Trapping microvesicles for diagnostics

Abstract

The present invention relates to a functionalized support and its use for disease diagnosis. It discloses a method for characterizing microvesicles present in a sample of biological fluid from a subject, comprising contacting the sample with a solid support having a binuclear metal complex compound of formula (I) or (II) covalently grafted to its surface, capturing the microvesicles, and characterizing the captured microvesicles. The method can also be used to diagnose diseases (nephropathy, Parkinson's disease, or diabetic nephropathy) by detecting the markers podocalyxin, α-synuclein, and CD41. The support is made of poly(vinyl chloride) (PVC), poly(ethylene terephthalate) (PET), or polystyrene (PS), and is prefunctionalized with glutaraldehyde, NHS functional groups, or N-oxysuccinimide functional groups, respectively. The metal M is selected from Zn, Cu, Mn, Co, Ni, and Fe.

Description

[Technical field]

[0001] The present invention relates to functionalized supports and their use for the diagnosis of disease. [Background technology]

[0002] Cellular microvesicles are small vesicles released into the extracellular matrix by cell budding upon activation after stress. Microvesicles released into biological fluids (blood, urine, tears, etc.) contain and / or carry on their surface components of the parent cell (lipids, proteins, and RNA or mitochondrial DNA) and can therefore be considered as early markers of tissue pathology.

[0003] The method described in International Application No. PCT / FR2012 / 050610 uses synthetic ligands to capture phosphatidylserine-positive microvesicles.

[0004] The present invention relates to improvements to the capture means and methods presented in International Application No. PCT / FR2012 / 050610. The present invention also relates to the identification of biomarkers for diseases of interest using functionalized supports according to the embodiments presented below. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Application No. PCT / FR2012 / 050610 Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to a functionalized solid support useful for the capture, detection and / or characterization of cell microvesicles. The support according to the invention is also useful in the medical field for carrying out diagnostic and / or prognostic methods, in particular for the early diagnosis of diseases.

[0007] The support according to the invention is a solid support, in particular a polymer, metal or ceramic, functionalized with a compound of formula (I) or (II) as follows: According to a particular embodiment, the support is a polymeric support, in particular a support made of poly(vinyl chloride) (or PVC), poly(ethylene terephthalate) (or PET) or polystyrene (or PS), functionalized with a compound of formula (I) or (II) as follows:

[0008] The present invention also relates to a method for the diagnosis of a disease, comprising the detection of the presence or absence of specific markers identified by the inventors, which detection can be carried out in particular after capturing microvesicles according to the embodiments presented in this application. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 illustrates steps in the functionalization of PET. [Diagram 2] FIG. 1 illustrates steps in the functionalization of PVC. [Diagram 3] Schematic representation of a PVC surface at different steps in its functionalization with complex 1. (A) Step 1: Untreated PVC. (B) Step 2: Addition of ethylenediamine (EDA). (C) Step 3: Addition of glutaraldehyde (Glu.). (D) Step 4: Addition of complex 1 (C1). [Figure 4] Schematic representation of the surface of DNA-BIND® at different steps in its functionalization with complex 1. (A) Step 1: Untreated DNA-BIND®. (B) Step 2: Addition of complex 1 (C1). [Diagram 5]Figure 1: Graphical representation of podocalyxin / β-actin ratios in microvesicles derived from urine of healthy donors and diabetic patients. Each point represents the ratio obtained for one patient. The mathematical mean values ​​are shown above each patient group. Statistical data were analyzed with Tukey's ANOVA test. *: p<0.05; **: p<0.01, and ***: p<0.001. Legend: n=number of patients, Healthy: healthy donors; Normo: normoalbuminuric patients, Micro: microalbuminuric patients; Macro: macroalbuminuric patients. [Figure 6] Figure 1: Graphical representation of podocalyxin / annexin A5 ratio in microvesicles derived from urine of healthy donors and diabetic patients. Each point represents the ratio obtained for one patient. The mathematical mean values ​​are shown above each patient group. Statistical data were analyzed with Tukey's ANOVA test. *: p<0.05; **: p<0.01, and ***: p<0.001, and ****: p<0.0001. Legend: n=number of patients, Healthy: healthy donors; Normo: normoalbuminuric patients, Micro: microalbuminuric patients; Macro: macroalbuminuric patients. [Figure 7] Detection of the CD41 platelet marker in microvesicles derived from the plasma of diabetic patients and graphical representation of the CD41 / Annexin A5 ratio. A. Electrophoresis of proteins of microvesicles derived from the plasma of two diabetic patients: one normoalbuminuric (Normo) and one macroalbuminuric (Macro). Revelation of the Western blot with antibodies against CD41 and β-actin and Annexin A5 reference proteins. B. Quantification of the signal from the bands associated with CD41 and Annexin A5 using ImageJ software and graphical representation of the CD41 / Annexin A5 ratio. [Figure 8]Detection of enzymatic activity of uPA in microvesicles isolated from cultured HUVEC cells. Graphical representation of the mean initial velocity measured spectrophotometrically in mOD / min. Legend: control no rinse condition = 100%; rinsed condition without complex = no material; rinsed condition with complex = with material. [Figure 9] Figure 1 shows immunological detection of podocalyxin in microvesicles isolated from human urine and captured by the material: Graphical representation of absorbance obtained by spectrophotometry at 450 nm in different conditions: 1 - microvesicles + TMB, 2 - primary antibody + TMB, 3 - secondary antibody + TMB, 4 - primary antibody + secondary antibody + TMB, 5 - primary antibody + secondary antibody + TMB. [Figure 10] 1 shows the immunological detection of podocalyxin as a function of the concentration of microvesicles from human urine captured by the material. Graphical representation of the absorbance obtained by spectrophotometry at 450 nm as a function of the concentration of microvesicles expressed in ng / μL. The equation of the line and R2 were calculated using Excel software. [Figure 11] Figure 1 shows the immunological detection of CD41 in microvesicles isolated from human plasma and captured by the material. Graphical representation of the signal detected for the presence of CD41 in microvesicles of normoalbuminuric diabetic patients P1 and P2. The control condition (Ctl) represents the background noise recorded in wells without microvesicles in the presence of the reagent TMB. [Figure 12] Figure 1 shows the immunological detection of CD41 in human plasma after capture of microvesicles by the material. Graphical representation of the signal detected for the presence of CD41 in microvesicles of normoalbuminuric diabetic patients P3, P4 and P5. The signal has been calculated after subtracting the signal obtained for the background noise (i.e. in the presence of all reagents except without the primary antibody). [Figure 13]CryoMEB analysis of the capture of microvesicles derived from cell activation of HUVEC cells by material PVC+C1. (A) Scale bar 10 μm. (B) Scale bar 2 μm. (C) Scale bar 500 nm. [Figure 14] CryoMEB analysis of the capture of microvesicles derived from cell activation of HUVEC cells by the material "DNA-BIND®" + C1. (A) Scale bar 500 nm. (B) Scale bar 500 nm and measurement of the diameter of the microvesicles. [Figure 15] CryoMEB analysis of the capture of microvesicles from human urine samples from healthy subjects by material "DNA-BIND®"+C1. (A) Scale bar 5 μm. (B) Scale bar 500 nm. (C) Scale bar 500 nm and measurement of the diameter of the microvesicles. [Figure 16] FIG. 1 shows detection of marker Annexin A5 on microvesicles of HUVEC cell model at two doses (5 μg and 10 μg) in duplicate, without capture (Control, Ctrl) and after capture on the kit at 37° C. for 1 h (Kit). [Figure 17] FIG. 1 shows detection of the marker, Annexin A5, on microvesicles in urine samples from two healthy subjects without capture (Control, Ctrl) and after capture with the kit overnight at 4° C. (Kit). [Figure 18] FIG. 1 shows detection of the pathological biomarker, podocalyxin, and the microvesicle reference marker, annexin A5, on microvesicles in urine samples from healthy subjects and from diabetic patients with nephropathy at the microalbuminuric stage (Micro) without capture (Control, Ctrl) and after overnight capture with the kit at 4° C. (Kit). [Figure 19] FIG. 1 shows a comparison of podocalyxin / annexin A5 ratios corresponding to microvesicles of urine samples from healthy subjects and from diabetic patients with nephropathy at the microalbuminuric stage (Micro) without capture (Control, Ctrl) and after overnight capture with the kit at 4° C. (Kit). [Figure 20] FIG. 1 shows a comparison of podocalyxin / annexin A5 ratios corresponding to microvesicles of urine samples from healthy subjects and from diabetic patients with nephropathy at Normo, Micro, and Macro stages without capture (Control, Ctrl) and after overnight capture with the kit at 4° C. (Kit). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] According to a first aspect, the invention relates to a solid support, characterized in that it has on its surface grafted a compound of formula (I) or (II) as follows:

[0011] [ka]

[0012] During the ceremony, - M +i represents a metal ion, and i is 1, 2, or 3; - L represents an exchangeable ligand; - X is -(CH2) m represents a -NH2 group or a -CH2-NHC(O)-R-NH2 group, where R is a substituted or unsubstituted, linear or branched C2-C 10 , especially C5~C 10 is an alkyl group of the formula: - m=1 to 12; - n=1, 2, or 3; - Y is H or (CH2) p —NH2, where p=0 to 12.

[0013] According to the invention, the compounds of formula (I) or (II) are covalently grafted onto the solid support. The method described in International Application No. PCT / FR2012 / 050610 does not teach the covalent grafting of the complexes onto the surface of the support. Rather, it describes the non-covalent adsorption of polyglutaraldehyde onto the support and the subsequent immobilization of the complexes in polyglutaraldehyde via the NH2 functional groups of said complexes. Advantageously, thanks to the method of the invention, the complexes are more accessible to phosphatidylserine. Furthermore, the method according to the invention gives a better reproducibility of the functionalization. The method according to the invention therefore allows for a precise control of the density of the covalently immobilized complexes and has, in particular, the following advantages: - Better control of the presentation of the complexes for better interaction with the phosphatidylserine present in the microvesicles - Better control of the density of grafting of complexes - Desorption cannot occur in the medium - Better reproducibility of functionalization between materials and batches.

[0014] The compounds of formula (I) or (II) are cationic and their counterions may be selected, for example, from anions tosylate, nitrate, sulfate, sulfonate, thiosulfonate, halides, hexafluorophosphate, tetraphenylborate, tetrafluoroborate, perchlorate, etc., in particular from anions of perchlorate, nitrate, sulfate, halide, and carboxylate.

[0015] According to a particular embodiment, M is selected from Zn, Cu, Mn, Co, Ni and Fe, with Zn or Cu being preferred and more particularly Zn.

[0016] According to a particular embodiment, Y represents H.

[0017] According to another particular embodiment: - X is -(CH2) m -NH2, m=1, n=1 and Y represents H; or - X is -(CH2) m-NH2, m=1, n=2 and Y represents H; or X represents -CH2-NHC(O)-R-NH2, R represents C5H10, n=1 or 2, and Y represents H.

[0018] According to a particular embodiment, the compound to be grafted is a compound of formula (I).

[0019] According to another particular embodiment, the compound of formula (I) is selected from:

[0020] [ka]

[0021] [ka]

[0022] It should be understood that the present application also describes compounds of formulae (Ia), (Ib), (Ic) and (Id) associated with a counterion different from the perchlorate ion, in particular selected from the anions tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate and tetrafluoroborate, more particularly from the anions nitrate, sulfate, halide and carboxylate. Furthermore, the salt used to generate the counterion may in particular be a zinc salt, a copper salt, a magnesium salt, a cobalt salt, a nickel salt or an iron salt, more particularly a zinc salt, in particular zinc perchlorate, zinc nitrate, zinc sulfate, zinc halide or zinc carbonate.

[0023] According to certain embodiments, the compound is of the following formula:

[0024] [ka]

[0025] It should be understood that the counterion may be different from the perchlorate ion, and may in particular be selected from the anions tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate, and tetrafluoroborate, more in particular from the anions nitrate, sulfate, halide, and carboxylate. According to a particular embodiment, the counterion is the perchlorate anion. Furthermore, the salt used to generate the counterion may in particular be a zinc salt, a copper salt, a magnesium salt, a cobalt salt, a nickel salt, or an iron salt, more in particular a zinc salt, in particular zinc perchlorate, zinc nitrate, zinc sulfate, zinc halide, or zinc carbonate.

[0026] Compounds of formula (I) or (II) may be prepared according to the embodiments presented in International Application No. PCT / FR2012 / 050610.

