In vivo micro-invasive investigation device including a metal guide

DEVICE FOR INVESTIGATION MICRO-INVASIVE IVD VIVO

INCLUDING GUIDE METAL

DESCRIPTION

Technical area

The present invention relates to the functionalization of a transparietal investigation device, an ex vivo method for analyzing a substrate using a functionalized device of the invention, and the use of such a device for the manufacture of a tool for diagnosing cancer, an infection, inflammation, a neurodegenerative disease or graft rejection in a patient.

PRIOR ART

It is known in the prior art devices of investigation or treatment in vivo. Such devices take the form of a rigid endoscope tube type or a catheter consisting of a flexible tube that is inserted in the body, especially by natural means or vessels, and which allows to reach an organ or a specific tissue. These devices including the elimination of blood clots or, when associated with optical fibers, and viewing the in vivo monitoring of the state of a system, such as the gastrointestinal tract, or body, as the colon. Trauma to the patient resulting from the use of such devices is then minimized but still to be improved. However, it is not always possible to perform an analysis of organs or tissues in a patient with these devices. Such impossibility may result from the reduced availability of that tissue or organ under the bloodstream or natural means, or so difficult to make a reliable diagnosis without resorting to a detailed study of cells of said organ or tissue. In these cases, it is routinely used in the prior art and to date, the removal of a fragment of said tissue or organ (biopsy) for controlling ex vivo the morphology of these tissues or organs, or more precisely the cells which constitute them, including using fine needles (FNA or "Fine needle Aspiration" Engelstein et al, Br J Urol, 7: 210-213, 1994; Rodrigues et al, J.... ... Am Acad Dermatol 42: 735-740, 2000; Ariga et al, Am J Surg 184:..... 410-413, 2002; Pérez-Guillermo et al, Diagn Cytopathol, 32.. 315-320, 2005; Fernandez-Esparrach et al, Arch Bronconeomol, 43. 219-224, 2007)... In addition, it is also possible to analyze the expression status of a number of markers whose expression is correlated with a specific medical condition (cancer, inflammation, infection, neurodegenerative disease or graft rejection in particular).

However, these different methods, because of the removal of a biopsy or aspiration of cells in situ, are sometimes undergo significant to said tissue or organ and trauma, consequently, to the patient. The body of the latter can thus be strongly felt due to hemorrhage or the subsequent healing in the collection, especially for certain organs (brain, pancreas, liver or lung).

It therefore still exists a need to identify new investigation methodologies for performing reliable and accurate diagnosis, particularly with regard to the expression of markers specifically associated with different pathologies like including cancer, inflammation, infection or neurodegenerative disease, while minimizing trauma inflicted on the patient.

EP 1,358,481 describes an in vivo assay or treating device comprising (i) an investigative micro-system of a substrate other than by analysis of a fluorescent signal, (ii) a flexible rod at one end of which is fixed said microsystem and the other end is designed to maneuver the said micro-system, (iii) a medical device having an inner lumen into which said flexible rod can slide, (iv) providing a system of protection removable micro-system at the substrate, and (v) at said micro-system, a laceration of tissue system or cell, optionally combined with one or more devices selected from a remote monitoring device by sensory receptors ( tactile, optical, physicochemical including electronic or computer scanned) biopsy of implementation, processing, local injection of biological or chemical products.

To allow the intrusion of blood vessels and the shredding tissue or cells, the micro-system is then associated with a more rigid system performing this function, preferably in the distal position.

However, in the case of investigation of in vivo device, the inquiry device is mostly directed towards the organ or tissue target using endovascular or intracavitary route. Coupling the microsystem with a shredding system then increases to a significant extent the diameter at the end of device described. This then proves to be a complex use to be properly directed to its target site without simultaneously altering the circulation routes. Simultaneously, the coupling of multiple elements affect the overall flexibility of the device, and hence its correct guiding, and the obtaining of the necessary rigidity to allow the perforation of an organ or tissue.

Description of the invention

Following extensive research, the inventor has now succeeded in developing a device comprising a guide wire to a perforating end which are directly coupled reactive groups, including antibodies or antibody fragments specific to one substrate test, and the other end is designed to maneuver said insertion site to guide microanalysis site and / or micro-sampling said substrate. Said guide can be inserted into a removable protection system, such as a flexible catheter, thereby protecting the functionalized end of said guide until microanalysis site and / or micro-sampling of the test substrate constituent fabric , organ, or cell thereof.

This is in accordance with the present invention, to structure the surface of the guide in order to define locations on the guide, for example, wells, where the reactive groups will be deposited and where the biochemical interactions will take place. Said "sink" can be made by various processes, for example by lithography, focused ion beam (FIB or

"Focused Ion Beam" Xie et al, Nuclear Instruments &.

Methods in Physics research Section B-beam interactions with Materials and Atoms, 211 (3): 363-368, 2003), by laser lithography followed by electrochemical etching and laser ablation.

Alternatively, the structure of the guide wire can be carried out in order to define locations on said guide, for example at least one groove in which is fixedly assigned at least one biochip linear miniaturized, circular or band, where the reactive groups will be deposited and where biochemical reactions take place. Locations formed by the structuring of the surface of the guide, for example, wells do not form a relief on the surface of the guide, but the grooves, or hollow. Places formed by structuring the surface of the guide therefore does not increase the diameter of the guide.

Advantageously, locations formed by structuring of the surface of the guide are located on the surface of the guide, which means that these areas are in direct contact with the fluids, tissues and cells.

Said device can further be inserted into a medical instrument having an internal lumen in which said guidewire is slidable, and in particular in a transparietal aspiration needle including transcutaneous or transmucosal, or an endoscope, including an endovascular navigation system.

The device may further be associated with an optical fiber, or the guide wire of said device may be replaced by an optical fiber to a perforating end of which is associated a metallic ring on which are directly coupled reactive groups, in particular antibodies or antibody fragments, specific for a substrate to be tested, allowing a thin display in situ, for the in vivo capture elements necessary for diagnosis and possibly prognostic evaluation. the optical fiber properties are used for tracking imaging purposes and setting up the device from the in situ visualization.

