IN VIVO MICRO-INVASIVE INVESTIGATION DEVICE INCLUDING A METAL
GUIDE
DESCRIPTION
Technical field
The present invention concerns the functionalization of a
transparietal investigation device, a method of ex vivo
analysis of a substrate using a functionalized device of the
invention, and the use of such a device for the fabrication of
a tool for the diagnosis of a cancer, an infection, an
inflammation, a neurodegenerative disease or graft rejection
in a patient.
State of the art
Investigation or in vivo treatment device are known of the
state of the art. Such devices take the form of a rigid tube
of endoscope type or of a catheter constituted of a flexible
tube which is inserted in the organism, especially by natural
pathways or the vessels, and which allows to reach an organ or
a specific tissue. These devices allow especially the
elimination of blood clot or, when they are associated to
optical fibres, the visualisation and the in vivo control of
the state of a system, as the digestive tube, or of an organ,
as the colon. The traumatism for the patient resulting from
the use of such devices is then minimized but still stay to
ameliorate. However, it is not always possible to carry out an
analysis of the organs or the tissues in a patient with these
devices. Such impossibility may result from the reduced
accessibility of said tissue or organ compared to the blood
circulation or the natural pathways, or from the difficulty to
carry out a reliable diagnostic without having recourse to a
fine study of the cells of said organ or tissue. In these
cases, it is generally used in the state of the art and by

now, the sampling of a fragment of said tissue or organ
(biopsies) in order to ex vivo control the morphology of these
tissues or organs, or more finely the one of their
constitutive cells, especially by mean of 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 ; Perez-Guillermo et al., Diagn. Cytopathol., 32 :
315-320, 2005 ; Fernandez-Esparrach et al., Arch.
Bronconeomol., 43 : 219-224, 2007). Moreover, it is also
possible to analyse the expression status of a certain number
of markers, the expression of which is correlated with a
specific pathologic state (especially cancer, inflammation,
infection, neurodegenerative disease or graft rejection).
However, these different methods, because of the sampling
of a biopsy or of the aspiration of cells, involve a
traumatism, sometimes important, to said tissue or organ and,
consequently, to the patient. The organism of the latter may
so be sorely afflicted because of haemorrhages or of the
cicatrisation consecutively to the sampling, especially for
certain organs (brain, pancreas, liver or lung).
It so exists still now a need for the identification of
new investigation methodologies allowing to carry out a
reliable and accurate diagnostic, especially compared to the
expression of markers specifically associated to different
pathologies such as cancer, inflammation, infection or
neurodegenerative disease, while limiting the traumatisms
inflicted to the patient.
The patent EP 1 358 481 describes an device of analysis or
of in vivo treatment comprising (i) a micro-system of
investigation of a substrate other than by analysis of a
fluorescent signal, (ii) a flexible butt to an extremity of
which is fixed said micro-system and the other extremity of
which is intended to the control of said micro-system, (iii) a

medical instrument having an internal opening in which said
flexible butt may slide, (iv) a sliding system of protection
of the micro-system that is removable on the level with the
substrate, and (v) on the level of the said micro-system, a
system of dilacerations of tissue or cell, eventually
associated with one or several devices chosen among a device
for monitoring by remote control by means of sensorial
receptors (tactile, optical, physico-chemical or numerized
informatic), for carrying out biopsies, for treatment, for
local injection of biological or chemical products.
In order to allow the effraction of blood vessels and the
dilacerations of tissues or cells, the micro-system is so
associated with another more rigid system ensuring this
function, preferably in distal position.
However, in the case of an in vivo investigation device,
the investigation device is the more often directed to the
target organ or tissue by using the endovascular or
endocavitary pathway. The coupling of the micro-system with a
dilaceration system then increases in a non-negligible manner
the diameter at the extremity of the described device. The
latter then reveal itself to be of a too complex use to be
correctly directed to its target site without altering
simultaneously the blood circulation pathways. Simultaneously,
the coupling of multiple elements do harm to the general
flexibility of the device, and so to its correct guidance, as
to the obtention of the rigidity, necessary to allow the
perforation of an organ or a tissue.
Description of the invention
Following important researches, the inventor has now
managed to develop an device comprising a metallic guide to a
perforating extremity of which are directly coupled reactive
groups, especially antibodies or antibody fragments, specific

of a substrate to be tested, and the other extremity of which
is intended to the manoeuvring of said guide from the
insertion site to the site of micro-analysis and/or micro-
sampling of said substrate. Said guide may be inserted into a
removable protection system, for example a flexible catheter,
then allowing to protect the functionalised extremity of said
guide to the site of micro-analysis and/or micro-sampling of
the substrate to be tested constitutive of the tissue, the
organ or the cells of these ones.
The invention is to structure the surface of the guide in
order to define some spots on the guide, for example wells,
where the reactive groups will be deposited and where the
biochemical interactions will occur. Said "wells" may be
carried out by different methods such as by lithography by
Focused Ion Beam (FIB or for « 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 an
electrochemical attack and a laser.
Alternatively, the structuration of the metallic guide may
be carried out in order to define some spots on said guide,
for example at least one furrow in which is jointly associated
at least one miniaturized biochip, linear, circular or in the
form of a ribbon, where the reactive groups will be deposited
and where the biochemical reactions will occur. The spots
formed by structuration of the surface of the guide, for
example wells, do not form relief at the surface of the guide,
but some furrows, or hollows. The spots formed by the
structuration of the surface of the guide so do not increase
the diameter of the guide.
Advantageously, the spots formed by the structuration of
the surface of the guide are situated at the surface of the
guide, which means that these spots are directly in contact
with the fluids, the tissues and the cells.

Moreover, said device may be inserted in a medical
instrument having an internal opening in which said metallic
guide may slide, and especially in a transparietal needle
punction, especially transcutaneous or transmucosal, or an
endoscope, including an endovascular system of navigation.
Moreover, said device may be associated with an optical
fibre, or the metallic guide of said device may be replaced by
an optical fibre, a perforating extremity of which is
associated to a metallic ring on which are directly coupled
reactive groups, especially antibodies or antibody fragments,
specific of a substrate to be tested, so allowing a fine in
situ visualization, in view of the in vivo capture of the
elements necessary to the diagnostic and eventually to the
prognosis evaluation. The properties of the optical fibre are
used for purposes of marking imagery and of setting of the
device from the in situ visualization.
The device according to the invention, because of its
simplification presents, while staying micro-invasive, a
flexibility or elasticity improved compared to the devices of
the prior art. The latter then may be used much more
efficiently for an investigation by endocavitary pathway,
especially by endovascular pathway, or by transparietal
pathway, especially by transcutaneous or transmucosal pathway.
Moreover, the device according to the invention, because of
the use of a metallic guide, an extremity of which is coupled
to specific reagents, is rigid enough at its functionalized
extremity to efficiently perforate a tissue or an organ.
Finally, the diameter of the device according to the invention
at its extremity is weak enough to allow the simplified
navigation in the blood circulation or in the natural
cavities, and particularly to minimize the traumatism at the
level of the tissue or the organ that the latter must
perforate.

