold) and semiconductors (eg silicon, germanium, ITO).
  • the solid support or the surface of said solid support is made of an organic material such as a polymer or a resin including nylon, polyethylene glycol, polycarbonates, polyfluoropolymers or composites.
  • Said solid support can be in various forms of variable size. By way of examples and non-exhaustively, it may be in the form of slides, chips, particles, beads or microchannels capillary type. These different types of support can have sizes ranging from a few hundred micrometers to several centimeters.
  • the solid support has a surface bearing functional groups (hereinafter referred to as "functionalized surface").
  • these functional groups are chosen from hydroxyl groups, radical entities, alcohol, amine or thiol functions.
  • This functionalization can be intrinsic to the nature of the surface material of the solid support used. Alternatively, this functionalization can be obtained by cleaning said surface by means of at least one solvent, detergent, radiation or oxygen plasma or any other method allowing the formation of functional groups as defined above.
  • the fusogenic compound may be directly bonded to the functionalized solid support or not.
  • the bond between the fusogenic compound and the functionalized or non-functionalized solid support is indirect and carried out through a coupling agent.
  • a functionalized solid support is advantageously chosen.
  • at least one joining agent is grafted beforehand to the surface of said solid support. This (or these) agent (s) joining then ensue the attachment of fusogenic compounds on the solid support and in particular on solid supports whose surface is inorganic.
  • Those skilled in the art know different usable bonding agents.
  • this form of implementation corresponds to the case of a support covered with a thin layer of a polyethylene glycol polyethylene glycol siloxane polymer or polylysine (D or L) for fixing the fusogenic compound.
  • polymer is meant a repetition of a number of monomeric units advantageously between 2 and 30.
  • the indirect fixation of the fusogenic compound on the solid support is carried out by means of an organized self-assembled monolayer of one or more organic or organometallic compounds (Si, Sn, Ge) having a chain alkyl terminated with a functional group.
  • These functional groups are, for example, a hydroxyl, an amino, a carboxyl, a halogen or a thiol and their modified forms, especially activated or protected forms.
  • the organized self-assembled monolayer comprises one or more organosilicon compounds corresponding to the following formula I:
  • n is between 3 and 40
  • - Xi, X2, X3 which may be identical or different from each other are selected from the group consisting of linear or branched, saturated C 1 to C 6 alkyl groups, and hydrolysable groups, at least one of X 1, X 2 and X 3 representing a hydrolysable group,
  • A represents the group -O- (CH 2 CH 2 O) k - (CH 2 ) x - in which k is between 1 and 100, and i is an integer greater than or equal to 0;
  • B represents a group -Ri, -CORi, -COORi, -CONRiR 2 ,
  • B represents a group -ORi, -OCORi, -NR x R 2 , -COORi, -CONRiR 2 , -SRi or a halogen atom.
  • R 1 and R 2 may be identical or different, representing a hydrogen atom, an optionally substituted, saturated or unsaturated and linear or branched hydrocarbon-based chain comprising 1 to 24 carbon atoms or an aromatic group.
  • B can represent any group resulting from the protection of a hydroxyl or carboxylic acid function such as the protective groups described in Protective groups in organic System (TW GREENE et al., 2 ed, Wiley Interscience), for example a cyclic protecting group.
  • i is between 0 and 100, in particular between 0 and 50, in particular between 0 and 10 and, more particularly, i is 0 or 1.
  • hydrolyzable means any group capable of reacting with an acid in an aqueous medium so as to give the compounds XiH, X 2 H or X 3 H, X 1 , X 2 , X 3 being as defined in formula I.
  • said hydrolysable group is selected from the group consisting of halogen atoms, the group -N (CH 3 ) 2 and the groups -OR ', R' being a linear or branched C 1 -C 6 saturated alkyl group.
  • halogen means fluorine as well as chlorine, bromine or iodine.
  • organosilicon compound (s) of formula I have an ethylene glycol to be used in the context of the present invention.
  • An organized self-assembled monolayer formed on a solid support makes it possible to obtain a dense, homogeneous organic surface and well defined parameters both chemically and structurally.