The device of the invention due to its present simplification while remaining micro-invasive, improved flexibility or elasticity compared to the devices of the prior art. The latter can then be used more effectively to an investigation by intracavitary way, including endovascular, or transparietal way, especially not transcutaneously or transmuqeuse. In addition, the device of the invention because of the use of a guide wire, one end is coupled to specific reagents, has sufficient rigidity to its functionalized end to efficiently perforate a tissue or organ. Finally, the diameter of the device according to the invention at its end is low enough to allow easy navigation through the bloodstream or in natural cavities, especially minimize the trauma to the tissue or organ that it should perforating.

Furthermore, after withdrawal of the device, it is then possible to identify ex vivo using conventional techniques such as an enzyme immunoassay or by immunofluorescence, for example on a solid support [ELISA, protein chip (ESPINA et al ._r J. Iwmunol. Methods, vol.290, p: 121-133, 2004)], the presence and relative concentration of said one or more specific substrates to the end of the device according to the invention and thus the level of the micro-site analysis and / or of microsampling (organ or tissue). Finally, and due to the metallic nature of the guide, the device has an improved signal imaging (angiography, ultrasound, CT, MRI, etc.). This property greatly simplifies the radio control device according to the invention during the intervention to targeted tissue or organ. It is the same for coupling said device with an optical fiber locating imaging purposes, and allows the fine display in situ.

Accordingly, a first object of the invention is a device for analysis of a substrate, characterized in that it comprises a micro-invasive investigation system and / or micro-sampling of said substrate, said system being consisting of at least one metal guide at one end Ea which is provided with at least a series of wells to which is directly coupled at least one reactive group specific of said substrate, said functionalized end Ea being perforating, and whose other end is intended Em to maneuver the said guide wire and possibly combined with a suction system.

Advantageously, the surface of the end Ea can be structured so as to define at least a series of wells to which is directly coupled at least one reactive group specific of said substrate, said end Ea being perforating.

The device of the invention, because of the low trauma it causes the patient, allows for more micro-analysis or micro-samples from a patient at regular intervals (eg, analysis and / or charges under floors of the prostate). Such analyzes and / or allow successive samples thus, besides the diagnosis, monitor cancer, inflammation, infection, a neurodegenerative disease or good decision for an organ transplant in a patient.

Advantageously, the functionalized Ea end can have a length of about 0.5 to 2 cm, at least a series of wells 1 to 25, preferably 2x25 wells, having an average diameter of about 30 to 80 .mu.m, preferably about 40 to 60 .mu.m, and most most preferably about 50 microns, a depth of about 20 to 30 .mu.m, preferably 25 .mu.m, said wells being spaced from each other of about 60 to 120 .mu.m. Preferably, the wells have a smooth or rough wall, an oval or round shape, with a flat or concave bottom.

Advantageously, the functionalized end can have a number of wells as high as possible. The number of wells may be such that the guide wire is of a rigidity to puncture tissue or organs. In this embodiment, the mean diameter of the functionalized end can be about 0.3 to 3.5 mm, preferably about 0.35 mm. Advantageously, the guide wire can be structured on a length of 0.5 to 2 cm, preferably about 1 cm, to form the wells. Advantageously, the diameter of the wells may be about 30 to 80 .mu.m, preferably about 30 to 60 .mu.m, for example 35 microns. Advantageously, the depth of the wells may be about 20 to 30 .mu.m, preferably about 25 .mu.m. The spacing of the wells may be from 20 to 120 .mu.m, preferably 25 .mu.m.

Advantageously, the functionalized end Ea may be cylindrical, flat, spiral or modified so as to increase the surface in contact with the well fluids, tissues and cells.

In a particular embodiment of the device according to the invention, the device further includes a removable protection system at the functionalized end Ea.

In yet another particular embodiment of the device according to the invention, said device further comprises a medical instrument with an internal lumen in which said at least one metal guide can slide.

By "guide wire" is meant for example a flexible solid metal rod or a hollow rigid metal rod, having a diameter ranging from 0.2 to 3.5 mm and a length of from 5xlO^{~ 2} 2 m, and being insertable into a blood vessel, a small cavity or through an organ or tissue, to be directed from the insertion site to the micro-site analysis and / or micro sample in situ.

In particular, a flexible solid metal rod may be comprised of an optical fiber to an end Ea of which is associated a metal ring provided with at least one series of wells to which is directly coupled at least one reactive group specific of said substrate, said functionalized end Ea being perforating.

Advantageously, said metal ring has a width of about 0.5 to 2 cm, and may have at least a series of wells 1 to 25, preferably 2x25 wells, having an average diameter of about 30 to 80 .mu.m, preferably about 30 microns or from 40 to 60, and so entirely preferably from about 50 .mu.m to 35 .mu.m, a depth of about 20 to 30 .mu.m, preferably 25 .mu.m, said wells being spaced from each other by about 20 to 120 microns, for example about 60 to 120 microns. Preferably, the wells have a smooth or rough wall, an oval or round shape, with a flat or concave bottom.

By "guide wire" a flexible solid rod further means or hollow, rigid or flexible consists wholly or part of a metal alloy, the characteristics of flexibility, rigidity, oxidation and immunogenicity are compatible with a such use in the living and particularly in animals, especially humans. Such biocompatible alloys can be simply identified by the skilled person in the light of his general knowledge and include stainless steels, titanium alloys, nickel, cobalt, or mixtures thereof.

The inventor has demonstrated that a guide based on an alloy of nickel titanium (Nitinol alloy) exhibited particularly advantageous properties in terms of overall flexibility and stiffness at its end to be effectively used for the endovascular or endocardial channels and also to efficiently perforate a tissue or organ while minimizing the trauma (the size of the perforation at said tissue or organ is for example of the order of 0.05 to 0.5 mm²And preferably of the order of 0.07 mm²).

Advantageously, the guide wire is made of an alloy of nickel and titanium, preferably based Nitinol (guidewires Euroflex marketed by the company).

Said metal guide can be covered in except for the functionalized end Ea, a hydrophilic polymer, preferably a hydrogel, or a porous polymeric protective layer having a thickness of from about 0.1 to 51 microns. Advantageously, the protective polymer comprises a parylene film, TiO₂ or OptoDex® (Arrayon Biotechnology, Switzerland), and more preferably a parylene film.

The removable protection system into which is inserted the metal guide can take many forms, including that of a flexible catheter, which can be simply determined by the skilled person, such as forms used for the endovascular, endocardial , transmural, including transcutaneous.