Moreover, and after the withdrawal of the device, it is so
possible to ex vivo identify by means of standard methods, as
an immunoenzymatic dosage or by immunofluorescence, for
example on a solid support [ELISA technique, protein chips
(ESPINA et al., J. Immunol. Methods, vol.290, p :121-133,
2004)], the presence and the relative concentration of said
specific substrate (s) at the extremity of the device according
to the invention and so at the level of the site of micro-
analysis ad/or micro-sampling (organ or tissue).
Lastly, and because of the nature of the metallic guide,
the device presents an improved imagery signal (arteriography,
echography, scan, MRI, etc..) . This property allows to
considerably simplify the radioguidance of the device
according to the invention at the time of the intervention to
the target tissue or organ. It is the same thing for the
coupling of said device with an optical fibre for the purpose
of marking imagery, which allows the fine in situ
visualization.
Consequently, a first object of the invention is a device
for the analysis of a substrate, characterized in that it
comprises a micro-invasive system of investigation and/or of
micro-sampling of said substrate, said system being
constituted of at least one metallic guide to an extremity Ea
of which is provided with at least one series of wells to
which is directly coupled at least one reactive group specific
of said substrate, said functionalized extremity Ea being
perforating, and the other extremity Em of which is intended
to the manoeuvring of said metallic guide and eventually
associated with an aspiration system.
Advantageously, the surface of the extremity Ea may be
structured so as to define at least one series of wells to
which is directly coupled at least one reactive group specific
of said substrate, said extremity Ea being perforating.

The device according to the invention, because of the weak
traumatism that it causes to the patient, allows to carry out
several micro-analyses or micro-samplings to a patient at
regular intervals (for example, analyses and/or samplings
carried out in stages within the prostate). Said analyses
and/or successive samplings so allow, in addition to the
diagnosis, to follow the evolution of a cancer, an
inflammation, an infection, a neurodegenerative disease or the
good graft retention of an organ in a patient.
Advantageously, the functionalized extremity Ea may
present on a length of about 0.5 to 2 cm, at least a series of
1 to 25 wells, preferably 2x25 wells, having a mean diameter
of about 30 to 80 µm, preferably of about 40 to 60 µm, and
with a very preferred diameter of about 50 µm, a depth of
about 20 to 30 µm, preferably of 25 µm, said wells being spaced
the ones of the others of about 60 to 120 µm. preferably, the
wells have a sleek or rough wall, an ovale or round shape,
with a flat or concave bottom.
Advantageously, the functionalized extremity may present a
number of wells as elevated as possible. The number of wells
may be such as the metallic guide is rigid enough for
perforating the tissues or organs. In this embodiment, the
mean diameter of the functionalized extremity may be of about
0,3 to 3,5 mm, preferably of about 0,35 mm. Advantageously,
the metallic guide may be structured on a length of 0,5 to 2
cm, preferably of 1 cm, to form wells. Advantageously, the
diameter of wells may be of about 30 to 80 µm, preferably of
about 30 to 60 µm, for example 35 µm. Advantageously, the depth
of the wells may be of about 20 to 30 fim, preferably of about
25 µm. The spacing of the wells may be of 20 to 120 µm,
preferably 25 µm.
Advantageously, the functionalized end Ea may be in a
cylindrical, plane, or spiral form or in a form modified so as

to increase the well surface in contact with the fluids, the
tissues and the cells.
According to a particular embodiment of the device
according to the invention, said device further comprises a
removable protective system at the functionalized extremity
Ea.
According to a particular embodiment of the device
according to the invention, said device further comprises a
medical instrument possessing an internal opening in which
said at least one metallic guide may slide.
By "metallic guide", it is meant for example a metallic
butt full supple or a hollow rigid metallic butt, having a
diameter from 0,2 to 3,5 mm and a length from 5x10-2 to 2 m,
and that may be inserted in a blood vessel, a little cavity or
through an organ or a tissue, making it possible to be
directed from the insertion site to the site of micro-analysis
and/or micro-sampling in situ.
Particularly, a full flexible metallic butt may be
constituted of an optical fibre, to an extremity Ea of which
is associated a metallic ring supplied with at least one
series of wells to which is directly coupled at least one
reactive group specific of said substrate, said functionalized
extremity Ea being perforating.
Advantageously, said metallic ring has a width of about
0,5 to 2 cm, and may presents at least a series of 1 to 25
wells, preferably 2x25 wells, having a mean diameter of about
30 to 80 urn, preferably of about 30 or 40 to 60 µm, and much
preferably of about 50 µm or 35 µm, a depth of about 20 to
30 µm, preferably of 25 µm, said wells being spaced the ones
of the others of about 20 to 120 µm , for example of about 60
to 120 µm. Preferably, the wells have a sleek or rough wall,
an ovale or round shape, with a flat or concave bottom.
By "metallic guide", it is further meant a full or hollow,
rigid or flexible butt, constituted of all or part of a

metallic alloy, the features of flexibility, rigidity,
oxidation and immunogenicity of which are consistent with such
a use in the living being and particularly in the animal, and
much particularly in human. Such biocompatible alloys may
simply be identified by the man skilled in the art by means of
his general knowledge and comprise notably the inoxidizable
steels, the titanium-based alloys, the nickel, the cobalt, or
mixtures of these ones.
The inventor have managed to demonstrate that a titanium
and nickel alloy-based guide (Nitinol alloy) presents some
particularly interesting properties in terms of general
flexibility and rigidity at its extremity to be efficiently
used in the endovascular or endocavitary pathways and also to
efficiently perforate a tissue or an organ while minimizing
the traumatism (the size of the perforation at the level of
said tissue or organ is for example of the order of 0,05 to
0,5 mm2, and preferably pf the order of 0,07 mm2) .
Advantageously, the metallic guide is a titanium and
nickel alloy-based guide, preferably Nitinol-based (metallic
guide sold by the Euroglex society).
Said metallic guide may be covered, except at the
functionalized extremity Ea, with a hydrophilic polymer,
preferably a hydrogel, or with a porous protective polymer
layer, having a depth from about 0.1 to 51 µm. Advantageously,
the protective polymer is constituted of a film of parylene,
of TiO2 or of OptoDex® (Arrayon Biotechnology, Switzerland),
and more preferably of a film of parylene.
The removable protective system in which the metallic
guide is inserted may take multiple forms, and notably the one
of a flexible catheter, that may be determined simply by the
man skilled in the art, for examples forms usable for the
endovascular, endocavitary, transparietal, and notably
transcutaneous pathway.
Said removable protective system may be inserted by

endovascular pathway notably to reach the vessels of the
heart, brain, lung, pancreas, kidney and liver.
Said removable protective system may be inserted by
endocavitary accesses, notably by mean of an endoscope, by
oral, anal, urogenital and respiratory pathway, or ENT by
transmucosal pathway, or also by transcutaneous pathway by
mean of a puncture at the level of the skin, to reach for
example the mammary gland, and notably to the kidney, in an
articulation, in the vertebral canal, by lumbar puncture or
transparietally in the liver, the lung or the kidney, but also
by transmucosal pathway, notably for the digestive tract.
The inventor has evidenced that the device according to
the invention then allows to reach the tissues or organs,
usually difficult to reach, by the endovascular, endocavitary
or transparietal pathways, and notably transmucosal ou
transcutaneous, usually used.
More generally, the inventor has evidenced that the device
according to the invention allows, because of its specific
features of general flexibility and of rigidity at its
extremity, to reach and to perforate by transparietal
(transcutaneous, transmucosal), endovascular or endocavitary
pathway some organs and tissues being part of the digestive
system from the oropharynx to the rectum (comprising the liver
and the pancreas), of the urogenital system (comprising
bladder, kidney, prostate, testis, ovary and mammary gland),
of the tracheobronchial system (comprising lung), of the ENT
system (comprising ear and rhinopharynx), of the osteo-
articulary (comprising synovial cavities), of the endocrine
system, of neurocerebral system or the tegumentary system,
then making possible the realization of diagnostic on
pathologies that necessitated since then the realization of
biopsies, nay, deep aggressive punctures as the transhepatic
biopsies, comprising the sampling of cells by fine needle
aspiration (also named FNA).

