EP2139999A1 - Method for preparing a substrate for immobilising a cell, said substrate and uses thereof - Google Patents

Abstract

The invention relates to a method for preparing a solid substrate capable of immobilising at least one cell and/or at least one cell portion, said method comprising the step of binding to said solid substrate at least one fusogenic compound capable of insertion into the cell membranes. The invention also relates to a method for immobilising at least one cell and/or at least one cell portion using the solid substrate thus obtained, to said solid substrate, and to the uses thereof in the field of biomedical diagnosis or sanitary monitoring of biological fluids for human or animal use.

Description

 PROCESS FOR PREPARING A SUPPORT FOR

IMMOBILIZATION OF A CELL, THIS SUPPORT AND ITS USES

DESCRIPTION

TECHNICAL AREA

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.

STATE OF THE PRIOR ART During blood transfusion exchanges, a conflict may arise between the donor product and the recipient organism. Transfusion safety must ensure immediate immunological compatibility but also prevent the incompatibility of future transfusions; this is essential from a point of view public health. Poorly controlled transfusions can cause antibodies to the surface structures of the red blood cells (antigen) and lead to haemolysis, which can lead to death in a severe immune reaction. Similarly, it is essential to prevent or monitor any unwanted immunization. This applies especially to fetal-maternal immunizations. Indeed, following a first delivery, a woman who does not have the Rhesus D antigen (RH D- phenotype) on her erythrocytes can develop antibodies directed against this antigen present on the surface of the erythrocytes of her first child (phenotype RH D +). This immunological reaction occurs when the mother's blood comes into contact with the child's blood during delivery. In the case of a second pregnancy (if the second child also has Rhesus D antigen (RH D +)) a serious immunological conflict for the mother and the fetus can occur, sometimes resulting in fetal death or problems in the child. newborn (cyanosis: oxygenation disorders). Thus the test of looking for irregular antibodies acquired secondarily (transfusion, pregnancy, transplant) by an individual is of considerable importance. Thus, to ensure the smooth running of a blood transfusion, systematic irregular antibody screening (RAI) is performed in addition to accurate phenotyping.

These exams are practiced in all medical laboratories and blood centers. Also, immunoassays haematological tests must be rapid and as reliable as possible.

Since its introduction in 1945, the Coombs test based on haemagglutination (aggregation of red blood cells due to the multifunctionality of antibodies) remains the standard technique for the detection of irregular antibodies. Although the tube technique was the first described, it is gradually replaced by less subjective techniques, more sensitive and more standardized. Gel techniques were introduced in 1990 and are now marketed by Diamed and Ortho. Solid phase techniques such as Capture-R (Immucor), Ready Screen (Biotest) or Biloba (Scibiex) are also available on the market. Currently, gel technology dominates the immuno-hematology market. However, it presents a major disadvantage because of the difficulties of automation.

In fact, the sharp increase in blood transfusions in hospital settings and the considerable increase in the number of blood tests in private laboratories have led manufacturers to automate their methods of blood tests. In order to improve these systems, it would be particularly interesting to develop a new immunological tool that would present the qualities of current tests with maximum improvements:

• easy, direct and rapid visualization of antigen-antibody binding

• high reliability, • the best possible sensitivity,

• a possibility of miniaturization and automation to compile multiple tests in a single analysis, • finally a low cost able to compete with its most often disposable analogues.

The use of spectral methods could respond to these different points especially in terms of speed, simplicity and sensitivity. In addition, direct visualization of the interaction would allow an evaluation of antibody affinity constants for antigens and thus allow for more precise determination and quantification of immunoglobulins. Today, most of these requirements can be overcome by the use of technologies to immobilize biomolecules. The latter then allow the detection of other target analytes. These analytical systems, known as biosensors, are highly studied in the fields of agribusiness, environment and especially biomedical with microarrays or protein chips, for example.