Said removable protection system can be inserted endovascularly in particular to achieve the vessels of the heart, brain, lung, pancreas, kidney, and liver.

Said removable protection system can be inserted through access intracavitary, in particular through an endoscope, oral, anal, urogenital and respiratory or ENT transmucosal, or transcutaneously through a puncture at the skin, reaching for example the mammary gland, and particularly to the loins in a joint, in the spinal canal, or by lumbar puncture in transparietal in the liver, the lung or the kidney, but also by way transmucosal, especially for the gastrointestinal tract.

The inventor has shown that the device according to the invention then achieves tissues or organs, normally difficult to access by endovascular tract intracavitary or transparietal and including transmucosal or transcutaneous, conventionally used.

More generally, the inventor has demonstrated that the device according to the invention due to its specific characteristics of overall flexibility and stiffness at its end to reach and perforate transparietal route (transdermal, transmucosal) endovascular or endocardial certain organs and tissues belonging to the digestive system of the oropharynx to the rectum (including liver and pancreas), the urogenital system (including bladder, kidney, prostate, testis, ovary and mammary gland), the tracheobronchial system ( including lung), the ENT system (including ear and nasopharynx), the musculoskeletal system (including synovial cavities), the endocrine system, the cerebral neural system or the integumentary system, making possible the realization of diagnostics of diseases that required up 'then biopsies see aggressive deep punctures like trans-liver biopsies, including cell removal by suction using fine needles (technique called "fine needle aspiration" or FNA).

Advantageously, said removable protection system into which is inserted the guide wire takes the form of a flexible catheter adapted for endovascular or intracavitary route.

The device according to the invention is particularly adapted to perform an investigation, for example, the arteries and veins, vessels of the heart, prostate, mammary gland, pancreas, kidney, cardiac muscle, CNS and its cavities or channels, brain or liver. According to a first particular embodiment of the device according to the invention, the removable protective system in which is inserted the guide wire itself is inserted into an endoscope. The device according to the invention is particularly adapted for administration by the intracavitary route.

The device according to the invention is particularly adapted to perform an investigation, for example, at the level of tracheo-bronchial system (including lung), digestive system of the pharynx to the rectum (including liver and pancreas), the urogenital system (including bladder, kidney, prostate, testis, ovary and mammary gland), the ophthalmic system (tear ducts), the ENT system (including ear and nasopharynx), the musculoskeletal system or the central nervous system, including endo way - spinal or of the mammary gland by the endogalactophorique way.

According to a second particular embodiment of the device according to the invention, the metal guide constituted of a thin puncture needle transmural, including transcutaneous or transmucosal aspiration, can be inserted in a removable protection system, for example a flexible catheter . The device according to the invention is particularly adapted for administration specifically by transcutaneous or transmucosal route.

The device of the invention thus is particularly adapted to perform an investigation, for example, at the level of the integument (skin, scalp, etc. ..) ^ breast, kidney, lung, liver, muscle, the 'musculoskeletal apparatus or musculoskeletal, central or peripheral nervous system or the endocrine glands (including thyroid, parathyroid, adrenal glands, testicles, mammary glands or ovaries).

As regards the functionalized end Ea of the device according to the invention, it is provided with at least one series of wells to which is directly coupled specific reactive groups of a substrate to be tested.

By "specific reactive groups," a nucleic acid sequence [DNA (amplification product, gene fragment, EST, SNP) or RNA] is meant for example a complementary nucleic acid sequence to detect a specific antigen of an antibody to be detected or an antibody or antibody fragment specific to an antigen to be detected, preferably an antibody or antibody fragment.

Advantageously, said specific reactive groups are arranged in the microwells of the functionalized end of the device according to the invention according to an increasing or decreasing range. The skilled artisan can determine simply by using his general knowledge and routine experimentation, said range depending on the affinity of the reactive group to its substrate. For example, the range of the reactive group is of the order of 50 to 500 micrograms / ml, preferably from 10 to 100 mcg / ml, for a reagent, including an antibody having an affinity for its substrate, including an antigen, of the order of 10^"9.

By "antibody" is meant preferably an immunoglobulin to be alive, mammalian, including human, and particularly preferably IgG.

By "antibody fragments" refers to fragments of antibodies capable of maintaining a specific binding of their antigen. For example such antibody fragments include Fab, Fab ', F (ab')₂ or Fv.

reactive coupling methods, particularly protein on a metal support are well known to those skilled in the art. Such methods, because of low chemical reactivity of the amino acids, generally require the activation of the metal surface or by oxidation mechanisms, or by covering said at least one layer of bonding molecules, the usually polymers (it may, for example thiol groups, carboxylic acids and / or amino) .In examples of such methods include adsorption on metal supports functional molecules s' organizing self-assembled monolayers (self-assembled monolayer SAMs) including alkanethiols (see especially WITTSTOCK and SCHUHMANN, Anal Chem, vol.69, p-5059-5066. 1997;.. and international patent application WO 03/006948) or pyrrole (electrochemical polymerization of pyrrole biotinylated; FILLIARD Dupont et al, Anal Chim Acta, vol.449, p: 45-50. 2001...).

By coupling the antibody or antibody fragment to said layer of functional molecules, covalent bonding means (such as disulfide bridges between the free thiol groups alkanethiols) or non-covalently (as a streptavidin-biotin binding between biotin a polymer layer of a pyrrole complex / biotin and streptavidin a streptavidin / antibody complex or antibody fragment) streptavidin).

According to a first preferred embodiment, the device according to the invention comprises at least one metal guide which an Ea end is coupled with at least one reactive group, preferably an antibody or a antibody fragment, specific for a marker (antigen) of a cancer, including breast cancer, ovarian, prostate, colon, intra-abdominal, kidney, liver, lung, pancreas, central or peripheral nervous system or an endocrine gland (including thyroid, testes or ovaries).