Advantageously, said removable protection system into
which the metal guide is inserted is in the form of a flexible
catheter adapted for endovascular or endocavitary delivery.
The device according to the invention is then particularly
adapted for carrying out an investigation, for example in
arteries and veins, heart vessels, the prostate, the mammary
glands, the pancreas, the kidneys, the heart muscle, the
central nervous system and the cavities or canals thereof, the
brain or the liver.
According to a first particular embodiment of the device
according to the invention, the removable protection system
into which the metal guide is inserted is itself inserted into
an endoscope. The device according to the invention is then
particularly adapted for an endocavitary delivery.
The device according to the invention is then particularly
adapted for carrying out an investigation for example, on the
tracheo-bronchial system (among which the lungs), the
digestive system from the pharynx to the rectum (including the
liver and the pancreas), the uro-genital system (among which
the bladder, the kidneys, the prostate, the testicles, the
ovaries and the mammary glands), the ophthalmic system
(lachrymal canals), of the otho-rhino-laringologic system
(among which the ear and the nasopharynx) , the osteo-articular
system, or the central nervous system, more particularly
through endo-spinal delivery or the mammary gland through
endogalactophoric delivery.
According to a second particular embodiment of the device
according to the invention, the metal guide composed of a fine
transparietal aspiration needle and more particularly
transcutaneous or transmucosal aspiration needle may be
inserted into a removable protection system, for example a
flexible catheter. The device according to the invention is
then particularly adapted for a specific transcutaneous or
transmucosal delivery.

The device according to the invention is then particularly
adapted for carrying out an investigation, for example, as
regards the teguments (the skin, the scalp, etc.)/ the breast,
the kidneys, the lungs, the liver, the muscle, the osteo-
muscular or osteo-articular system, the central or the
peripheral nervous system or the endocrine glands (more
particularly the thyroid, the parathyroid, the adrenal glands,
the testicles, the mammary glands or the ovaries.
As regards the functionalized extremity Ea of the device
according to the invention, it is provided with at least a
series of wells to which are directly coupled reactive groups
specific to a substrate to be tested.
"Specific reactive groups" means, for example, a nucleic
acid sequence [DNA (amplifiat, fragment of a gene, EST, SNP)
or NRA] complementary to a nucleic acid sequence to be
detected, and an antigen specific to an antibody to be
detected or an antibody or a fragment of antibody specific to
an antigen to be detected, preferably an antibody or a
fragment of antibody.
Advantageously, said specific reactive groups are
positioned in the micro-wells of the functionalized extremity
of the device according to the invention, according to an
increasing or decreasing range. The man skilled in the art
can determine simply, using their general knowledge and
routine experiences, said range as a function of the affinity
of the reactive group for the substrate. For example, the
range of the reactive group is of the order of 50 to 500ug/ml,
preferably to 10 to 100µg/ml, for a reagent, more particularly
an antibody having an affinity for its substrate, more
particularly an antigen of the order of 10-9.
"Antibody" preferably means an immunoglobulin of a living
organism, a mammal, more particularly a human being and more
preferably an IgG.

"Fragments of antibodies" means fragments of antibodies
capable of maintaining a specific fixation of their antigens.
The fragments Fab, Fab', F(ab')2 or Fv can be cited as examples
of such fragments of antibodies.
Methods for coupling reagents, more particularly proteins
once on a metal support are well known to the man skilled in
the art. Such methods, because of the low chemical reactivity
of amino acids, generally require the activation of the metal
surface either through oxidation mechanisms or by covering the
latter with at least one layer of link molecules, most often
of polymers (the latter may have for example thiol groups,
carboxylic acids, and/or amines). As examples of such
methods, the adsorption on metal supports of functional
molecules organized in self-assembled monolayers (SAMs) and
more particularly alcanethiols (refer to WITTSTOCK and
SCHUHMANN, Anal. Chem., vol.69, p: 5059-5066, 1997; and the
international application WO 03/006948) or pyrrole
(electrochemical polymerization of biotinylated pyrrole;
DUPONT-FILLIARD and al., Anal. Chim. Acta., vol.449, p :45-50,
2001) can be mentioned.
Coupling antibodies or a fragment of the antibody to this
layer of the functional molecules means a covalent link (like
disulfide bridges between the free thiols groups of the
alcanethiols) or non covalent link (like the streptavidine-
biotin link between the biotin of a layer of polymer of the
pyrrole/biotin complex and streptavidine of a
streptavidine/antibody complex or antibody/streptavidine
fragment) .
According to a first preferred embodiment, the device
according to the invention includes at least a metal guide, an
Ea extremity of which is coupled to at least one reactive
group preferably an antibody or a fragment of antibodies
specific to a marker (antigen) of cancer, more particularly
breast cancer, ovary cancer, prostatic cancer, cancer of the

colon, intra-abdominal cancer, kidney cancer, liver cancer,
lung cancer, pancreatic cancer, cancer of the central or
peripheral nervous system or an endocrine gland (more
particularly thyroid, the testicles or the ovaries).
As an example of a marker of breast cancer, marker CA 15-3
can be mentioned (Carcinoma-Associated Antigen 15-3; Duffy
M.J., Shering S., Sherry F., McDermott E., O'Higgins N., Int.
J. Biol. Markers, 2000 Oct.-Dec.;15(4): 330-3), CA 27-29
(Carcinoma-Associated Antigen 27-29; Kaohsiung J., J. Med.
Sci., 1999 Sept.; 15(9): 520-8), CEA (Carcinoembryonic
antigen; Soletormos G., Nielsen D., Schioler V., Mouridsen H.,
Dombernowsky P., Eur. J. Cancer, 2004 Mar.;40 (4): 481-6); TPA
(Tissue Polypeptide Antigen); TPS (Tissue Polypeptide Specific
Antigen; Given M. , Scott M. , Mc Grath J.P., Given H.F.,
Breast, 2000 Oct.; 9 (5):277-80), HER2 (Fehm T., Jager W.,
Kramer S., Sohn C, Solomayer E., Wallwiener D., Gebauer G.,
Anticancer Research, 2004 May-Jun; 24 (3b): 1987-92), ER
(Estrogene Receptor; Platet N., Cathiard A.M., Gleizes M.,
Garcia M., Crit. Rev. Oncol. Hematol., 2004 Jul.; 51(1): 55-
67), PR (Progesterone Receptor; Duffy M.J., Clin. Chem., 2005
Mar.;51(3): 494-503. Epub 2005 Jan. 6.), Ki-67 (cell
proliferation-associated antigen of antibody Ki-67; Schliiter
C, Duchrow M., Wohlenberg C, Becker M.H., Key G., Flad H.D.,
Gerdes J., J. Cell. Biol., 1993 Nov.; 123(3): 513-22) and UPA
(Urokinase Plasmogen Activator; Duffy M.J., Crit. Rev. Clin.
Lab. Sci., 2001 Jun.;38(3): 225-62).
As an example of a marker of the ovary cancer, marker
CA125 (Carcinom Antigen 125; Moss E.L., Hollingworth J.,
Reynolds T.M., J. Clin. Pathol., 2005 Mar.; 58(3): 308-12), CA
15-3 and CEA (Valenzuela P., Mateos S., Tello E., Lopez-Bueno
M.J., Garrido N., Gaspar M.J., Eur. J. Gyn. Oncol., 2003;
24(1): 60-2) can be mentioned.
As an example of a marker of the prostatic cancer, marker
PSA (Prostate-Specific Antigen; Gray M.A., Clin. Lab.,2005;