  • the formation of this monolayer obtained by virtue of the self-assembling properties of the compounds of formula I for well defined values of n, m, k, and i, is perfectly reproducible for one or each organosilicon compound or for mixtures of several compounds as well. in terms of quantity in terms of surface distribution of the support.
  • This functionalization is stable in time and the grafted molecules have a good orientation vis-à-vis the biological molecules.
  • These organosilicon distribution parameters on the solid support are determined and monitored by different optical methods such as vibrational imaging, atomic force microscopy, ellipsometry, etc.
  • organosilicon compounds of formula I used in the present invention advantageously have very varied functionalities and a high reactivity, having regard to the nature of the group A and the diversity of the terminal B groups which can be used, these groups B being of course able to be modified and functionalized to will according to organic chemistry reactions well known to those skilled in the art.
  • the bonds involved between the peptide compound, the solid support and / or the joining agent are chosen from covalent, ionic or electrostatic bonds or any strong chemical interaction without degradation. siloxane bonds developed between the organosilicon compounds and the solid support.
  • the process for preparing a solid support capable of immobilizing at least one cell or at least one cell part advantageously comprises the following steps: a) the preparation of a solid support such as above defined, modified by a self-assembled monolayer comprising at least one organosilicon compound corresponding to formula I as defined above, said organosilicon compound having at its end a halogen, a hydroxyl function, acid or amine, protected or not, activated or not, b) optionally, the deprotection of the terminal function of said organosilicon compound used in step (a), c) optionally, in the case where the modified solid support carries terminal carboxylic acid functions, the activation of these functions, d) optionally, the deprotection of the side chains and of the terminal amine of the fusogenic peptide compound as defined above, e) optionally, in the case where the modified solid support bears terminal hydroxy or amino functions, the activation of the terminal carboxylic acid function of the fusogenic peptid
  • the treatment step (b) may for example be a basic treatment and possibly ultrasound in order to eliminate the organosilicon compounds only adsorbed on the surface.
  • the steps (c) and (e) of activation of the carboxylic acid functions may, for example, be carried out using a solution of N-hydroxysuccinimide or of carbodiimide, or else any other suitable activating reagent known in the art. skilled person.
  • Step (f) is carried out under temperature conditions of 0 to 70 ° C. and in a satisfactory pressure range.
  • step (f) it is understood that, to solubilize the peptide compounds, any solution allowing a good solubility of the latter and a control of the evaporation of the solution will be used.
  • the fixation of the peptide compounds during step (f) may be followed by different optical or spectroscopic methods (vibrational, visible UV), infrared or Raman imaging, atomic force microscopy, ellipsometry, etc.
  • the fusogenic peptide compounds that can be used in steps (d), (e) or (f) can be used alone or as a mixture.
  • step (g) of washing in particular in an osmosis water bath, the support on which the peptide compounds are grafted can be subjected to ultrasound and this, to eliminate, without weakening the grafted layer, the peptide compounds only adsorbed on the support.
  • the present invention also relates to a method for immobilizing at least one cell and / or to least one cell part.
  • This process comprises the following steps: a ') the preparation of a support according to a process as defined above, b') the preparation of a cell suspension containing at least one cell or at least one cell part, c ') contacting the solid support as prepared in step (a ') by immersion for an indefinite period in the cell suspension prepared in step (b'), d ') at least one wash of the support obtained in the step (c ') on which said cell or said cell part is immobilized.
  • step (b ') The preparation of a cell suspension in step (b ') is advantageously carried out by diluting the cells or parts of cells in a buffer capable of preserving the integrity of the cells and cell membranes. Prior to this step (b '), the cells may be subjected to different treatments such as centrifugation, washing or concentration. These cells and cell parts as cell membranes are as described below. Step (c ') is advantageously carried out under conditions of temperature ranging from 0 to 50 ° C. and of suitable pressure.
  • the cell may be a yeast such as a yeast of the genus Saccharomyces or Candida, a mammalian cell, a plant cell or an insect cell.