The major problem in the case of immobilization of cells on a bioreceptor is the preservation of their biological properties. The use of covalent coupling of one of the surface biomolecules of the cell is rarely used because grafting techniques affect cell viability. Most often the cells are only adsorbed on the surface of the support. Sometimes the Cell growth is performed directly on the measurement electrode. Finally, a more complex technique is to use an antibody directed against a recognized cellular element and to bind this antibody to the support. This technique is of specific use because of the chosen antibody which determines the type (s) of immobilized cells.

STATEMENT OF THE INVENTION

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

(transducer). 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. Thus, 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.

In addition, 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.

By "fusogenic compound" is meant 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. In the context of the present invention, the fusogenic compound may be chosen from the compounds non-peptide fusogenic and peptic fusogenic compounds.

By "non-peptide fusogenic compound" is meant 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. Among these non-peptide fusogenic compounds, 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. Indeed, many proteins of various functions, ranging from enzymatic catalysis to adhesion, are attached to the outer face of the plasma membrane of eukaryotic cells by anchoring to a GPI. 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).

By "peptide compound" is meant 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:

A AI alanine

Cysteine Cysteine

D Asp aspartic acid

E Glu glutamic acid

F Phenylalanine

G GIy glycine

H his histidine

I Isoleucine Island

K Lily lysine

Leucine leucine

M Met methionine

N Asn asparagine

P Pro proline

Q Gin glutamine

R Arg arginine

Serin serine

TH threonine

Valine Valley

W Trptophan Trp

Tyr Tyrosine Y

In a first embodiment of the present invention, the fusogenic peptide compound used is a basic peptide derived from viral proteins, transcription factors or toxins.

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

(International application WO 97/12912). This peptide is derived from the homeodomain of an Antennapedia drosophilia transcription factor. Its particular properties allow its use as a carrier vector of hydrophilic active substances inside the cells. Several other internalisation vectors are also known such as those described in international application WO 99/07728.

In a second embodiment of the present invention, 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. Advantageously, 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.

The fusogenic peptide compound according to the present invention is advantageously chosen from the group consisting of the peptides having the following sequences:

- AVGIGALFLGFLGAAGSTMGARS (SEQ ID NO: 1 in the attached sequence listing) corresponding to the sequence between amino acids 519 and 541 at the N-terminus of the HIV Gp41 protein,

- RQIKIWFQNRRMKWKK (SEQ ID NO: 2 in the attached sequence listing) corresponding to the sequence of the 3 ^rd helix of the homeodomain of the transcription factor Antennapedia, also known as Penetratin peptide,

TAALRLGIKLTQHYFGLLTAFGSNFGTIG (SEQ ID

NO. 3 in the attached sequence listing) corresponding to the sequence of the internal fusogenic domain of the Sendai virus F1 protein, SV201 peptide in the article by Ghosh et al., 2000, Biochem., Vol.

39, pages 11581-92;

- MMIMLGAICIVIVIVIVIVFFT (SEQ ID NO: 4 in the list of sequences in the appendix) corresponding to the transmembrane sequence of the Vamp protein of the SNARE system involved in exocytosis,

- RGGRLSYSRRRFSVSVGR (SEQ ID NO: 5 in the attached sequence listing) corresponding to a peptide derived from the protegrins and, more particularly, to the peptide SM1738 of the international application WO 99/07728, - C (Acm) GRKKRRQRRRQC with C (Acm) = Cys-acetamidomethyl (SEQ ID No. 6 in the attached sequence listing) corresponding to the TA basic peptide TA-T internalization, their derivatives and their fragments.

The present invention contemplates using derivatives of the fusogenic peptides defined above. By "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.

It is clear that the derivatives and fusogenic peptide fragments that may be used in the context of the present invention must also have a fusogenic activity. It will be easy for those skilled in the art to check the presence of this activity, in particular by using the technique described in the experimental section below.

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. In the context of the present invention, the solid support on which the fusogenic compound is attached is in particular an inorganic solid support. Advantageously, 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). In another variant of the invention, 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.