51(3-4) :127-33) ; PMSA (Prostate-Specific Membrane Antigen) and
AR (Androgen Receptor; Birtle A.J., Freeman A., Masters J.R.,
Payne H.A., Harland S.J., BJU Int., 2005 Aug.; 96(3): 303-7)
can be mentioned.
As an example of a marker of the colon cancer, marker CEA
(Duffy M.J., Clin. Chem., 2001 Apr.; 47(4): 624-30), CA 19-9
(Carcinom Antigen 19-9), CA242 (Carcinom Antigen 242), CA 72-4
(Carcinom Antigen 72-4), TPA, TPS (Duffy M.J., van Dalen A.,
Haglund C, Hansson L. , Klapdor R. , Lamerz R. , Nilsson 0.,
Sturgeon C, Topolcan 0., Eur. J. Cancer, 2003 Apr.; 39 (6):
718-27) can be mentioned.
As an example of a marker of intra-abdominal cancer,
marker CEA or CA 19-9 (Coban E., Samur M., Bozcuk H., Ozdogan
M., Int. J. Biol. Markers, 2003 Jul-Sep; 18(3): 177-81) can be
mentioned.
As an example of a marker of the cancer of pancreas, the
marker TA90-IC (a 90-kDa immugenic Tumor-associated Antigen),
CA-19-9 (Chung M.H., Gupta R.K., Bilchik AJ, Ye W, Yee R.,
Morton D.L., Curr. Surg.,2002 March-April;59(2): 194-198),
TPS, HCG beta (hCG beta , Human Chorionic Gonadotropin beta),
CA 72-4, CEA, CA 19-9, CA 242 (Louhimo J., Alfthan H., Stenman
U.H., Haglund C, Oncology, 2004; 66(2): 126-31) can be
mentioned.
As an example of a marker of the liver cancer, the alpha-
fetoprotein marker can be mentioned.
As an example of a marker of lung cancer, the marker Cyfra
A41 (Cytokeratin fragment 41) , SCC (Squamous Cell Carcinoma
antigen), ACE (Angiotensin Converting Enzyme), CA 19-9, CA
125, NSE (Neuron Specific Enolase), chromogranine A, CYFRA 21-
1 (Cytokeratin fragment 21-1,) CA 15-3 can be mentioned.
According to a second preferred embodiment, the device
according to the invention includes at least one metal guide
whose an Ea extremity is coupled to at least one reagent,
preferably an antibody or a fragment of an antibody, specific

to a specific marker of an inflammation, more particularly
rheumatoid arthritis.
As an example of a marker of rheumatoid arthritis, IL-1β,
IL-IRα, IL-2, IL-2R, IL-4, IL-5, IL-6, IL7, IL8, IL10,
IL12p40P70, IL-13, IL-15, IL-17, le TNFα, IFNα, IFNγ, le GM-
CSF, le MIP-1, IP-10, the MIG, Eotaxine, the RANTES and the
MCP-1 (COCKRUM and al., Lab Automation, BTi, October 2005,
p: 19-21) can be more particularly mentioned.
According to a third preferred embodiment, the device
according to the invention comprises at least one metal guide,
an Ea extremity of which is coupled to at least one reagent
preferably an antibody or a fragment of an antibody,
preferably specific to a specific marker of an infection, more
particularly a viral, bacterial or parasitic infection.
Many infectious markers are known to the man skilled in
the art and this can identify very easily the specific marker
or markers associated to a given infection.
According to a fourth preferred embodiment, the device
according to the invention includes at least one metal guide,
an Ea extremity of which is coupled to at least a reagent,
preferably an antibody or a fragment of an antibody, specific
to a specific marker of the graft rejection.
Numerous markers of a graft rejection are known to the man
skilled in the art. As an example, MIP-1β and the VE-
cadherine for heart transplantations (ROUSSOULIERES and al. ,
Circulation, vol.111 (20), p: 2636-2644, 2005) can be
mentioned.
As regards the specific markers which have been described
above, the man skilled in the art will be able to use their
general knowledge and to easily identify, without any
excessive experiment, the antibodies or specific fragments of
antibodies which can be used in the device according to the
invention. As an example of such antibodies, the antibodies
available at TEBU or AXXORA can be mentioned. The man skilled

in the art can also obtain such antibodies using well-known
immunization methods.
Similarly, the man skilled in the art will be able to
identify adapted specific nucleic acids which can be used in
the device according to the invention.
According to a fifth preferred embodiment, the device
according to the invention includes at least one metal guide,
an Ea extremity of which is coupled to at least one reagent,
preferably an antibody or a fragment of an antibody, which is
specific to a specific marker or a set of specific markers of
neurodegenerative pathologies, such as for example Alzheimer's
disease (MA), Parkinson's syndrome, amyotrophic lateral
sclerosis (SLA), with this list not being exhaustive.
Several markers are known and used by the man skilled in
the art for studying such pathologies. As an example for
insane or pre-insane conditions, total protein Tau (MAPT-
Microtubule Associated Protein Tau), amyloid peptide ABETA1-
42, hyperphosphorylated protein Tau (p. Tau phosphorylated in
128), described for example by Waldemar G., Dubois B., Emre M.
and al, Eur. J. Neurol., 2007, 14, pp 1-26; Dubois B.,
Feldmann H.H., Jacova C, Dekosky S.T. and al, Lancet Neurol.
2007, 6, pp 734-746; Krolak-Salmon P. and al. "Vers un
diagnostique biologique de la maladie d'Alzheimer et des
syndromes apparentes", La Revue de medecine interne (2008),
doi:10.1016/J. revmed. 2008.01.029. These markers can be
determined by a technique Antigen-Antibody of the
ELISA.INNOTEST B-Amyloïd type (1-42), INNOTEST hTAUAg,
INNOTEST PHOSPHOTAU (181p); Innogenetics, Ghent, Belgium.
Other markers also make it possible to test the
deteriorated condition of the brain and more particularly
Visinin-like protein (VLP6 or VILIP-1 or VSNL), as described
for example by Ref: Lee J.M. et al, Clin. Chem., 2008, 54, pp

1617-1623. These markers can be used while implementing the
present invention.
As regards Parkinson disease and multi-systematised
(synucleopathies) atrophies, marker Alpha-SYNUCLEINE
(Mollenhauer B., Cullen V., Khan I., Experimental Neurology,
2008,213, pp 315-325) can be mentioned.
Finally, markers which are non specific to affections
concerning the central nervous system (SNC) exist, which can
be used within the scope of the present invention. These are
proteins originating from SNC such as protein GFAP, myelin,
neuropeptides and neurotransmitters. Protein BNDF (brain
derived neurotrophic factor), more particularly described in
the document by the University of California, San Diego,
Medicine & health/diseases February 2009, is also of
particular interest. Besides, immune response proteins, for
example IgG, albumin, complement protein, reactive protein C,
as well as inflammation protein, such as for example,
Transferrin, Haptoglobine, Ceruloplasmine, Lysozyme, Enolase
can be used when implementing the invention.
Whatever the embodiment of the invention, several
different markers, i.e. markers of various pathologies, can be
placed in wells of the same metal guide. Then, markers can be
placed in the wells so that the various markers do not
interact with each other.
Advantageously, the device according to the invention or
at least in its functionalized terminal part in contact with
the substrate to be analyzed shows a level of assurance of
sterility (SAL for Sterility Assurance Level) of the order of
10-6. Various alternative solutions can be considered so as to
reach this level of sterility. A possibility consists in
sterilizing a device in the absence of the reactive groups
specific to the substrate to be detected, and then in adding
the latter under sterile conditions. Another possibility
consists in sterilizing the device after adding reactive