  • Mammalian cells can be, for example, tumor cells, normal somatic line cells or stem cells. It may be non-exclusively red blood cells, osteoblasts, neuronal cells, hepatocytes, muscle cells, lymphocytes or progenitor cells.
  • Prokaryotic cells are bacteria that can be gram + OR -.
  • bacteria belonging to the branches of spirochetes and chlamydiae mention may be made, by way of examples and in a non-exhaustive manner, of the bacteria belonging to the branches of spirochetes and chlamydiae, the bacteria belonging to the families of Enterobacteriaceae (such as Escherichia coli), Streptococcaceae (such as streptococcus), microbacteria (such as staphylococcus), legionellae, mycobacteria, bacillaceae and others.
  • Enterobacteriaceae such as Escherichia coli
  • Streptococcaceae such as streptococcus
  • microbacteria such as staphylococcus
  • legionellae mycobacteria
  • mycobacteria bacillaceae and others.
  • the cells used in the context of the present invention may be obtained from a primary cell culture or from a cell line culture or from a sample of a fluid such as water. or a biological fluid previously extracted from a human or animal body, said sample may have undergone various prior treatments such as centrifugation, concentration, dilution ....
  • part of cell is meant in the context of this particular invention the whole or a portion of the cell membrane in which the fusogenic compound and in particular the fusogenic peptide, its derivatives or fragments as defined above are anchored.
  • cell membrane is intended to mean both the phospholipid-rich plasma membrane of eukaryotic cells (also called the cytoplasmic membrane, plasma membrane or plasma membrane) and the plasma membrane and the carbohydrate cell wall (containing peptidoglycan). ) bacteria or plant cells.
  • the cell parts used in the context of the present invention may be obtained from cells derived from a cell culture or from a sample of a fluid as previously defined.
  • Those skilled in the art know various techniques for obtaining, from cells or cell cultures, cell membranes, parts of cell membranes, fractions rich in cell membranes such as the phase-sharing technique.
  • the present invention also relates to a solid support capable of being prepared by the preparation method according to the invention and / or capable of being obtained after immobilization of a cell and / or part of cell on the latter.
  • the present invention also relates to a diagnostic kit containing at least one solid support according to the invention.
  • the present invention relates to a solid support such as than previously defined on which is fixed a fusogenic compound capable of being inserted into the cell membranes, said compound being optionally anchored in at least one cell and / or at least one cell part as previously described.
  • the support according to this first form of implementation is remarkable in that it can be stored before any use. It can especially be frozen, dried or freeze-dried. Those skilled in the art know different preservation techniques that do not affect the protein structure of the fusogenic compound attached to said support.
  • the present invention relates to a solid support as defined above on which is fixed a fusogenic compound capable of being inserted into the cell membranes, said compound being anchored in at least one cell and / or minus a cell part as previously described.
  • a fusogenic compound capable of being inserted into the cell membranes, said compound being anchored in at least one cell and / or minus a cell part as previously described.
  • the support object of the present invention described allows rapid immobilization, simple, reproducible, aspecific, homogeneous, given cellular elements and can be used for the detection of cells coming to rest with the fusogenic compounds decorating the solid support (first form of implementation above), of antibodies or ligands respectively specific for antigens or receptors present on the cells or parts of cells immobilized on the solid support (second embodiment above), of compounds with potential therapeutic activity by testing them on cells or parts of cells immobilized on the solid support, for example, cancer cells (second embodiment above).
  • the present invention finds a particularly interesting application in the field of biomedical diagnosis or sanitary monitoring of biological fluids or intended for human or animal use.
  • the present invention relates to the use, for the immobilization of biomolecular or cellular elements, of a solid support optionally modified by an organized monolayer of one or more organometallic compounds such as, for example, organosilicon compounds and methods of analysis by optical or spectroscopic methods of these biological elements. Therefore, the present invention relates to the use of a solid support as previously defined in the context of a health watch. Indeed, the various fusogenic compounds that can be used fit into the cell membrane in a non-specific manner. It is therefore possible to use the support having the compound fusogen as part of a health watch to verify the presence or absence of contaminating cells of the bacteria type in a fluid. The present invention allows to focus the target to make it detectable. This health watch can include the control of the microbiological quality of the water or an industrial microbiological control.