For example breast cancer marker include the marker CA 15-3 (Carcinoma-Associated Antigen 15-3; Duffy MJ, Schering S Sherry F, E McDermott, N O'Higgins, J / it J Biol Markers 2000 Oct-Dec; 15 (4): 330-3), CA 27- 29 (Carcinoma-Associated Antigen 27-29; Kaohsiung J Med Sci J 1999 September; 15 (9): 520-8 ), CEA (Carcinoembryonic antigen; Soletormos G, D Nielsen, Schioler V Mouridsen H, P Dombernowsky, Eur J Cancer 2004 March; 40 (4): 481-6); TPA (Tissue Polypeptide Antigen); GST (Tissue Polypeptide Antigen Specifies; Given M, Scott M, McGrath JP, Given HF, Breast, October 2000; 9 (5): 277-80), HER2 (Fehm T, W Jager, Kramer S, Sohn C, Solomayer E, D WALLWIENER, G. Gebauer, Anticancer Research, 2004 May-Jun; 24 (3b): 1987-1992), ER (estrogen Receptor; Platet N, Cathiards AM, Gleizes M, Mr. Garcia, Crit Rev Oncol Hematol, JuI 2004; 51 (1): 55- 67), PR (Progesterone Receptor; MJ Duffy, Clin Chem 2005 March; 51 (3). 494-503 Epub 2005 January 6), Ki-67 (cell proliferation- associated antigen of antibody Ki-67; Schliiter C Duchrow M, Wohlenberg C, Becker MH, Key G, Flad HD Gerdes J, J Cell Biol 1993 November; 123 (3): 513-22) and UPA (Urokinase Plasmogen Activator; MJ Duffy, Crit Rev Clin Lab Sci, 2001 Jun; 38 (3): 225-62).

For example marker of ovarian cancer include CA125 marker (carcinom Antigen 125; EL Moss,

Hollingworth J, Reynolds TM, J Clin Pathol, 2005 March;

58 (3): 308-12), CA 15-3 and CEA (Valenzuela P, S Mateos, Teho E, Lopez-Bueno MJ, N Garrido, Gaspar J., Eur J Gyn Oncol 2003; 24 (1): 60-2).

For example prostate cancer marker include the marker PSA (Prostate-Specific Antigen, Gray MA, Clin Lab 2005; 51 (3-4):. 127-33); PMSA (Prostate- Specific Membrane Antigen) and AR (Androgen Receptor; AJ Birtle, Freeman A, Masters JR, HA Payne, Harland SJ, BJU Int 2005 Aug; 96 (3): 303-7).

For example colon cancer marker include the marker CEA (MJ Duffy, Clin Chem 2001 Apr; 47 (4): 624-30), CA 19-9 (19-9 carcinom Antigen) CA242 (carcinom Antigen 242), CA 72-4 (carcinom Antigen 72-4), TPA, TPS (Duffy MJ, van Dalen A, C Haglund, Hansson L, R Klapdor, Lamerz R, Nilsson O, C Sturgeon, Topolcan O Eur J Cancer 2003 Apr; 39 (6): 718-27).

For example marker of intra-abdominal cancer include CEA or CA 19-9 marker (Coban E, M Samur, Bozcuk H, Ozdogan M, Int J Biol Markers 2003 Jul-Sep; 18 (3): 177-81).

For example pancreatic cancer marker include the TA90-IC tag (90-kDa immugenic Tumor- associated Antigen), CA-19-9 (Chung MH RK Gupta, Bilchik AJ, Ye W, Yee R, Morton DL, Curr Surg 2002 March- April; 59 (2): 194-198), TPS, hCG beta (hCG beta Human Chorionic Gonadotropin beta), CA 72-4, CEA, CA19-9, CA 242 (Louhimo J, H Alfthan, Stenman UH, Haglund C, Oncology, 2004; 66 (2): 126-31).

For example liver cancer marker include alpha-fetoprotein marker.

For example lung cancer marker, we include the marker Cyfra A41 (Cytokeratin fragment 41), SCC (Squamous Carcinoma Antigen CEIL), ACE (Angiotensin Converting Enzyme), CA 19-9, CA 125, NSE (Neuron Specifies Enolase), chromogranin A, CYFRA 21-1 ( cytokeratin fragment 21-1) CA 15-3.

According to a second preferred embodiment, the device according to the invention comprises at least one metal guide which an Ea end is coupled with at least one reagent, preferably an antibody or antibody fragment, specific for a specific marker an inflammation, including rheumatoid arthritis.

As an example of marker of rheumatoid arthritis include in particular IL-lβ, IL-lRα, IL-2, IL-2R, IL-4, IL -5, IL-6, IL7, IL8, the Illo, the ILl2p40P70, IL-13, IL-15, IL-17, TNFa, IFNa, the IFN.gamma, GM-CSF, MIP-I, IP-10, MIG, the eotaxin, RANTES and MCP-I (Cockrum et al, Lab Automation, BTI, October 2005, p.: 19-21).

According to a third preferred embodiment, the device according to the invention comprises at least one metal guide which an Ea end is coupled with at least one reagent, preferably an antibody or antibody fragment, specific for a specific marker an infection, in particular a viral, bacterial or parasitic infection.

Many infectious markers are known to those skilled in the art and it can easily identify the one or more specific markers associated with a given infection.

According to a fourth preferred embodiment, the device according to the invention comprises at least one guide Ea metallic one end is coupled with at least one reagent, preferably an antibody or antibody fragment, specific for a specific marker of graft rejection.

Many transplant rejection markers are known to the skilled person. For example, include MIP-lβ and VE-cadherin to heart transplantation (ROUSSOULIÈRES et al., Circulation, vol.111 (20), pp .2636-2644, 2005).

In view of the specific markers described above, the skilled person in the light of his general knowledge and can easily identify without undue experimentation antibodies or antibody fragments specific for use in the device according to the invention. For example, such antibodies include the antibodies available from TEBU or AXXORA. The skilled artisan may also obtain such antibodies by well known methods of immunization.

Similarly, those skilled in the art can without difficulty to identify the specific nucleic acids suitable for use in the device according to the invention.