groups, which requires the use of sterilization techniques
which do not significantly reduce the activity of said
reactive groups (for example sterilization using ethylene
oxide or radiation).
A second object of the invention is an ex-vivo detection
method for a substrate existing in a tissue or an organ,
characterised in that it includes the following steps:
a) incubation of the functionalized Ea extremity of a device
according to the invention with a solution comprising at least
one detection agent specific to said substrate, when said
extremity is in contact with said tissue or organ to be
examined.
b) detection of said substrate.
The step of incubation is performed during a time
sufficient so that the detection agent in the solution, more
particularly an antibody, can specifically get fixed to the
substrate (marker, antigen, antibody, etc.), more particularly
an antigen and optionally existing at the end of the device.
The man skilled in the art can easily determine, using their
general knowledge and routine experiment, this incubation time
as a function of the affinity of the detection agent in
solution, more particularly an antibody for its substrate,
more particularly an antigen. This incubation time also
depends on the temperature of the solution during the
incubation. As an example, the incubation time is of the
order of 1 minute to 2 hours, preferably 5 minutes to 1 hour
and particularly preferably from 10 to 30 minutes for a
temperature between 20°C (room temperature) and 37°C.
Advantageously, the detection agent in solution is
different from the specific reagent coupled to the
functionalized extremity of the device according to the
invention.
Preferably, the detection agent is an antibody.

Advantageously, the antibody in solution and the antibody
coupled to the functionalized extremity of the device
according to the invention are each a polyclonal antibody
preferably said antibodies are identical.
Advantageously, the antibody in solution and the antibody
coupled to the functionalized extremity of the device
according to the invention are each a monoclonal antibody
preferably said antibodies are different.
Advantageously, the antibody in solution is marked and
more particularly, it is coupled to an enzyme, for example
peroxidase or phosphatase alkaline.
According to a first particular embodiment of the method
according to the invention, the method further includes a step
a' ) of incubation of said extremity in a solution comprising
at least one detection agent specific to the detection agent
of step a) interposed between steps a) and b).
The man skilled in the art can simply identify using their
general knowledge, the antibodies adapted to the method
according to the invention. As an example, it is possible to
use in this second step, an antibody specifically recognizing
mouse immunoglobulins if such mouse immunoglobulin
specifically directed against the substrate (marker, antigen,
antibody, etc.) to be identified are used at step a).
According to a second particular embodiment of the method
according to the invention, the method according to the
invention includes a step of washing, following the step of
incubation a) and possibly step a' ) which washing step makes
it possible to eliminate antibodies which are not specifically
fixed to a marker (the antigen).
The protocol of such a washing step also belongs to
general knowledge and can be simply determined by routine
experiments. As an example, such a step is performed with a
solution including a more or less important concentration of
detergent (0.05 to 1%) such as TRITON X100® or the TWEEN 20®,

as a function of the affinity of the antibody in solution for
its specific antigen.
The step of detection is carried out by evidencing an
activity, more particularly an enzymatic activity coupled to
the antibody used in step a) or optionally step a').
The protocol used for this step of detection depends on
the marker used and more particularly the enzyme used, for
example peroxidase and alkaline phosphatase, and belongs to
the general knowledge of the man skilled in the art.
This detection step makes it possible to deduce the
quantity of specific substrate (for example of antigen) fixed
to the functionalized extremity of the device and finally the
quantity of the specific substrate existing at the level of
the organ or of the tissue where the micro-analysis and/or
micro-sampling was carried out.
Finally, the various reactive groups which can be used for
carrying out the method according to the invention are well
known to the man skilled in the art and more particularly
include the reagents used for the immuno-enzymatic or immuno-
fluorescence dosage technique, for example on a solid support
[ELISA technique, protein chips (ESPINA and al., mentioned
above, 2004) ] .
A third object of the invention consists in using a device
according to the invention for manufacturing a tool intended
for diagnosing cancer, an inflammation, an infection, a graft
rejection or a neurodegenerative pathology in a patient.
According to a particular embodiment of the invention,
said diagnostic tool may include at least a metal guide
inserted into a flexible catheter inserted into an endoscope.
According to another particular embodiment of the
invention, said diagnostic tool may include at least one metal
guide consisting into a transparietal aspiration needle and
more particularly transcutaneous or transmucosal aspiration
which can be inserted into a removable protection system for

example a flexible catheter. In addition, the removable
protection system and the metal guide cooperate so as to
enable the contact of the functionalized Ea extremity of said
guide with the micro-analysis or and/or micro-sampling site.
In these two particular embodiments of the invention, said
at least one metal guide may be associated with at least a
part of the length of an optical fiber with a view to
identification and positioning.
Advantageously, said diagnosing instrument is delivered by
endocavitary route.
Said tool thus makes it possible to carry out a micro-
analysis and/or a micro-sampling in the digestive system from
the pharynx to the rectum (including the liver and the
pancreas), of the uro-genital system (including the bladder,
the urether, kidney, prostate), tracheo-bronchial system
(including the lungs), the ORL system (including the ear and
the nasopharynx), and the osteo-articular system (including
synovial cavities).
Preferably, said diagnostic tool is delivered by a
transparietal route, more particularly by transmucosal or
transcutaneous route.
Such a diagnostic tool also makes it possible to analyze
tissues or organs which can hardly be reached through the
endocavitary or endovascular routes usually used. Such a
diagnostic tool also makes it possible to carry out a micro-
analysis and/or micro-sampling using the transparietal route
at the level of the skin, the testicles, the prostate, the
ovaries, the mammary glands and also the kidney and the liver,
the peripheral nervous system as well as the central nervous
system, more particularly by endo-spinal route as well as the
endocrine system (for example thyroid).
Examples hereinunder make it possible to illustrate the
invention and are given as non-limitative examples.
Brief description of the Figures

- Figure 1 shows various possibilities for structuring metal
guides.
- Figure 2 shows scanning electronic microscopy photography of
various holes made by FIB (important surface roughness
resulting from etching non-homogeneity can be observed).
- Figure 3 shows an explanation and an observation of the
shadowing effect of the milling technique assisted by
fluorine.
- Figure 4 shows the advance of micro-shaping operations.
- Figure 5 shows (a) hemispheric holes observed by optical
microscope (b) series of holes observed on a guide using
Nitinol by scanning electronic microscopy (c) a detail of a
cavity (d) comparison of the surface roughness in the cavity
with the surface roughness of the guide.
- Figure 6 shows pictures of scanning electronic microscopy
showing the same hemispheric hole as in Figure 5 after the
electrochemical polishing treatment.
- Figure 7 shows a diagram of the device of the invention for
the immuno-capture with a first monoclonal antibody (AcM 1)
and the revelation of the antigen ACE with a second monoclonal
antibody (AcM 2).
Figure 8 shows the results of ELISA with the capture
antibody 5910 and the revelation with antibody 5909
(absorbance obtained with an antigen ACE positive serum).
Figure 9 shows the results of ELISA with the capture
antibody 5910 and the revelation with 5909 antigen (absorbance
obtained with a antigen ACE negative serum).
Figure 10 shows the results of ELISA with the capture
antibody 5905 and the revelation with the antibody 5909
(absorbance obtained with an antigen ACE positive serum).
Figure 11 shows the results of ELISA with the capture
antibody 5905 and the revelation with the antibody 5909
(absorbance obtained with an antigen ACE negative serum).