  • the present invention relates to the use of a solid support as defined above and / or a method for immobilizing at least one cell and / or part of a cell on a solid support in the search for antibodies and / or ligands respectively specific for antigens or receptors present on the surface of the cells or cell parts attached to said support.
  • This form of implementation is particularly interesting in the case of the search for autoantibodies when there is suspicion of autoimmune disease such as Hashimoto's disease or in the case of the search for irregular antibodies acquired secondarily. especially for blood transfusion.
  • two variants can be envisaged:
  • the detection of an antibody on the cell or part of the cell can be carried out by different optical or spectroscopic methods (vibrational, visible UV), Infra-Red or Raman imaging, atomic force microscopy, ellipsometry ...
  • a simple infra-red transmission assembly calibrated in the characteristic frequency domain of the amide groups will make it possible to rapidly obtain the percentage of fixed biomolecules in comparison with unexposed or unrecognized samples. It is also possible via Infra-Red microscopy to perform imaging mapping of the support thus treated. It is therefore possible to make a chip by nanotechnology and thus obtain a multitude of detections to target a very large number of samples.
  • FIG. 1 is a schematic representation of the solid phase synthesis of peptides 1 and 2. The deprotection and coupling steps are repeated for each amino acid to be incorporated. The duration of a cycle is 2 to 3 hours.
  • Figure 2 shows the ratio between the infrared spectrum of glass materials grafted by two joining agents (compounds or organic arms C and D) before and after treatment with potassium after subtraction of the spectrum of the raw glass.
  • Figure 3 is a schematic representation of the indirect binding of a peptide on a solid support via a joining arm.
  • FIG. 4 shows the ratio between the infrared spectrum of glass materials grafted by the joining agents C and D and then by the peptide 1 after subtraction of the spectrum of the raw glass.
  • Figure 5 shows the ratio between the Infra-Red spectrum of the different materials (glass + compounds C and D (arm), glass + arm + peptide 1, glass + peptide 1) whether or not treated with ultrasound after subtraction of the spectrum of the raw glass.
  • FIG. 6 shows the ratio between the infrared spectrum of glass materials grafted by the linking agents C and D and then by the peptide 2 after subtraction of the spectrum of the raw glass.
  • FIG. 7 presents photonic microscopy images of different glass supports brought into contact with a cell suspension and then rinsed.
  • the snapshots of FIGS. 7A and 7B respectively correspond to:
  • FIG. 8 is a schematic representation of the bioreceptor as visualized on the plate of FIG. 7B on which antibodies specific for membrane antigens of erythrocytes are fixed.
  • Figure 9 shows the scanning electron micrographs of various oxidized silicon supports brought into contact with a cell suspension and rinsed.
  • the snapshots of FIGS. 9A to 9D respectively correspond to:
  • peptide 2 Two peptides synthesized in the laboratory were used: the fusogenic peptide 519-541 corresponding to the NH 2 end of the HIV virus Gp41 protein (peptide 2) whose sequence is as follows: AVGIGALFLGFLGAAGSTMGARS (SEQ ID NO: 1 in the list of sequences in the appendix), the synthetic peptide, derived from the HTLV-1 virus protein Gp46 (peptide 1) whose sequence (sequence 242-261) is the following: SPNVSVPSSSSTPLLYPSLA (SEQ ID NO: 7 in FIG. list of sequences in annex).
  • Peptide 1 is particularly interesting. Indeed, there is an antibody specifically directed against this peptide 1 (called DB4), which makes it possible to evaluate the preservation of its functionality after grafting.
  • Solid phase peptide synthesis (S.P.S.) according to the Fmoc strategy was used to synthesize these two peptides.
  • the synthesis is carried out recurrently from the first amino acid, anchored on the solid support by its function carboxylic acid (step 1, Figure 1).
  • the 9-fluorenylmethoxycarbonyl group Fmoc (baso-labile) is used for the temporary protection of the ⁇ -amino function.