According to a fifth preferred embodiment, the device according to the invention comprises at least one metal guide which an Ea end is coupled with at least one reagent, preferably an antibody or antibody fragment, specific for a specific marker or a set of specific markers of neurodegenerative diseases, such as Alzheimer's Disease (AD), Parkinson's syndrome, amyotrophic lateral sclerosis (ALS), this list is not exhaustive. Several markers are known and used in the art for the study of these pathologies. Examples include for dementia or prédémentiels states, total tau (MAPT Microtubule-Associated Protein Tau), the peptide Amyloid ABETA1-42, hyperphosphorylated Tau protein (phosphorylated Tau P 128), or example described by Waldemar G, Dubois B, Emre M. et al, Eur. J.Neurol. 2007, 14, pp 1-26; B. Dubois, Feldmann HH, Jacova C, DeKosky ST et al, Lancet Neurol.2007, 6, pp 734-746;. Krolak-Salmon P. et al, Towards a biological diagnosis of Alzheimer's disease and related syndromes La Revue de Médecine Interne (2008), doi: 10.1016 / J.revmed.2008.01.029. These markers may be assayed by an assay technique ELISA Antigen-Antibody. Innotest B-Amyloid (1-42) Innotest hTAUAg, Innotest PHOSPHOTAU (18Ip); Innogenetics, Ghent, Belgium.

Other markers also allow testing the brain state of degradation including the Visinin-like protein (or VLP6 VILIP-I or VSNL), as described for example by .REF Lee JM et al, Clin Chem, 2008, 54, pp 1617-1623. These markers can be used in the implementation of the present invention.

For Parkinson's disease and multiple system atrophy (synucleopathies) include the marker, alpha-synuclein (Mollenhauer B., V. Cullen, I. Khan, Experimental Neurology, 2008, 213, pp 315-325).

Finally, there are also non-specific markers of disorders of the central nervous system (CNS) which may be used in the context of the present invention. These proteins from CNS such as GFAP, myelin, neuropeptides and neurotransmitters. The protein BDNF (brain derived neurotrophic factor), more particulièreemnt disclosed in University of California San Diego Medicine & health / diseases February 200, is of particular interest 9. In addition, proteins of the immune response, e.g., IgG, albumin, complement proteins, the C-reactive protein, and protéinesde inflammation, such as transferrin, haptoglobin, Ceruloplasmin, Lysozyme, Enolase can be used in the implementation of the invention.

Whatever embodiment of the invention, several different markers, that is to say, the markers of various diseases, may be placed in wells of a same metal guide. In this case, the markers may be placed in the manner well as the different labels do not interact on each other.

Advantageously, the device according to the invention, at least in its terminal part functionalized in contact with the substrate to be analyzed, has a sterility assurance level (SAL Sterility Assurance Level) of the order of 10^"6. Various alternatives are possible to achieve this level of sterility. One possibility is to sterilize the device in the absence of specific reactive groups of the substrate to detect and then add them under sterile conditions. Another possibility is to sterilize the device after adding reactive groups, which requires the use of sterilization techniques do not significantly reduce the activity of said reactive groups (eg sterilization with ethylene oxide or radiation) .

A second object of the invention is a method of ex-vivo detection of a substrate present in a tissue or organ, characterized in that it comprises the following steps: a) incubating the functionalized end Ea of a device according to the invention with a solution comprising at least one specific detecting agent of said substrate, after said end has been brought into contact with said tissue or organ to be examined. b) detecting said substrate. The incubation step is carried out for a time sufficient for the solution in detection agent, in particular an antibody, can bind specifically to the substrate (label, antigen, antibody, etc.), in particular u antigen, if present in the end of the device. Those skilled in the art can simply determine by using his general knowledge and routine experimentation, this incubation time based on the affinity of the solution in detection agent, in particular an antibody, for its substrate including an antigen. This incubation time is also dependent on the temperature of the solution during this incubation. For example, the incubation time is of the order of 1 minute to 2 hours, preferably 5 minutes to 1 hour, and particularly preferably 10 to 30 minutes, to a temperature between 2O⁰C (room temperature) and 37 ° C.

Advantageously, the detection agent in solution is different from the specific reagent coupled to the functionalized end of the device according to the invention.

Preferably, the detecting agent is an antibody.

Advantageously, the antibody solution and the antibody coupled to the functionalized end of the device according the invention are each a polyclonal antibody, preferably said antibodies are identical.

Advantageously, the antibody solution and the antibody coupled to the functionalized end of the device according to the invention are each a monoclonal antibody, preferably said antibodies are different.

Advantageously, the solution antibody is labeled, and in particular is coupled to an enzyme, for example peroxidase or alkaline phosphatase.

According to a first particular embodiment of the method according to the invention, the method further comprises a step a ') of said end of incubation in a solution comprising at least one specific detecting agent of the detecting agent of the step a), interposed between steps a) and b).

The skilled person can easily identify and using his general knowledge, the appropriate antibodies the method of the invention. For example, it is possible to use in this second step an antibody specifically recognizing mouse immunoglobulins if such mouse immunoglobulins specifically directed against the substrate (label, antigen, antibody, etc ..) to be identified are used in step a).

According to a second particular embodiment of the method according to the invention, the method of the invention comprises a washing step after the incubation step a) and optionally of step a '), which step washing helps eliminate antibodies that are not specifically attached to the marker (antigen).

The protocol of such a washing step is again the general knowledge or can simply be determined by routine experiments. For example, such a step is performed with a solution comprising a higher or lower concentration of detergent (0.05 to 1%) such as TRITON XlO0® or TWEEN 20®, and this according to the affinity of the antibody in solution for its specific antigen.

The detection step is performed by highlighting activity, particularly enzyme coupled to the antibody used in step a) or, optionally in step a ').

The protocol used for this detecting step depends on the marker used, in particular the enzyme used, such as peroxidase and alkaline phosphatase, and is within the general knowledge of those skilled in the art.

This detection step can be deduced the amount of specific substrate (e.g., antigen) attached to the functionalized end of the device and finally the amount of specific substrate present at the organ or tissue which has been performed (an ) micro-analysis and / or micro-sampling.

Finally, the various reactive groups suitable for carrying out the method according to the invention are well known to those skilled in the art and include, in particular, the reagents used for the technique of immunoassay or by immunofluorescence, for example on a solid support [ELISA , protein chip (ESPINA et al., supra, 2004)].

A third object of the invention is the use of a device according to the invention for the making a tool for diagnosing cancer, inflammation, infection, graft rejection or a neurodegenerative disease in a patient.

In a particular embodiment of the invention, the diagnostic tool may comprise at least one metal guide inserted into a flexible catheter inserted through an endoscope.