[0027] The solid supports used in the context of the present invention may in particular be microtiter plates, sheets, cones, tubes, wells, beads, particles, strips, films, threads, screws or needles. In general, the solid supports according to the invention may be used to create any type of material of variable morphology and porosity.

[0028] In certain embodiments, the solid support is a polymeric, metallic or ceramic support functionalized with a compound of formula (I) or (II). According to certain embodiments, the support is a polymeric support, in particular a support made of poly(vinyl chloride) (or PVC), poly(ethylene terephthalate) (or PET) or polystyrene (or PS).

[0029] In the context of the present invention, "grafted to its surface" refers to a covalent bond between the support and the compound of formula (I) or (II) in relation to the grafting of the compound of formula (I) or (II) onto the support. As described below, the compound of formula (I) or (II) can be directly or indirectly covalently bonded to the support. In the case of indirect covalent bond, the compound of formula (I) or (II) is covalently bonded to the reactive functional group provided on the surface of the support by a prefunctionalizing agent, which is also covalently bonded to the support.

[0030] The solid support may be pretreated before grafting the compound of formula (I) or (II). The pretreatment may have the purpose, in particular, of destroying undesired functional groups, in particular ester functional groups, on the surface of the support, increasing the density of desirable functional groups and / or of prefunctionalizing the support by making it more electrophilic. In the context of the present invention, when the support is prefunctionalized, said prefunctionalization is carried out by covalent bonding using a prefunctionalizing agent. According to a particular embodiment, the support used is prefunctionalized. Thus, the support may be prefunctionalized, for example, using glutaraldehyde, N-hydroxysuccinimide (NHS) or N-oxysuccinimide (NOS). According to a representative embodiment, in which the support is made in particular of PET, the pretreatment may include hydrolysis of the ester functional groups present in particular on the surface of the support, increasing the density of COOH functional groups, in particular by oxidation of the support, in particular with potassium permanganate, and increasing the electrophilicity of this surface by modification with electron-withdrawing groups and good leaving groups such as NHS groups. According to a particular embodiment, the PET used (oxidized PET) has a viscosity of 1×10 as measured using toluidine blue (TBO). 12 COOH / cm 2 From 1×10 18 COOH / cm 2 Especially during 1×10 13 From 1×10 17 More specifically, between 1.2×10 15 to 1.2 × 10 17According to one variant, the support is made of PET and has a density of 5×10 functional groups on its surface before pre-functionalization. 15 Pieces to 5×10 16 COOH functional groups / cm 2 between 1.26×10 and, more particularly, before its pre-functionalization with NHS groups. 16 ±10% COOH functional groups / cm 2 Includes.

[0031] Thus, according to one embodiment, the support is a support made of PET, in particular a pre-functionalized PET, in particular with NHS functional groups. According to a particular embodiment, the pre-functionalized PET is a 1×10 13 COOH / cm 2 From 1×10 18 COOH / cm 2 Especially during 1×10 14 1×10 17 Between pieces, more specifically 8.1×10 14 Pieces to 8.2 x 10 16 According to one variant, the support made of pre-functionalized PET has a concentration of COOH functional groups on its surface comprised between 5×10 15 1×10 16 COOH functional groups / cm 2 More specifically, 8.18×10 15 ±10% COOH functional groups / cm 2 Includes.

[0032] According to another particular embodiment, the support is made of PVC, in particular pre-functionalized PVC, especially with glutaraldehyde.

[0033] According to a particular embodiment, the solid support is a support made of PS, in particular a pre-functionalized PS, in particular with N-oxysuccinimide (NOS) functional groups. In particular, the density of NOS functional groups of such a support made of pre-functionalized PS is 10 13 pcs to 10 16 NOS / cm2 During the 10 14 pcs to 10 15 NOS / cm 2 and more particularly, the density may be between about 68×10 14 NOS / cm 2 It is.

[0034] The compound of formula (I) or (II) is covalently immobilized on a solid support, optionally pre-functionalized, by introducing said support into a solution containing the compound of formula (I) or (II) to be grafted. According to an embodiment in which the support is pre-functionalized, the pre-functionalizing agent is covalently bound to the support. According to a particular embodiment, the solution is dissolved in 10 -5 M to 10 -2 During M, especially 10 -4 M to 5×10 -2 More specifically, between M and 5×10 -4 M to 5×10 -3 M. The compounds of formula (I) or (II) are particularly preferred in solution at about 10 -3 The immobilization time can be a concentration of M. The immobilization time can vary widely. However, according to a particular embodiment, the support is contacted with the compound of formula (I) or (II) for a period of between 1 hour and 72 hours, in particular between 5 hours and 48 hours, more particularly between 10 hours and 24 hours, and even more particularly for about 16 hours.