- Figure 12 shows the results of ELISA with the rigid plastic
butts with capture antibody 5910 and the revelation with
antibody 5909 (absorbance obtained with a positive antigen ACE
serum).
The figures are illustrations of the sensitivity and
specificity of the method used.
EXAMPLE 1: PREPARATION OF A NITINOL-BASED METAL GUIDE AND THE
ACTIVATION THEREOF
The surface of a Nitinol-based metal guide (Euroflex) is
structured for defining locations, for example wells, where
the reactive groups will be placed and where the biochemical
interactions will occur (Figure 1).
Said "wells" can be obtained using various methods such
as, for example focus ion beams lithography (FIB, Xie and al,
Nuclear Instruments & Methods in Physics research Section B-
beam Interactions with Materials and Atoms, 211(3): 363-368,
2003), by a laser lithography followed by an electrochemical
etching and a laser ablation.
Using the FIB technique, the machine creates an ion beam
which is focused onto the surface which must be structured.
Under the mechanical action of the ion beams, the atoms of the
surface material are eliminated from the surface. Holes
having a diameter of 20µm can be formed with the technique FIB
in a reasonable time with an etching reader of 8um3s-1 in a
beam current of 20nA. Figure 2 shows holes having a diameter
of 5, 20 and 40µm with a depth of 10 and 20µm. The surface of
the bottom of the hole is rough because of the re-deposition
of the pulverized material during the etching. The etching
rate has been measured at 200nm min-1 on a circular area, 40µm
in diameter and a beam current of 20nA. This results in an
etching rate 0.2µm3 nC-1 (approximately 5µm3s-1) which
corresponds to a processing time of 20 minutes to make a hole

20um in diameter and 20um in depth. In order to improve the
surface roughness, a fluorine (XeF2) assisted milling technique
was used; a very low surface roughness was then obtained but
as the XeF2 was not exactly along the axis of the etching beam,
a shadowing effect was noted (Figure 3).
The laser lithography technique and electrochemical
etching consist in a first step of coating the surface with a
layer of polymer. In a second step, the polymer layer is
machined using the laser ablation. In a third step, the
surface is etched using an isotropic electrochemical etching
through the opening provided in the polymer layer (Figure 4) .
Figure 5 shows the result of various structuring tests on
Nitinol based metal guides.
In addition, Nitinol based metal guides which are used in
vivo are usually processed through an electrochemical
polishing which replaces the native NiTi oxide layer with a
biocompatible TiO2 layer. The machined guides having holes
have to undergo this method in order to estimate the influence
of the method on the structure of the holes (Figure 6).
Another way to prepare cavities on the surfaces of the
Nitinol based guides uses laser ablation. The utilization of
short laser impulsion enables the local evaporation of metal
without affecting the surrounding metal because of the heat
generated. The smallest reported dimensions are of the order
of 20µm.
If the three methods mentioned hereabove make it possible
to create wells, the electrochemical etching method gives the
best results.
EXAMPLE 2: TRAUMA. CONSECUTIVE TO THE IN VIVO INSERTION OF A
METAL GUIDE INTO A PARTICULAR ORGAN
For these experiments, metal micro-guides (MTI 0.012"
Silver speed) were used and said metal guides were inserted
into micro-catheters.

The device was introduced in pigs under general anesthesia
at the level of an aspiration, then at the level of the Scarpa
up to the kidney through the endovascular route (via the
femoral artery). This guide was provided by the follow-up of
said device in the femoral artery through an arteriography.
When the device is positioned at the entrance of the
kidney, it is introduced into the kidney by endo-arterial
invasion. This penetration into the tissue was a few
millimeters deep and said device was kept there for about ten
minutes.
Finally, the device was removed.
Animals were euthanized and their kidneys were sampled to
estimate their conditions after the penetration of the device
according to the invention.
The result showed that no important hemorrhage in the
kidney was associated to the invasion. The most important
damage noted showed a dimension of 3xlmm at the level of the
invasion site.
The device according to the invention thus makes it
possible to have access to an organ while being very slightly
invasive.
EXAMPLE 3: MICRO TRAUMA FOLLOWING THE INSERTION OF A DEVICE
INTO THE LIVER
Metal micro-guides (MTI 0,012" Silver speed) were used
which metal micro-guides were placed in a fiberscope which is
different from example 1.
The device was introduced into pigs under general
anesthesia at the level of an aspiration, at the level of the
Scarpa then up to the liver through endo-arterial navigation
(via the femoral artery) . This guiding was provided by a
control of said device in the femoral artery through
arteriography.

When positioned close to the liver, the device was
introduced into this organ. This penetration into the tissue
was a few millimeters deep and said device was kept there
again for approximately ten minutes.
Finally, the device was removed.
As before, the liver sampling of the operation made it
possible to estimate the aggressivity of the operation on the
organ.
No macroscopically visible damage could be seen at the
surface of the liver. When cut, the presence of two intra-
parenchymatous hemorrhagic nodes of the sub-capsular seat
1.5x0.4cm and 1.8x0.5cm in dimension could be seen.
Histologically, the hepatic architecture is kept in every
aspect with a congestion of sinusoids, portal venules and
centro-lobular veins without any other remarkable anomaly.
Conclusion
The results showed that hemorrhagic lesions were minimum:
two minor microscopic damage could be seen without any
destruction of parenchymatous cells and with a simple
congestion of capillaries and centro-lobular veins.
The utilization of a metal guide thus makes it possible to
obtain a minor trauma and, in any case, largely smaller than
that resulting from a biopsy.
EXAMPLE 4: STUDY OF THE PARAMETRES FOR THE CONCEPTION AND
PRODUCTION OF A DEVICE ENABLING IN VITRO IMMUNOCAPTURE AND
DETECTION OF ANTIGEN ACE ON SOLID SUPPORTS
The device uses the principle of the ELISA technique
making it possible to show antigen ACE. Two monoclonal
antibodies recognizing different epitopes on this antigen were
used for the capture (AcM1) and revelation of the antigen ACE
(AcM2). These monoclonal antibodies having the same isotope
(IgG1) the revelation of the ACE antigen was made using a

monoclonal antibody coupled to biotin and a streptavidine-
peroxidase complex (Figure 7).
Two types of support were used, either plates for ELISA or
rigid plastic butts.
Plates for ELISA (Greiger)
100µl of a monoclonal antibody directed against the
antigen ACE (clone 5910 or clone 5905, produced in mice and
marketed by Medix Biochemical) diluted (1/5000 and 1/128000)
in carbonate/bicarbonate buffer were placed in each well, and
the plate was placed for 1 hour at 37°C. One negative control
was obtained by replacing the antibody by
carbonate/bicarbonate control solution.
After three washings with 250ul of PBS per well, the free
sites of the plate have been saturated with 200ml of 3% PBS-
BSA (bovine serum albumin) for 2 hours at 37°C.
Then the wells were washed three times with 250ul of 0.5%
PBS-Tween prior to adding lOOul per well of an antigen ACE
positive serum diluted within 1/10, 1/100, 1/1,000 with PBS-
Tween and the plate was incubated for 1 hour at 37°C.
Three washings with 250µl with PBS-Tween per well were
carried out prior to adding 100µl per well of a monoclonal
antibody directed against antigen ACE (clone 5909 produced in
mice and marketed by Medix Biochemical, which is different
from the previous capture antibodies used because of its
constant affinity and the recognized epitopes) biotynylated
within 1/500 with PBS-Tween and the plate was incubated again
for 1 hour at 37°C.
After three washings using PBS-Tween, lOOul of
streptavidine complex coupled to peroxidase diluted within
1/2000 were added in each well and incubated for 1 hour at
37°C.
After three washings with PBS-Tween, the revelation was
carried out by adding 200µl per well of the substrate (H2O2)