According to another particular embodiment of the invention, said diagnosis tool may comprise at least one metal guide comprising a transparietal aspiration needle, including transcutaneous or transmucosal aspiration, which can be inserted in a removable protection system, by such a flexible catheter. In addition, the removable protective system and the metal guide cooperate to allow the contacting of the functionalized end Ea of said guide with micro-site analysis and / or micro-sampling.

In these two particular embodiments of the invention, said at least one metal guide can be associated to at least a portion of its length to an optical fiber for location and positioning.

Advantageously, the diagnostic tool is administered by intracavitary way.

The tool thus enables to perform a micro-analysis and / or micro-sampling in the digestive system of the pharynx to the rectum (including liver and pancreas), the urogenital system (including bladder, urethra, kidney, prostate) of tracheobronchial system (including lung), ENT system (including ear and nasopharynx), the musculoskeletal system (including synovial cavities). Preferably, said diagnostic tool is administered by transparietal route, in particular by transmucosal or transdermal route.

Such diagnostic tool allows to analyze tissues or organs difficult to reach by road or endocardial endovascular usually used. Such a diagnostic tool thus enables a microanalysis and / or micro-sampling transparietal route at the skin, testicular, prostate, ovarian or mammary gland, but also kidney or liver, peripheral nervous system and central nervous system, including endo-spinal pathway and the endocrine system (eg thyroid).

The following examples illustrate the invention and are given és non-limiting.

Brief Description of the Figures

- Figure 1 shows various possibilities for structuring metal guides.

- Figure 2 shows electron micrographs scanning different holes made by FIB (large surface roughness due to etching inhomogeneities can be observed).

- Figure 3 shows an explanation and observation of the shading effect of the milling technique assisted by fluorite.

- Figure 4 shows the result of microfaçonnage operations.

- Figure 5 shows (a) hemispherical holes observed by optical microscope (b) of the holes series observed on a nitinol-based guide by scanning electron microscopy (c) detail of a cavity (d) comparing the surface roughness in the cavity with the surface roughness of the guide.

- Figure 6 shows scanning electron microscopy images showing the same hemispherical hole as in Figure 5 after the electrochemical polishing treatment.

- Figure 7 shows a diagram of the device of the invention for immunocapture with a First Prev monoclonal antibody (Mab 1) and the revelation of the antigen ACE with a second monoclonal antibody (mAb 2).

- Figure 8 shows the results of ELISA with the capture antibody 5910 and the revelation with the antibody 5909 (absorbance obtained with positive serum CEA antigen)

- Figure 9 shows the results of ELISA with 5910 capture antibody and revelation with the antibody 5909 (absorbance obtained with a negative serum antigen ACE)

- Figure 10 shows the results of ELISA with 5905 capture antibody and revelation with the antibody 5909 (absorbance obtained with a positive serum CEA antigen).

- Figure 11 shows the results of ELISA with 5905 capture antibody and revelation with the antibody 5909 (absorbance obtained with a negative serum antigen ACE) - Figure 12 shows the results of ELISA rigid plastic rods with 5910 capture antibody and revelation with the antibody 5909 (absorbance obtained with a positive serum antigen ACE)

These figures are illustrations of the sensitivity and specificity of the method used.

EXAMPLE 1; PREPARATION OF METAL GUIDE BASED Nitinol AND ACTIVATION

The surface of a metal guide based Nitinol (Euroflex) is structured in order to define locations such wells, where the reactive groups will be filed and where the biochemical interactions will occur (Figure 1).

Such "sink" can be achieved by various methods such as lithography by focused ion beam (FIB or "Focused Ion Beam". Xie et al, Nuclear Instruments and Methods in Physics research

Section B-Beam Interactions with Materials and Atoms,

211 (3): 363-368, 2003), by laser lithography followed by electrochemical etching and laser ablation.

With the FIB technique, the machine creates an ion beam, which is focused on the surface to be structured. Under the mechanical action of the ion beam, the atoms of the surface material are removed from the surface. Holes with a diameter of 20 microns can be formed with the FIB technique in a reasonable time with a bite factor s ^ 8 μπ^'1 under a beam current of 20 nA. Figure 2 shows the hole diameters of 5, 20 and 40 microns with a depth of 10 and 20 .mu.m. The surface of the hole bottom is rough due to the re-deposition of sputtered material during the attack. The etching factor was measured Min 200 nm^"1 on a circular area of ​​40 mm in diameter and with a 20 nA beam current. This gives an etching factor of 0.2 .mu.m³ nC^'1 (About 5 ^ s μπ^"1), Which corresponds to a procedure time of 20 minutes to perform a hole of 20 mm in diameter and 20 microns deep. To improve the surface roughness, a milling technique assisted by fluorine (XeF₂) Was used; a very low surface roughness was then obtained, but as the source of XeF₂ was not exactly in the axis of the beam etching, a shading effect was observed (Figure 3).

The laser lithography technique and electrochemical etching consists in a first step to cover the surface with a polymer layer. In a second step, the polymer layer is formed using laser ablation. In a third step, the surface is etched using an isotropic electrochemical etching through the opening made in the polymer layer (Figure 4). Figure 5 shows the results of various structuring tests on metal guides based Nitinol.

Moreover, the metal guides based Nitinol that are used in vivo are usually treated by electrochemical polishing, replacing the native oxide layer with a NiTi layer TiO₂ biocompatible. The guides fashioned with holes undergo this process in order to evaluate the influence of the process on the structure of the hole (Figure 6).

Another way to prepare cavities on the surfaces of Nitinol-based guides uses laser ablation. The use of short laser pulses allows local evaporation of the metal without affecting the metal surrounding due to the heat generated. Smaller dimensions are reported in the order of 20 microns.

If the three above described methods allow the production wells, it is the process by electrochemical etching which provides the best results.

EXAMPLE 2; TRAUMA TO INSERT CONSEQUENTIAL IVD VIVO OF METAL GUIDE FOR A PARTICULAR BODY

For these experiments, metal micro-guides (MTI 0.012 '' Silver speed) were used, which guide wires were inserted in micro-catheters.

The device was introduced in pigs under general anesthesia, at a puncture and then at the Scarpa to kidney through the endovascular route (via the femoral artery). This guidance was provided by monitoring said device in the femoral artery by arteriography.

Once positioned kidney input, the device was introduced in the kidney by breaking endoartérielle.

This penetration into the tissue is made to a depth of a few millimeters and said device has been maintained for about ten minutes.