In a first embodiment of the present invention, the solid support has a surface bearing functional groups (hereinafter referred to as "functionalized surface"). Advantageously, 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.

In a first variant of the present invention, the fusogenic compound may be directly bonded to the functionalized solid support or not.

In a second variant of the present invention, the bond between the fusogenic compound and the functionalized or non-functionalized solid support is indirect and carried out through a coupling agent. In this form of implementation, a functionalized solid support is advantageously chosen. In this second variant, 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. By way of example and non-exhaustively, 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. By "polymer" is meant a repetition of a number of monomeric units advantageously between 2 and 30. In a very particular variant of the present invention, 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.

Ulman described in Chem. Rev., 1996, vol. 96, pages 1533-54, the formation of self-assembled monolayers of functionalized alkyltrichlorosilane type. Their use for the fixation of nucleic acids has been described in the patent application FR 2 804 129. Thus, in the context of the present invention, the organized self-assembled monolayer comprises one or more organosilicon compounds corresponding to the following formula I:

in which

n is between 3 and 40,

m is 1,

- 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;

m being equal to 1 and k ≥ 1 • if i = 0, then B represents a group -Ri, -CORi, -COORi, -CONRiR ₂ ,

If i ≥ 1, then 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. When B represents a group -ORi,

-OCORi or -COORi, regardless of the values of i and when k ≥ 1 then it is understood that 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.

Advantageously, 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.

For the purposes of the present invention, the term "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. According to a method of implementation Advantageously, 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.

The term "halogen" means fluorine as well as chlorine, bromine or iodine.

Surprisingly, it is appropriate that the 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. In addition, the formation of an organized self-assembled monolayer, very dense protects the siloxane bonds vis-à-vis chemical treatments (acid or basic in particular), which allows for various chemical reactions on the surface of the support. The 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.

Whether the binding of the fusogenic compound to said solid support is direct or indirect, 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.

Thus, in the context of the present invention, 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 peptide compound as defined above, f) bringing into contact the modified solid support obtained in steps (a), (b) or (c) by immersion, for a specified time, with one or more solutions, in one or more polar solvents, of the compound (s) (s) peptide (s) such (s) as previously defined (s) to immobilize, g) optionally, the washing of the support on which are immobilized said (or said) compound (s) peptide (s) following the step (f).

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. During 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.

During 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. 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.

In the context of the present invention, the term "cell", both a prokaryotic type cell and eukaryotic type. Among the cells eukaryotes, 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 -. Among these bacteria, 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.

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 .... By "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. For the purposes of the present invention, the term "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. In a first embodiment, 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.

In a second embodiment, 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. In this embodiment, it is possible to speak of an activated solid support i.e. activated by the cells or parts of cells anchored on said support by means of the fusogenic compound.

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. In this particular form of implementation, two variants can be envisaged:

or are fixed, to the solid support via the fusogenic compound, erythrocytes whose group and / or phenotyping are known and the solid support thus activated is brought into the presence of a fluid previously extracted from an individual such as a mammal whose group and / or phenotyping is to be defined, said fluid being advantageously blood serum, or are fixed to the solid support via the fusogenic compound, erythrocytes of an individual such as a mammal whose group and / or blood phenotyping is to be defined and the solid support thus activated is placed in the presence of a fluid containing at least one anti-rhesus antibody or directed against the defined antigen.

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 ...

Indeed, all these different components have characteristic spectral signatures for their detection. It is possible to realize a differential detection system for observing the presence or absence of biomolecules fixed, specifically, on the cellular elements or cells themselves anchored to the solid support via the fusogenic compound. The implementation of such detection techniques is routine work for those skilled in the art.

By way of example, 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.

Other features and advantages of the present invention will become apparent on reading the examples given below by way of illustration and not limitation and with reference to the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS 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:

to a glass functionalized with the coupling agent, which are the linking agents C and D and then with the peptide 1, to a glass functionalized with the coupling agent, which are the coupling agents C and D, and then with the peptide 2.