and chromogen (OPD, Sigma) mixture in a citrate-phosphate
control (pH 5).
In parallel, the same operation was carried out using a
"normal" patient serum as an antigen (negative control with an
ACE dosage of <5 UI/ml).
The reaction was then stopped by the addition of 50ul of
1M sulfuric acid per well. The absorbance was read at 492nm
on a plate reader (ref: ELX. 800UV).
The results obtained by using the monoclonal antibody 5910
for immuno-capture (diluted within 1/500 then half by half up
to 1/128000) and the revelation through the biotinylated
monoclonal antibody 5909 are shown in Figure 8 for the antigen
ACE positive serum, and in Figure 9 for the antigen ACE
negative serum.
The results obtained using the monoclonal antibody 5905
for immuno-capture (diluted within 1/100, 1/200, 1/500 then
half by half up to 1/32000) and the revelation using the
biotinylated monoclonal 'antibody 5909 are shown in Figure 10
for the antigen ACE positive serum and in Figure 11 for the
antigen ACE negative serum.
Captions of Figures 8 to 11:
Ordinate: absorbance (DO) at 4 92nm
Abscissa: dilutions of capture antibodies (5910 or 5905)
♦ ACE positive serum dilution within 1/10
■ ACE positive serum dilution within 1/100
∆ ACE positive serum dilution within 1/1000
x = no serum
The results show that the antigen ACE positive serum
within 1/10 gives an absorbance (DO) above 0.5 when the
capture monoclonal antibodies used within 1/500 (Figure 8) .
Under the same conditions, the antigen ACE negative serum
gives a DO of less than 0.15 (Figure 9).
It should however be noted that better results were
obtained with the capture monoclonal antibody 5905 and

detection monoclonal antibody 5909 couple (Figures 10 and 11)
than with the capture monoclonal antibody 5910 and detection
monoclonal antibody 5909 couple (Figures 8 and 9) . As a
matter of fact, a DO of 1 was noted with the antigen ACE
positive serum diluted within 1/10 (Figure 10) whereas the
antigen ACE negative serum gives a DO of 0.1 (Figure 11) under
the same conditions. These results were confirmed using a
capture monoclonal antibody 5910 in various dilutions (data
not shown).
Rigid classic supports
In a first step, rigid plastic supports in the form of
butts 2 to 3cm in length and 0.5 to 1mm in diameter were
activated.
In a second step, the thus activated supports were placed
in 1ml haemolyse micro-tubes (Fisher) and were functionalized
with a monoclonal antibody directed against the antigen ACE
(clone 5910 produced in mice and commercialised by Medix
Biochemical) and diluted within 1/50, 1/100, 1/250, 1/500 in a
carbonate/bicarbonate (250 µl/tube) control for 1 hour at
37 °C. A negative control was made by replacing a monoclonal
antibody with a carbonate/bicarbonate control. After a
fixation and washings, saturation was obtained with 500ul of
3% PBS-BSA overnight with a temperature of +4°C.
Then the supports were incubated with 250ul of antigen ACE
positive serum diluted within 1/10, 1/100 with PBS or with
serum from a "healthy" subject (antigen ACE negative control)
with the same dilution for 1 hour at 37°C.
A monoclonal antibody directed against the antigen ACE
(clone 590 9 produced in mice and marketed by Medix
Biochemical, which is different from the purified clone 5910
by its constant affinity and by the acknowledged epitopes)
within lmg/ml was dialysed overnight at 4°C against a 0.1 M
borate control, pH 8.8. A 10mg/ml DMSO biotin solution was
then added in 50µg/mg of antibodies. After an incubation of 4

hours at room temperature and under stirring, 1 M ammonium
chloride was added, in a quantity of 20µl/250 ug of biotin and
the obtained solution was incubated again for 1 minute at room
temperature. Upon the stopping of the reaction, the marked
antibody was dialysed for 24 hours at +4°C against PBS and
this marked antibody was kept as aliquots at -20°C.
After 3 washings with PBS-Tween, the supports were
incubated with 250µl of biotinylated antibody 590 9 and diluted
within 1/500 with PBS-Tween for 1 hour at 37°C.
Detection of biotin (ester of 6-biotinamidocaproylamido-
caproic acid and N-hydroxysuccinimide, Sigma) was evidenced
using a steptavidine-peroxidase complex (Amersham Biosciences)
diluted within 1/2000 in PBS for 1 hour at 37°C.
The revelation of enzymatic activity was carried out by
adding 750µl of the substrate (H2O2) and chromogen (OPD, Sigma)
mixture per tube in a citrate-phosphate control (pH 5).
The reaction was then stopped by adding 1M sulfuric acid.
Absorbance was read at 492nm.
ELISA results on plastic butts are shown in Figure 12.
Captions of Figure 12:
Ordinate: absorbance (DO) at 4 92nm
Abscissa: dilution of capture antibody 5910
♦ antigen ACE dilution within 1/10
■ antigen ACE dilution within 1/100
∆ negative control dilution within 1/10
x negative control dilution within 1/100
In general, the results show that DOs are 7 to 10 times
higher with antigen ACE positive serum than those obtained
with antigen ACE negative serum.
The best results were obtained with rigid plastic supports
on which capture monoclonal antibody 5910 diluted within 1/50
or 1/100 was fixed.
The best Antigen ACE concentration detected corresponds to
the patient's serum diluted within 1/100, i.e. 6UI/ml (close

to the rate considered as "normal" : <5UI/ml) and when the
dilution of the detection of monoclonal antibody 5909 is
within 1/500.
Using plastic supports makes it possible to validate the
specificity and sensitivity of immuno-capture processes on
functionalised metal butts according to the above-mentioned
protocol.
Conclusion
The good results obtained for the detection of antigen ACE
with the in vitro immuno-capture and revelation techniques
validate the evaluation of "functionalized butt" devices
making it possible to capture in vivo the antigen ACE followed
by the ex vivo revelation.
EXAMPLE 5: IDENFICATION OF THE EXPRESSION OF THE MARKER ACE IN
A BREAST TUMOR FOR EXAMPLE UNDER THE CONTROL OF IMAGING
TECHNIQUES AND MORE PARTICULARLY RADIOLOGICAL TECHNIQUES
According to the protocol described in the application PCT
WO 03/006948, in a first step, an alcanethiol layer is
absorbed on one of the ends of Nitinol-based metal guides
(Euroflex), in a first step. In a second step, the free thiol
functions of this layer make it possible to form disulfide
bridges with a monoclonal antibody directed against the
antigen ACE.
The metallic guide obtained is then introduced into a
biopsy needle adapted to be used in an animal or a human
being.
An extemporaneous anatomo-pathological examination is
performed using this device on a tumor to be operated (mammary
tumour) after it has been removed from a patient suffering
breast cancer. According to an alternative solution, when the
medical ethic conditions are present, the micro-incision is
performed in the breast under a local or general anaesthesia
in a patient suffering from breast cancer. The needle in