Finally, the device was then removed.

Animals were then euthanized, and kidneys of these were taken to evaluate the condition thereof after penetration of the device according to the invention.

The results showed no significant bleeding kidney was associated with burglary. The most important lesion observed had a size of 3 x 1 mm at the break-site.

The device according to the invention thus allows access to an organ while being very slightly invasive.

EXAMPLE 3; MICRO-CONSEQUENTIAL INJURY TO INSERT THE DEVICE AT THE LIVER

Metal micro-guides (MTI 0.012 '' Silver speed) were used, which metal micro-guides were placed in a fiberoptic unlike Example 1.

The device was introduced in pigs under general anesthesia, at a puncture at the scarpa then to the liver by endoartérielle navigation (via the femoral artery). This guidance was provided by monitoring said device in the femoral artery by arteriography.

Once positioned adjacent to the liver, the device has been introduced therein. This penetration into the tissue is made to a depth of a few millimeters and said device has been maintained again for ten minutes.

Finally, the device was then removed.

As before, the removal of the liver after surgery allowed to judge the aggressiveness of the intervention on the organ.

No gross lesions were observed in the liver surface. When cut, the presence of two intra-parenchymal hemorrhagic foci of subcapsular seat 1,5x0,4 cm and 1,8x0,5 cm were observed. Histologically, the hepatic architecture is preserved at any point with a sinusoidal congestion, venules doors and central veins without any significant abnormality.

Conclusion The results showed that hemorrhagic lesions are minimal: two minor macroscopic lesions were observed without any destruction of parenchymal cells and with a simple capillary congestion and central veins.

The use of a guide wire thus produce minor trauma and, in all cases significantly lower than that resulting from a biopsy.

EXAMPLE 4; STUDY PARAMETERS FOR THE DESIGN AND REALIZATION OF A DEVICE FOR THE DETECTION AND immunocapture ANTIGEN ACE IN VITRO ON SOLID SUPPORTS

The device uses the principle of the ELISA technique to highlight the CEA antigen. Two monoclonal antibodies recognizing this antigen different epitopes were used to catch (MLF) and the revelation of the CEA antigen (ACM2). These monoclonal antibodies having the same isotype (IgG), the revelation of the antigen CEA was performed using a monoclonal antibody coupled to biotin and a complex streptavidne peroxidase (Figure 7).

Two types of media were used, either ELISA plates or rigid plastics rods.

ELISA plates (Greiger)

100 .mu.l of a monoclonal antibody directed against the antigen ACE (5910 or 5905 clone clone, produced in mice and marketed by Medix Biochemical) diluted (1/5000 to 1/128000) in carbonate / bicarbonate buffer were deposited per well, and the plate was placed for 1 hour at 37 ° C. A negative control was carried out by replacing the antibody by the carbonate / bicarbonate buffer.

After three washes with 250 .mu.l per well of PBS the free sites of the plate were saturated with 200 ml of PBS-BSA (bovine serum albumin) 3% for 2 hours at 37 ° C.

The wells were then washed three times with 250 .mu.l of PBS-Tween 0.5% before the addition of 100 .mu.l per well of a positive serum CEA antigen diluted to 1/10, 1/100, 1/1000 in PBS-Tween, and the plate was incubated for 1 hour at

37 ° C.

Three washes of 250 .mu.l per well were carried out in PBS-Tween prior to the addition of 100 .mu.l per well of a monoclonal antibody directed against the antigen ACE (5909 clone produced in mice and marketed by Medix Biochemical, which differs from previous capture antibody used by its affinity constant and by recognized epitopes) biotinylated at 1/500 in PBS-Tween, and the plate was again incubated for 1 hour at 37 ° C.

After three washes with PBS-Tween, 100 .mu.l of streptavidin coupled to peroxidase diluted 1/2000 were added to each well and incubated for 1 hour at 37 ° C.

After three washes with PBS-Tween, revelation was done by addition of 200 .mu.l per well of substrate mixture (H₂O₂) And chromogen (OPD, Sigma) in citrate-phosphate buffer (pH 5).

In parallel, the same operation was carried out using as antigen, patient serum "normal" (negative control with an ACE assay <5 IU / ml). The reaction was then stopped by adding 50 .mu.l of IM sulfuric acid per well. Absorbance was read at 492 nm on a plate reader (ref: ELX.800 UV).

The results obtained using the monoclonal antibody 5910 for the iπununocapture (diluted 1/500 in half and half to 1/128000) and revelation by biotinylated monoclonal antibody 5909 are shown in Figure 8 for the positive serum CEA antigen, and in Figure 9 for the negative serum ACE.

The results obtained using the monoclonal antibody 5905 for immunocapture (diluted 1/100, 1/200, 1/500 and half in half to 1/32000) and revelation by monoclonal antibody biotinylated 5909 are shown in Figure 10 for the positive serum CEA antigen, and in Figure 11 for the negative serum ACE.

Legends of Figures 8-11:

Northing: absorbance (OD) at 492 nm

X-axis: dilutions of the capture antibody (5910 or 5905)

^ = Dilution positive serum ACE 1/10

M = ACE positive serum dilution 1/100

Δ = positive serum dilution in ACE 1/1000

x = no serum

The results show that the positive serum antigen ACE 1/10 gives an absorbance (OD) greater than 0.5 when the monoclonal capture antibody is used at 1/500 (Figure 8). Under the same conditions, serum negative antigen ACE gives an OD less than 0.15 (Figure 9).

However, it is noted that better results were obtained with the monoclonal antibody pair 5905 of capture and detection monoclonal antibody 5909 (Figures 10 and 11) with monoclonal antibody torque catch 5910 and 5909 monoclonal antibody detection ( figures 8 and 9). Indeed, an OD of 1 was observed with positive serum diluted in ACE antigen 1/10 (Figure 10) while the negative serum CEA antigen under the same conditions gives an OD of 0.1 (Figure 11). These results were confirmed using the 5910 monoclonal capture antibody at different dilutions (data not shown).

Rigid plastic supports

In a first step, rigid plastics materials in the form of rods of 2 to 3 cm in length and 0.5 to 1 mm in diameter have been activated.