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:

an oxidized silicon support after UV treatment, a silicon support functionalized by the C and D junction agents, a silicon support functionalized with the linking agents C and D and then with the peptide 1,

a silicon support functionalized by the linking agents C and D and then by the peptide 2.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

I. Peptide synthesis.

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.

According to the SPPS strategy, 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 α-amino function. The release of this function is the next step in the synthesis (step 2). The second amino acid, whose α-carboxylic function has been activated beforehand, is coupled with the free α-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. This resin has a presubstitution rate of approximately 0.5-0.75 μmol · 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.

II. Preparation of the functionalized support.

II.1. Preparation of the glass support for grafting

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. For this, 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

(CH ₃ (2955 and 2875 cm ^-1 ) and CH ₂ (2920 and 2850 cm ^-1 ) groups).

II.2. Preparation of organic compounds After conditioning the solid support glass grafting, organic arms for functionalizing said support were synthesized. The use of arms longer than 17 carbons was chosen in order to obtain dense layers resulting in good protection of the siloxane bond (Si-O-Si) while retaining a good reactivity of the terminal function. Trichlorosilylated molecules of sufficient length and possessing a functional end are not commercially available. The synthesis of arms possessing such properties and in particular an alcohol function for the functional end allowing the subsequent fixing of a peptide has been undertaken. at. Synthesis of docos-21-enyl acetate. i. Docos-21-en-l-ol.

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:

OH

20

C ₂₂ H ₄₄ O MM = 324.60 g. mol ^"1

First step: Formation of 11-bromo magnesium-1-undecene

To a solution of anhydrous THF containing 2.7 g (107 mmol) of magnesium in the form of turnings is added, dropwise, a solution of 5 g (21.4 mmol) of 11-bromo-1-undecene in 21 ml of THF. This reaction is carried out under an inert atmosphere. The exothermicity of the reaction is regulated by means of an ice bath. Then, 5 ml of dibromoethane is added to this mixture. The reaction is kept active for one hour and the supernatant is removed using a syringe and placed in a flask under an inert atmosphere.

Second step: Formation of lithium alkoxide To 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 To the solution containing 11-bromomagnesium-1-undecene cooled to -78 ° C, 0.21 g (1.1 mmol) of copper iodide is added. 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. The remaining ethereal phase is then washed with water and then with a saturated solution of NaHCO 3. The organic phase is then recovered, dried over MgSO ₄ and then concentrated by evaporation of the solvents under reduced pressure. The remaining residue is finally reprecipitated in anhydrous acetone. After filtration on frit 4, 4.86 g (yield = 70%) of docos-21-en-1-ol are obtained.

ii. 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:

C ₂₄ H ₄₆ O ₂ MW = 366.63 g. mol ^"1

2 g (6 mmol) of docos-21-en-1-ol are added 20 ml of anhydrous pyridine. After stirring for a few minutes, the solution is cooled with an ice bath and 0.7 ml (7.2 mmol) of acetic anhydride is added. After returning to ambient temperature, the solution is stirred for 12 hours. The pyridine is then co-evaporated with anhydrous toluene and the residue is then immersed in 30 ml of dichloromethane. This solution is washed 3 times with a saturated aqueous solution of sodium hydrogencarbonate. The organic phases are combined after drying over MgSO ₄ , the solvents are evaporated under reduced pressure. A white powder is recovered. After purification by column chromatography on silica gel using a dichloromethane / methanol mixture (98/2, v / v) as eluent, 1.34 g (62%) of docos-21-enyl acetate, hereinafter designated organic compound A are recovered as a translucent oil that hardens and whitens at room temperature.

b. Synthesis of 1-O-acetyl-10-O- [1-docos-21-enyl] triethylene glycol. i. Tosylate of docos-21-ene.