[0035] The present invention further relates to a support on whose surface a compound of formula (I) or (II) is grafted, said support being included in a kit intended for the detection or characterization of cell microvesicles. Such a kit may advantageously be used for the diagnosis of a disease of interest, in particular in medical analytical laboratories. The kit according to the invention comprises the support according to the invention and may optionally comprise any means usable for carrying out the method according to the invention. The kit may therefore in particular comprise means for carrying out a preliminary purification of the microvesicles, if this proves to be useful or necessary. The kit may also comprise buffers usable during the performance of the method according to the invention, in particular buffers for suspending the microvesicles, washing buffers or buffers for storage. Means for detecting or quantifying one or more markers that may be present on or in the microvesicles, in particular means for detecting or quantifying protein markers or nucleic acids, may be included in the kit. To this end, the kit according to the invention may in particular comprise: - a support as described above, - a means for detecting or quantifying one or more markers, and - optionally, buffers that can be used to carry out the method according to the invention.

[0036] Furthermore, the kit may also comprise means for detecting or quantifying normalization markers, in particular normalization markers selected from Annexin A5 and β-actin.

[0037] Thus, according to a particular embodiment, the kit according to the invention may comprise: - a support as described above, - means for detecting or quantifying one or more markers - means for detecting or quantifying one or more standardization markers, and - optionally, buffers that can be used to carry out the method according to the invention.

[0038] According to another particular embodiment, the present invention relates to a kit comprising: - a support as described above, - Means for detecting or quantifying podocalyxin - means for detecting or quantifying a standardization marker, in particular a standardization marker selected from Annexin A5 and β-actin, more particularly Annexin A5, and - optionally, buffers that can be used to carry out the method according to the invention.

[0039] According to another particular embodiment, the present invention relates to a kit comprising: - a support as described above, - Means for detecting or quantifying alpha-synuclein - means for detecting or quantifying a standardization marker, in particular a standardization marker chosen from Annexin A5 and β-actin, more particularly Annexin A5, and - optionally, buffers that can be used to carry out the method according to the invention.

[0040] According to another particular embodiment, the present invention relates to a kit comprising: - a support as described above, - Means for detecting or quantifying CD41 - means for detecting or quantifying a standardization marker, in particular a standardization marker chosen from Annexin A5 and β-actin, more particularly Annexin A5, and - optionally, buffers that can be used to carry out the method according to the invention. "Means for detecting or quantifying" means any means known by a person skilled in the art for detecting or quantifying a marker. Naturally, the means used will depend on the nature of the marker, in particular protein markers are detected by immunological techniques (in particular ELISA and Western blot), while nucleic acid markers can be detected in particular using specific amplification techniques (in particular PCR / qPCR or RT-PCR / RT-qPCR, or sequencing), which can be qualitative or quantitative. Other means include chromogenic assays, depending on the nature of the biomarker to be detected.

[0041] Furthermore, a kit according to the invention may comprise a leaflet providing instructions to the user for using the kit to carry out a method according to the invention.

[0042] The present invention therefore also relates to the use of the support according to the invention for capturing microvesicles present in a sample of biological fluid from a subject. The data presented by the inventors show that it is possible to carry out the detection of biomarkers in various types of biological samples, in particular urine or blood, more particularly plasma, using the method presented in this application. The method according to the invention can therefore provide for the detection of microvesicles in a wide panel of biological fluid samples. The biological fluid may in particular be a sample of blood, serum, plasma, saliva, tears, urine, lymphatic fluid, cerebrospinal fluid or semen. The subject is a mammal of any age, sex or condition, in particular a human being. According to a particular embodiment, the subject is an individual suspected of a pathological condition, in particular based on a previously performed bioassay or a medical consultation. In another embodiment, the subject has not undergone a bioassay or a prior medical consultation.