In a second step, the thus activated substrates were placed into microtubes hemolysis 1 ml (Fisher) and were functionalized with a monoclonal antibody directed against the antigen ACE (5910 clone produced in mice and marketed by Medix Biochemical) and diluted 1/50, 1/100, 1/250, 1/500 in carbonate / bicarbonate buffer (250 .mu.l / tube) for 1 hour at 37⁰C. A negative control was achieved by replacing the monoclonal antibody by the carbonate / bicarbonate buffer. After fixation and washing, the saturation was carried out with 500 .mu.l of PBS-3% BSA overnight at 4 ° C.

The supports were then incubated with 250 ul of positive serum CEA antigen diluted to 1/10, 1/100 in PBS or with the serum of a "healthy" subject (negative control antigen ACE) at the same dilution for 1 hour at 37 ° C.

A monoclonal antibody directed against CEA antigen antibody (clone 5909 produced in mice and marketed by Medix Biochemical, which differs from the 5910 clone by its affinity constant and by recognized epitopes) purified, 1 mg / ml was dialyzed overnight at 4 ° C against 0.1 M borate buffer pH 8.8. Biotin solution at 10 mg / ml in DMSO was then added at 50 ug / mg antibody. After incubation for 4 hours at room temperature and under stirring, IM ammonium chloride was added at a rate of 20 .mu.l / 250 mcg of biotin, and the resulting solution was again incubated for 1 minute at room temperature. After blocking of the reaction, the labeled antibody was dialysed for 24 hours at + 4 ° C against PBS, and this labeled antibody was stored in aliquots at -20⁰C.

After 3 washes in PBS-Tween, the supports were incubated with 250 .mu.l of biotinylated antibody 5909 and diluted 1/500 in PBS-Tween for 1 hour at 37 ° C.

The detection of biotin (ester 6- biotinamidocaproylamido caproic and N-hydroxysuccinimide, Sigma) was demonstrated using a streptavidin-peroxidase complex (Amersham Biosciences) diluted 1/2000 in PBS for 1 hour at 37 ° C.

The revelation of the enzymatic activity was achieved by adding 750 .mu.l per tube substrate mixture (H₂O₂) And chromogen (OPD, Sigma) in citrate-phosphate buffer (pH 5).

The reaction was then stopped by adding sulfuric acid to IM. Absorbance was read at 492 nm. The results of ELISA on plastic rods are shown in Figure 12.

Legends Figure 12:

Northing: absorbance (OD) at 492 nm

Easting: 5910 dilutions of the capture antibody

^ = Dilution ACE antigen 1/10

M = dilution ACE antigen 1/100

Δ = negative control dilution 1/10

x = dilution negative control 1/100

In general, the results show that the OD are 7 to 10 times higher for the positive serum CEA antigen than those obtained with the negative serum CEA antigen.

The best results were obtained with the rigid plastic supports on which are fixed the capture 5910 monoclonal antibody diluted to 1/50 or 1/100.

The ACE antigen concentration have been better detected, corresponding patient serum diluted 1/100 is 6 IU / ml (close to the rate considered "normal" <5 IU / ml) and when the dilution of antibody monoclonal

detection 5909 is 1/500.

The use of plastic substrates used to validate the specificity and sensitivity of the process immunocapture on metal rod functionalized according to the protocol described above.

Conclusion The good results obtained for detection of the CEA antigen with the techniques of in vitro and iπununocapture revelation, validation of the evaluation devices

"Functionalized rods" allowing the in vivo capture of CEA antigen followed by ex vivo revelation.

EXAMPLE 5; IDENTIFICATION OF EXPRESSION IN TUMOR MARKER ACE BREAST FOR EXAMPLE UNDER CONTROL OF IMAGING TECHNIQUES, INCLUDING RADIOLOGICAL

According to the protocol described in PCT application WO 03/006948, in a first step, an alkanethiol layer is adsorbed on one end of guidewires made from nitinol (Euroflex) in a first step. In a second step, the free thiol functions of this layer allow disulfide bridge formation with a monoclonal antibody directed against CEA antigen.

The resulting guide wire is then inserted in a biopsy needle adapted for use in animals or humans.

A frozen section pathological examination is performed using this device on a specimen (breast tumor), after its withdrawal in a patient with breast cancer. Alternatively, when medical ethical conditions are met, a micro-incision in the breast is performed under local or general anesthesia in a patient with breast cancer. The needle, in which is inserted the guide wire coupled to the antibody directed against the CEA antigen, is introduced into the tumor or by micro-incision and directed to the tumor following its progress by imaging, and in particular ultrasound. Said micro-invasive guidance system is then used to pull out the end of the guide wire coupled to the antibody directed against the antigen ACE. The end of the guide wire is then introduced into the tumor (perforation) to a depth of about a few millimeters. After a low latency of the order of ten minutes, allowing immunocapture ACE antigen possibly expressed by the tumor, the device is removed.

The micro-sampling is limited to one immunocapture in vivo analyte and does not require a biopsy.

Finally, the device is removed and an ELISA assay ACE marker is formed on the end of the device with a monoclonal antibody directed against CEA antigen which is different from the capture antibody by its affinity constant-vis screw CEA antigen and epitopes recognized by, and coupled to biotin.

The revelation of enzymatic activity using a streptavidin-peroxidase complex to conclude with the expression of ACE marker by the tumor and to adjust accordingly the therapy to be used to best treat the patient.

EXAMPLE 6; SKIN CANCER

According to the protocol described in PCT application WO 03/006948, an alkanethiol layer was adsorbed on the end of a guidewire-based Nitinol

(Euroflex) in a first step. In a second step, the free thiol functions of this layer enabled disulfide bond formation with a monoclonal antibody directed against the FAP marker (fibroblast-activating protein; RETTIG et al, Proc Natl Acad Sci USA, Vol..... .85, p: 3110, 1988).

The resultant guide wire is then introduced at a skin tumor in animals or humans when medical ethical conditions are met, or at the level of a skin tumor in a patient with cancer skin after its removal to a classic or frozen section pathological examination.

The micro-sampling is limited to an in vivo immuno again and requires no specific biopsy.

Finally, the device is removed and an ELISA assay PAF marker is formed on the end of the device with a monoclonal antibody directed against the FAP marker coupled to peroxidase.

The revelation of the peroxidase activity will conclude with the expression of FAP by tumor marker and to adjust accordingly the therapy to be used to best treat the patient.