  • the release of this function is the next step in the synthesis (step 2).
  • the second amino acid whose ⁇ -carboxylic function has been activated beforehand, is coupled with the free ⁇ -amino group of the immobilized amino acid on the resin.
  • Step 3 Steps 2 and 3 are repeated for each residue to be incorporated.
  • the syntheses were carried out under the standard conditions of the Fmoc protocol using an automatic synthesizer and by starting the reaction chain with an amino acid grafted onto a Wang-type resin.
  • the coupling reactions were carried out in N-methylpyrrolidone (NMP), polar aprotic solvent which allows maximum solvation of the peptide-resin complex.
  • NMP N-methylpyrrolidone
  • This resin has a presubstitution rate of approximately 0.5-0.75 ⁇ mol ⁇ g -1, it consists of polystyrene beads crosslinked with 1% of divinylbenzene and functionalized with p-benzyloxybenzyl alcohol (arm) which allows binding to the first amino acid.
  • the solid support chosen is an inorganic glass substrate which has been subjected to a treatment which makes it possible to obtain a clean and reactive surface. Indeed, it is necessary to carry out an effective cleaning before any grafting, cleaning which must not however alter the nature of the surface.
  • an alkaline detergent "Hellmanex II” was used to optimize cleaning.
  • the surface condition of the material is observed in medium Infra-Red by transmission after immersion for 15 minutes at 50 ° C in an aqueous solution of Hellmanex 2%.
  • the material is then rinsed with osmosis water and then treated with jets of osmosis water is applied systematically.
  • the analysis of the plates after this cleaning made it possible to note the disappearance of the characteristic bands of the organic pollution
  • the reaction frequently used to obtain a long carbon chain is the coupling of two chains via two sp 3 carbons.
  • the heterocoupling reactions are carried out between a Grignard reagent and a halide using a copper catalyst, for example LiCuCl 4 or copper iodide I.
  • the docos-21-en-1-ol is of formula:
  • Second step Formation of lithium alkoxide
  • a solution of anhydrous THF is added 5.4 g (21.4 mmol) of 10-bromo-undecanol.
  • the solution is placed at -78.degree. C. under an inert atmosphere, then with an equi-pressure ampoule, 0.71 ml (23.45 mmol) of methyl lithium is added and the reaction is then allowed to gradually return to ambient temperature .
  • Step 3 Formation of docos-21-en-ol
  • 0.21 g (1.1 mmol) of copper iodide is added to the solution containing 11-bromomagnesium-1-undecene cooled to -78 ° C.
  • the solution containing the lithian derivative is added dropwise through a cannula.
  • the mixture is stirred for one hour at this temperature, then 15 hours at room temperature.
  • the reaction is stopped by the addition of 40 ml of absolute ethanol; a black precipitate is formed. The latter is accentuated by the addition of 3 ml of 10% HCl.
  • After filtration on frit 1, the clear solution obtained is extracted three times with diethyl ether.
  • docos-21-enyl acetate The OH end of docos-21-en-1-ol is then acetylated with acetic anhydride in dichloromethane to yield docos-21-enyl acetate of formula:
  • organic compound A docos-21-enyl acetate
  • organic compound B hereinafter designated organic compound B of formula:
  • organic compound A 200 mg (0.5 mmol) is placed in a Schenck tube having been previously purged by alternating switching between a vacuum ramp and an argon ramp. After addition of 2 ml of freshly distilled toluene, the solution is stirred under argon until complete dissolution of the solid. Then 300 ⁇ l of freshly distilled trichlorosilane are added as well as 2 drops of Krsted catalyst. The solution turned pale yellow is stirred for 2 hours at 40 ° C. After evaporation under reduced pressure, a crude solid is obtained and is then used in the state in the grafting step. This solid corresponds to 22- (trichlorosilanyl) -docosyl acetate hereinafter referred to as organic compound C of formula:
  • the grafting was carried out by mixing the two types of compounds and using an equimolar mixture in order to obtain a surface having an average density of active sites vis- with respect to the peptide.