Activation of the hydroxyl end of docos-21-en-1-ol by the action of tosyl chloride in the presence of triethylamine in dichloromethane at room temperature made it possible to obtain docos-21-enol tosylate of formula :

C ₂₉ H ₅₀ O ₃ MS = 478.77 g. mol -1

To a solution of 4 g (12 mmol) of docos-

21 g of 1-ol in 4 ml of anhydrous triethylamine and 30 ml of anhydrous dichloromethane are added, under nitrogen and at room temperature, 4.6 g (24 mmol) of tosylate chloride, which is itself diluted in 30 ml of dichloromethane. . The yellowish mixture is stirred for 12 hours. After filtration on frit 4 and evaporation of the solvents under reduced pressure, the residue is purified by column chromatography on silica gel with 100% dichloromethane as eluent. 3.4 g (yield = 60%) of docos-21-enosylate are recovered.

ii. 2- (2- (2- (docos-21-enyloxy) ethoxy) ethoxy) ethanol.

1.9 g (12.6 mmol) of triethylene glycol dissolved in 30 ml of anhydrous THF are added 83 mg (2.1 mmol) of 60% sodium hydride in the oil. After stirring for 15 minutes under nitrogen, Ig (2.1 mmol) of compound 6 dissolved in 40 ml of THF is added drop by drop. The mixture is then refluxed with THF for 12 hours. After returning to ambient temperature, the solution is filtered on frit 4, the solvents are evaporated and the residue is purified by column chromatography on silica gel with dichloromethane / methanol (98/2, v / v) as eluent. 0.64 g (yield = 67%) of 2- (2- (2- (docos-21-enyloxy) ethoxy) ethoxy) ethanol of formula:

C ₂₈ H ₅₆ O ₄ MW = 456.75 g. mol ^"1

iii. 2- (2- (2- (docos-21-enyloxy) ethoxy) ethoxy) ethyl acetate In a solution of 20 ml of dichloromethane is dissolved 0.5 g (1 mmol) of 2- (2- (2- 21-enyloxy) ethoxy) ethoxy) ethanol, then 0.26 ml (2.7 mmol) of acetic anhydride is added. The solution is stirred under nitrogen for one hour and then at reflux of dichloromethane for 12 hours. After returning to ambient temperature, the solution is extracted 3 times with 50 ml of dichloromethane. The organic phase is then washed 3 times with a saturated aqueous solution of sodium hydrogencarbonate. After drying over MgSO ₄ and evaporation of the solvents under reduced pressure of the organic phase, a yellowish paste is recovered. After purification by column chromatography on silica gel with a dichloromethane / methanol (98/2, v / v) is recovered 0.45 g (yield = 90%) of 10-acetyl-10-O- [1-docos-21-enyl] triethylene glycol (or 2- [2- (2-docos-21) acetate). -ényloxy-ethoxy) -ethoxy] -ethyl) hereinafter designated organic compound B of formula:

C 50 H 50 M = 498.79 g. mol -1

vs. Hydrosilylation of organic compounds A and B. The organic compounds A and B have at one of their ends an unsaturation. This double bond was functionalized with chlorosilane to fix in a second time these arms to the glass support. This hydrosilylation was carried out by reacting trichlorosilane on A and B in toluene in the presence of Kartsted catalyst.

The 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 μl 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:

C ₂₄ H ₄₇ Cl ₃ O ₂ Si 502.08 g. mol -1

From the organic compound B and under the same operating conditions is obtained the organic compound D corresponding to 1-O-acetyl-10-O- [22-

(trichlorosilanyl) -docosyl] triethylene glycol of formula:

C ₃₀ H ₅₉ Cl ₃ O ₅ Si 634.24 g. mol -1

d. Grafting organic compounds onto the glass support.

After having validated the exclusive grafting of compound C and that of compound D, 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. Thus, after cleaning, 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 chlorosilyl compounds C and D (final concentration of each compound 6.10 ^"3 M) are solubilized in the mixture hexane / chloroform, 90 / 10. This so-called" silanization "solution is then introduced into the reactor where the materials are located. Scheme 1 below shows the glass grafting reaction of the compound C.