which the metal guide coupled to the antibody directed against
the antigen ACE is inserted, is introduced into the tumour or
through the micro-incision then guided up to the tumour while
following the progression thereof by imaging, and more
particularly echographia.
Said micro-invasive guiding system then makes it possible
to take out the end of the metal guide coupled to the antibody
directed against the antigen ACE. The end of the metal guide
is then introduced into the tumour (by perforation) at a depth
of the order of a few millimetres. After a short waiting time,
of the order of ten minutes, which enables the immuno-capture
by the antigen ACE optionally expressed by the tumour, the
device is removed.
The micro-sampling is limited to an in vivo immuno-capture
of the analyte and does not request any biopsy.
Finally, the device is removed, then an ELISA dosage of
the marker ACE is carried out on the end of the device with a
monoclonal antibody directed against the marker ACE which is
differentiated from the capture antibody by its constant
affinity as regards the antigen ACE and by the recognised
epitopes, and which is coupled to biotin.
The revelation of the enzymatic activity using a
streptavidine-peroxidase complex makes it possible to deduce
the expression of the marker ACE by the tumour and to modulate
the therapy to be used to treat a patient at best accordingly.
EXAMPLE 6: SKIN CANCER
According to the protocol described in the application PCT
WO 03/006948, a layer of alcanethiol has been absorbed on the
end of a Nitinol-based metal guide (Euroflex) in a first step.
In a second step, free thiol functions of this layer enable
the formation of disulfide bridges with a monoclonal antibody
directed against FAP marker (Fibroblast-activation protein;

RETTIG et al., Proc. Natl. Acad. Sci. USA, vol.85, p: 3110,
1988).
The metal guide obtained is then introduced at the level
of a skin tumour in an animal or in a human being when medical
ethic conditions are present or at the level of a skin tumour
in a patient suffering from skin cancer after the ablation
thereof, for a conventional or extemporaneous anatomo-
pathological examination.
Micro-sampling is limited to an in vivo immuno-capture and
does not require specific biopsy.
Finally, the device is removed and an ELISA dosage of the
FAB marker is carried out on the end of the device with a
monoclonal antibody directed against the marker FAB coupled to
peroxidase.
The revelation of the peroxidase activity makes it
possible to deduce the expression of the marker FAP by the
tumour and to modulate the therapy to be used for treating the
patient at best accordingly.

CLAIMS
1. An analysis device characterised in that it comprises a
system for a micro-invasive and/or micro-sampling
investigation of a substrate, said system being composed of at
least one metal guide including:
- an Ea extremity the surface of which is structured so as to
define at least a series of wells to which is directly coupled
at least one reactive group specific to said substrate, said
Ea extremity being perforating, and
- another Em extremity intended for handling said metal guide.

2. A device according to claim 1, further comprising a
removable protection system at the level of the Ea extremity.
3. A device according to claim 2, wherein said removable
protection system is a flexible catheter.
4. A device according to any one of claims 1 to 3 further
comprising a medical instrument having an internal opening
wherein said at least one metal guide can slide.
5. A device according to claim 4, wherein said medical
instrument is selected in the group comprising a transparietal
aspiration needle and/or an endoscope, inclusive of an
endovascular navigation system.
6. A device according to claim 5, wherein said transparietal
aspiration needle is a transcutaneous or transmucosal
aspiration needle.
7. A device according to any one of claims 1 to 6, wherein
said at least one metal guide is selected in the group
comprising one or several flexible solid metal butts and/or

one or several hollow rigid metal butts having a diameter from
0.3 to 3.5mm and a length from 5x10-2 to 2m.
8. A device according to any one of claims 1 to 7, wherein
said at least one metal guide is associated with a visual
display system on at least one part of the length thereof.
9. A device according to claim 8, wherein said visual display
system is an optical fibre.

10. A device according to claim 7, wherein said at least one
flexible solid metal butt is an optical fibre at an Ea
extremity of which a metal ring provided with at least a
series of wells is associated, to which at least one reactive
group specific to said substrate is directly coupled, with
said Ea extremity being perforating.
11. A device according to any one of claims 1 to 10, wherein
said at least one metal guide is constituted, as a whole or in
part, of a metal alloy selected in the group comprising
stainless steel, titanium-, nickel-, cobalt-based alloys or a
mixture thereof.
12. A device according to claim 11, wherein said metal guide
is constituted, as a whole or in part, of a titanium and
nickel alloy.
13. A device according to any one of claims 1 to 12, wherein
said at least one metal guide is coated, except for the Ea
extremity, with a protective polymer layer having a thickness
of 2x10-3 to 1µm.
14. A device according to any one of claims 1 to 13, wherein
said at least one reactive group is specific to a substrate or

an antigen specific of cancer, an inflammation, an infection,
a graft rejection or a neurodegenerative pathology.
15. A device according to any one of claims 1 to 14, wherein
said at least one reactive group specific to a substrate is an
antibody or a fragment of antibody selected in the group
constituted of fragments Fab, Fab', F(ab')2 and Fv.
16. Use of a device according to any one of claims 1 to 15 for
manufacturing a tool intended for diagnosing a cancer, an
inflammation, an infection, a graft rejection or a
neurodegenerative pathology such as Alzheimer's disease,
Parkinson's disease or an amyotrophic lateral sclerosis.
17. The use according to claim 16, wherein said tool comprises
at least one metal guide inserted into a flexible catheter
inserted into an endoscope and is intended for diagnosing
cancer, an inflammation, an infection, a graft rejection or a
neurodegenerative pathology.
18. The use according to claim 16, wherein said tool includes
at least one metal guide constituted of a transparietal
aspiration needle and inserted into a flexible catheter and is
intended for diagnosing cancer, an inflammation, an infection,
a graft rejection or a neurodegenerative pathology.
19. The use according to claim 18, wherein said at least one
metal guide and the flexible catheter cooperate so as to place
the functionalized Ea extremity in contact with the micro-
analysis and/or micro-sampling site.
20. The use according to any one of claims 17 to 19, wherein
the transparietal route is selected in the group constituted
of the transmucosal and transcutaneous routes.

21. The use according to any one of claims 17 to 20, wherein
said at least one metal guide is associated with an optical
fibre on at least a part of the length thereof.
22. A method for detecting ex-vivo a substrate existing in a
tissue or an organ, characterised in that it includes the
following steps:
a) incubation of the functionalised Ea extremity of a device
according to any one of claims 1 to 15, with a solution
comprising at least one detection agent specific to said
substrate when said extremity is in contact with said tissue
or organ to be examined.
b) detection of said substrate.
23. A method according to claim 22, wherein said at least one
detection agent of step a) is a possibly marked antibody
specific to said substrate.
24. A method according to any one of claims 22 or 23, wherein
a step a' of incubation of said extremity in a solution
including at least one detection agent specific of the
detection agent of step a) is interposed between steps a) and
b) .
25. A method according to claim 24, wherein said at least one
detection agent of step a' ) is a marked antibody specific to
the detection agent of step a).
26. A method according to any one of claims 22 or 23, wherein
a step of washing follows step a).
27. A method according to any one of claims 24 or 25 wherein a
step of washing follows step a) and/or step a').

The present invention relates to an analysis device, said
system being composed of at least one metal guide at one
extremity of which is provided at least a series of wells to
which are directly coupled reagents specific to the
substrates, said extremity being a perforating one and the
other extremity of which is intended for controlling said
guide and optionally associated with a aspiration system.
Said guide may be inserted into a protection system that is
removable at the level of the functionalised extremity up to
the micro-analysis and/or micro-sampling site and/or into a
medical instrument having an inner opening in which said guide
may slide.
The present invention also relates to the use of such a
device for making a tool for diagnosing cancer, an
inflammation, an infection, a neurodegenerative disease or a
graft rejection in a patient, preferably by transparietal
route.
The present invention also relates to a method for the ex
vivo analysis of a substrate using such device.