  • the glass materials are introduced into a reactor.
  • This enclosure makes it possible to dry the material at a controlled temperature while avoiding any organic contamination after the cleaning; this is frequently encountered during drying in an oven.
  • This type of jacketed reactor will also be used to carry out the silanization step which must take place under an inert atmosphere and at a fixed temperature. This is possible using an external cooling system with thermal controller.
  • the materials are removed from the chamber and immersed in an osmosis water bath subjected to ultrasound over a period of five minutes.
  • This type of treatment makes it possible to eliminate the organosilicon compounds only adsorbed on the support without weakening the grafted layer.
  • the next step is the deprotection of the OH function by saponification of the compounds C and D mounted on support using 0.5M alcoholic potassium hydroxide.
  • the materials are immersed in this KOH solution for 20 minutes.
  • the supports are then removed and the impurities are removed by 3 successive treatments of 3 minutes by ultrasound in an osmosis water bath.
  • the materials are then dried on adsorbent paper.
  • Scheme 2 below corresponds to the deprotection reaction of the ester function after grafting of compound C.
  • the grafted layer is sufficiently compact and dense to prevent the penetration of the alcoholic potash within the layer.
  • the glass support was thus functionalized by the two types of compounds C and D so as to obtain a functional surface with a low density of active sites.
  • Each functionalized support is placed in a pillbox with a wide neck, in which the grafting takes place.
  • a micro bar magnet is added to ensure agitation.
  • These pill containers are themselves placed in the reactor which is then closed and then purged by alternating switching between a vacuum ramp and an argon ramp. The supports are therefore in an inert atmosphere. 8 ml of grafting solvent are added to each pill in the reactor and stirring is started.
  • activation solution comprising, per unit of support, 2 mmol of HOBt and 2 mmol of [3- (N-ethylcarbodiimide) -N-propyl] triethylammonium iodide (DiPC) dissolved in solution, under an inert atmosphere, in 1 ml of grafting solvent (osmosis water at 9 g / l NaCl).
  • grafting solvent osmosis water at 9 g / l NaCl
  • the peptide 2 synthesized according to the method described above was fixed using the same protocol as that for peptide 1. Only the osmosis water with 9 g / l NaCl present in the grafting and activation solvents was replaced by hexafluoropropan-2-ol which makes it possible to solubilize the hydrophobic peptides.
  • the glass supports thus obtained are analyzed by Infra-Red in specular reflection mode by the P.M. method. I .R. R. A. S. ( Figure 6).
  • the characteristic bands of the amides I and II confirm that the peptide 2 has been grafted according to the protocol described on a glass support.
  • ELISA tests were carried out in order to verify the biological properties and in particular the accessibility of the epitope and the specific recognition of antibodies for the fixed peptides. These tests were carried out in particular for the peptide 1 specifically recognized by the antibody DB4, using as negative control an antibody called
  • BF6 directed against a human complement system protein and not recognizing peptide 1.
  • the fusogenic peptide 2 was able to immobilize the red blood cells on the glass support.
  • red blood cells were not damaged and retained normal biconcave disk forms.
  • results quite similar to those previously presented were obtained using an oxidized silicon support instead of a glass support. Indeed, identical experiments to those previously described for the glass were carried out on oxidized silicon supports and the results obtained by scanning microscopy are presented in FIG. 9. No red blood cells are present on the plates without fusogenic peptide 2. ie no aspecific adsorption of these cells on silicon (FIG. 9A), coupling agents C and D

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EP08736151A 2007-04-12 2008-04-11 Verfahren zur herstellung eines substrats zur immobilisierung einer zelle, substrat und verwendungen davon Withdrawn EP2139999A1 (de)

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FR0754424A FR2914928B1 (fr) 2007-04-12 2007-04-12 Procede de preparation d'un support pour l'immobilisation d'une cellule, ledit support et ses utilisations
PCT/EP2008/054442 WO2008125637A1 (fr) 2007-04-12 2008-04-11 Procede de preparation d'un support pour l'immobilisation d'une cellule, ledit support et ses utilisations

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