After a night of immersion, 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.

Figure 2

In addition, in the presence of a heterogeneous grafting, KOH penetrates into the graft layer, and breaks the bonds binding the organosilicon arms to the surface. In the case of a homogeneous grafting, the molecules of this strong base do not find any space between the grafted molecules to reach the surface. The grafting of the inorganic material will therefore remain intact. The use of alcoholic potash thus also makes it possible to test the homogeneity of the grafting.

In order to validate the grafting of compounds C and D, the variations of hydrophobicity / hydrophilicity of the surface were first observed by studying the contact angle of a drop of water before, after silanization reaction and after saponification. The results of this wettability analysis are presented in Table 1.

Table 1

A sharp increase in the contact angle after grafting (raw glass versus treated glass) is obtained, which confirms the presence of a hydrophobic aliphatic layer appeared on the surface. In addition, after treatment with alcoholic potash, the decline of this angle validating the passage to a surface made more hydrophilic after removal of the ester function. This type of analysis is therefore a good indicator of the change in surface condition.

However, in order to obtain more precise information on the grafted elements, a transmission infrared analysis was carried out in order to characterize the wave number corresponding to the CH ₂ groups of the aliphatic chains of the compounds C and D grafted. The spectra obtained are shown in FIG.

The analysis confirms the presence on the surface of the long aliphatic chain by the observation of the characteristic bands of the CH ₂ bonds (V _as = 2923 cm ^-1 , V _s = 2853 cm ^-1 ). Moreover, the intensity of the bands obtained here (ΔDO = 3.10 ^"3) is comparable to the simulation performed with a monolayer of glass arachidate (ΔDO = 2.5x10 ^-3). This therefore seems to indicate the formation surface of the support of a monolayer compounds C and D. The grafted layer is sufficiently compact and dense to prevent the penetration of the alcoholic potash within the layer.

In Figure 3, there is also a spectrum shift after treatment with alcoholic potash at low frequencies. Thus, it appears that the presence of the ester group before saponification does not allow the chains to orient themselves relative to each other. After treatment with alcoholic potash, the disappearance of this terminal ester causes a reorganization of the chains 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.

II.3. Fixation of the peptide 1.

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.

Then added to the material is the so-called 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). The peptide solution (by support, 1 mmol of peptide 1 dissolved in argon in 1 ml of grafting solvent) is added, dropwise, very slowly, with stirring, to the support previously immersed in the activation solution. The reaction always follows the reaction protocol shown in Figure 3.

Stirring is maintained for 30 minutes and then stopped. The supports remain immersed for 20 hours. The materials are then removed from the pill containers and the molecules not fixed covalently are removed by ultrasound, by successive treatments of 2 minutes in osmosis water and are analyzed as above. The spectrum obtained is shown in FIG. 4. The observation of the spectrum shows the characteristic bands of the amide I (v _∞ ) and the amide II (combination between the V _CN and the δ _NH ). This suggests the presence of peptide 1 at the surface of the plates. The glass plates obtained during the various experiments were analyzed by Infra- Red in specular reflection mode and the spectra obtained are shown in Figure 5.

Thus the analysis of these spectra reveals the presence of peptides only on the plates previously grafted by the compounds C and D. This would confirm the hypothesis of a covalent grafting and not a simple absorption. Thus, the functional supports made allow the covalent attachment of peptides of biological interest.

II.4. Fixation of the peptide 2.

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.

III. Verification of the biological properties of the fixed peptides.

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.

In addition, the antigen-antibody interaction in the case of peptide 1 and the DB4 antibody has been observed by infrared transmission spectrometry.

IV. Association of erythrocytes in support.

Blood obtained from the French Blood Establishment was used, corresponding to a red blood cell pellet preserved in C.P. (Citrate Phosphate Dextrose) buffer. 1 ml of this blood is removed, dissolved in 15 ml of 1 × PBS buffer and then centrifuged for 5 min at 2500 rpm. The pellet recovered is then resuspended in this same buffer. This operation, repeated three times, allows the elimination of a large proportion of plasma proteins and this to promote cell immobilization.

Different glass supports placed in the wells of a 6-well plate were then immersed in a 1% erythrocyte solution in PBS buffer for 1 hour with gentle agitation. The supports are then held by a clamp to be stirred in 3 successive baths of PBS buffer.

The rinsed glass supports are preserved in PBS, before being placed between slide and coverslip and be observed under a light microscope NIKON optiphot 2 (Biocom visiol @ b). The snapshots obtained (x 650 objective 50) are shown in Figure 7.

This experiment was carried out on: Glass after treatment using a Hellmanex solution

Functionalized glass with organic compounds C and D,

Glass functionalized with organic compounds C and D then with peptide 1 (FIG. 7A). Glass functionalized with organic compounds C and D followed by peptide 2 (FIG. 7B).

No red blood cells are present on the glass supports after treatment using a Hellmanex solution and glass functionalized with organic compounds C and D. Furthermore, no red blood cells are observed on the plate which corresponds to the support without peptide. 2 fusogenic (Figure 7A). The erythrocytes thus do not exhibit aspecific adsorption on glass, organic compounds or peptide 1. On the other hand, the cliché carried out on a support comprising the fusogenic peptide 2 (FIG. 7B) has a high proportion of erythrocytes regularly distributed over the plaque. Figure 8 schematizes the bioreceptor as viewed in Figure 7B.

Thus, the fusogenic peptide 2 was able to immobilize the red blood cells on the glass support. In addition, red blood cells were not damaged and retained normal biconcave disk forms. It should be noted that 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

(Figure 9B) or peptide 1 (Figure 9C).

Claims

1) 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 fixing to said solid support a fusogenic compound capable of being inserted into the membranes cell. 2) Process according to claim 1, characterized in that said fusogenic compound is a non-peptide fusogenic compound such as the glycosyl phosphatidylinositol unit or the isoprene units. 3) Process according to claim 1, characterized in that said fusogenic compound is a peptide fusogenic compound. 4) Process according to claim 3, characterized in that said fusogenic peptide compound is a basic peptide derived from viral proteins, transcription factors or toxins. 5) Method according to claim 3, characterized in that said fusogenic peptide compound is a peptide whose amino acid composition comprises at least 40% of hydrophobic amino acids relative to the total number of amino acids in its sequence. 6) Process according to any one of claims 3 to 5, characterized in that said Fusogenic peptide compound is selected from the group consisting of peptides having the following sequences: - AVGIGALFLGFLGAAGSTMGARS (SEQ ID No. 1 in the attached sequence listing), - RQIKIWFQNRRMKWKK (SEQ ID NO: 2 in the attached sequence listing), TAALRLGIKLTQHYFGLLTAFGSNFGTIG (SEQ ID NO: 3 in the attached sequence listing), - MMIMLGAICAIIVVVIVIVFFT (SEQ ID NO: 4 in the attached sequence listing), - RGGRLSYSRRRFSVSVGR (SEQ ID NO 5 in the attached sequence listing), C (Acm) GRKKRRQRRRQC with C (Acm) = Cys-acetamidomethyl (SEQ ID No. 6 in the attached sequence listing), their derivatives and their fragments. 7) Method according to any one of the preceding claims, characterized in that said solid support is inorganic. 8) Process according to any one of the preceding claims, characterized in that said solid support is selected from the group consisting of glasses, quartz, silicas, ceramics, metals and semiconductors. 9) Process according to any one of claims 1 to 6, characterized in that said solid support or the surface of said solid support is an organic material such as a polymer or resin including nylon, polyethylene glycol, polycarbonates, polyfluoropolymers or composites. 10) Method according to any one of the preceding claims, characterized in that said solid support has a surface carrying functional groups. 11) Process according to any one of the preceding claims, characterized in that said fusogenic compound is bonded directly to said solid support. 12) Process according to any one of claims 1 to 10, characterized in that the connection between said fusogenic compound and said solid support is indirect. 13) Process according to claim 12, characterized in that said indirect fixing of said fusogenic compound on said solid support is carried out by means of an organized self-assembled monolayer of one or more organic or organometallic compounds having an alkyl chain terminated by a group functional. 14) Method according to claim 13, characterized in that said self-assembled monolayer compound comprises one or more organosilicon compounds having the following formula I: in which - n is between 3 and 40, m is 1, - Xi, X2, X3 which may be identical or different from each other are selected from the group consisting of linear or branched C 1 -C 6 saturated 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) 1} - wherein k is between 1 and 100, and i an integer greater than or equal to 0 where m is equal to 1 and k ≥ 1 If i = 0, then B represents a group -R ₁ , -CORi, -COORi, -CONRiR ₂ , If i ≥ 1, then 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. 15) Method according to any one of the preceding claims, characterized in that said method comprises the following steps: a) the preparation of a solid support as defined in any one of claims 7 to 10, modified by a self-assembled monolayer comprising at least one organosilicon compound corresponding to formula I as defined in claim 14, said organosilicon compound having at its end a halogen, a hydroxyl, acidic or amine functional group, protected or otherwise, activated or no, 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 the terminal amine of a peptide compound as defined in any one of Claims 3 to 6, e) optionally, in the case where the modified solid support bears hydroxyl functions or terminal amine, the activation of the terminal carboxylic acid function of a peptide compound as defined in any one of the claims 3 to 6, f) contacting the modified solid support obtained in steps (a), (b) or (c) by immersing, for a predetermined time, with one or more solutions, in one or more polar solvents, of the peptide compound (s) as (s) as defined in any one of claims 3 to 6 and optionally after steps (d) and / or (e), g) optionally, the washing of said support on which are immobilized said (or said) compound (s) ) peptide (s) following step (f). 16) Method for immobilizing at least one cell and / or at least one cell part, characterized in that said method comprises the following steps: a ') the preparation of a support according to a process as defined according to any one of of the preceding claims, b ') the preparation of a cellular solution containing at least one cell or at least one cell part, c') bringing the solid support as prepared in step (a ') into contact by immersion for an indefinite period in the cell solution prepared in step (b '), d') at least one washing of the support obtained in step (c ') on which said cell or said cell part is immobilized. 17) The method of claim 16, characterized in that said cell is a eukaryotic cell selected from yeast, a mammalian cell, a plant cell or an insect cell. 18) The method of claim 17, characterized in that said mammalian cell is selected from red blood cells, osteoblasts, neuronal cells, hepatocytes, lymphocytes, muscle cells and progenitor cells. 19) The method of claim 16, characterized in that said cell is a prokaryotic cell such as a bacterium. 20) Method according to any one of claims 16 to 19, characterized in that the cell part consists of all or a portion of a cell membrane. 21) solid support as defined in any one of claims 7 to 10, on which is optionally grafted an organized self-assembled monolayer as defined in any one of claims 13 or 14, and on which is fixed a fusogenic compound capable to be inserted into the cell membranes as defined in any one of claims 1 to 6, said fusogenic compound being optionally anchored in at least one cell and / or at least one cell part as defined in any one of Claims 17 to 20. 22) Solid support according to claim 21, characterized in that said fusogenic compound is anchored in at least one cell and / or at least one cell part as defined in any one of claims 17 to 20. 23) Diagnostic kit comprising at least one solid support according to any one of claims 21 or 22. 24) Use of a support according to claim 21 in the context of a health watch. 25) Use of a carrier according to claim 22 and / or a method of immobilizing at least one cell and / or at least one cell part as defined in any one of claims 16 to In the search for antibodies and / or ligands respectively specific antigens or receptors present on the surface of cells or cell parts attached to said support.