[0043] The present invention therefore relates to a method for capturing microvesicles, comprising a step of contacting a sample of biological fluid that may contain microvesicles with a support according to the present invention, after which the microvesicles thus captured can be characterized. According to one embodiment, before capture with the support according to the present invention, the microvesicles are first purified or isolated from the sample of biological fluid by procedures known by the skilled artisan. However, the experimental data presented below show that microvesicles can advantageously be captured directly in the sample of biological fluid using the support according to the present invention. Thus, in a particular embodiment, the microvesicles are captured directly from the sample of biological fluid, in particular from blood, serum, plasma, saliva, tears, urine, lymph, cerebrospinal fluid or semen, more particularly directly from plasma or urine.

[0044] According to certain embodiments, the characterization of the captured microvesicles allows the diagnosis of a disease, the assessment of the risk of developing a disease, the prognosis of a disease, the differential diagnosis of a disease, the monitoring of the progression of a disease, the monitoring of the effectiveness of a therapeutic treatment of a disease. As mentioned above, the composition of the cell microvesicles, in particular the lipid and protein composition, as well as the contents of the cell microvesicles (e.g. proteins and genetic material, in particular RNA or mitochondrial DNA), may vary depending on the type of cell from which they originate and the state of that cell. Thus, the microvesicles may allow the detection of the state of the cell from which they originate and therefore represent an optimal tool for the early detection of pathological conditions. Thus, according to one aspect, the present invention relates to the use of the support according to the present invention in a method for the diagnosis, differential diagnosis, risk assessment, prognosis, monitoring of the progression or monitoring of the effectiveness of a therapeutic treatment of various diseases, in particular thrombotic, inflammatory and / or metabolic diseases, or cardiovascular or neurovascular diseases or accidents, or diseases such as diabetes, cancer, Alzheimer's disease, Parkinson's disease, or any other disease.

[0045] The invention therefore also relates to a method for the diagnosis, differential diagnosis, risk assessment, prognosis, monitoring of the progression or monitoring the effectiveness of therapeutic treatment of various diseases, in particular thrombotic, inflammatory and / or metabolic diseases, or cardiovascular or neurovascular diseases or accidents, or diseases such as diabetes, cancer, Alzheimer's disease, Parkinson's disease or any other disease, and therefore we may refer to diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, multiple sclerosis, vasospasm after rupture of an aneurysm, Parkinson's disease or derivative conditions thereof. Depending on the particular purpose, the results of the characterization of the tested sample are compared to the results of a characterization carried out on a sample of a control biological fluid.

[0046] Advantageously, the control biological fluid is the same biological fluid as that being assayed, but originating from a subject considered to be healthy. Alternatively, the control biological fluid is from the same individual as the biological fluid being assayed, but from an earlier sampling. Controls according to this alternative may allow monitoring of the progression of a disease or its treatment.

[0047] The characterization of the microvesicles can be carried out by any means known by the skilled artisan, taking into account the nature of the characterization marker, as mentioned above. Thus, in the case of protein markers, immunological techniques can be used, using specific antibodies of said protein markers. We particularly want to mention ELISA or Western blot techniques. The detection and characterization of nucleic acids, in particular mitochondrial DNA or RNA (in particular messenger RNA or microRNA), includes the use of methods of detection of specific nucleic acids, such as PCR / qPCR, RT-PCR / RT-qPCR, and sequencing. Other assays, such as chromogenic assays, can be used depending on the nature of the biomarker to be detected. Advantageously, a standardization can be carried out based on the quantification of the markers present on or in the microvesicles. The standardization markers can be selected in particular from Annexin A5 and β-actin. For example, the experimental data presented below show that the markers podocalyxin, α-synuclein, and CD41 can be used for the diagnosis of certain diseases. According to certain embodiments, the quantification of these markers is performed on a relative basis by parallel quantification of standardized markers such as Annexin A5 and β-actin. Thus, according to certain embodiments, the method according to the invention comprises the detection of the amount of biomarkers present in or on the captured microvesicles and the detection of the amount of standardized markers. The standardized amounts are then compared for determining the diagnosis, differential diagnosis, for assessing the risk, prognosis, monitoring the progression or for monitoring the effectiveness of therapeutic treatments of various diseases.

[0048] According to a second aspect, the present invention relates to the use of biomarkers that allow the detection of the presence or absence of a particular disease. Characterizing biomarkers are selected in association with the disease of interest.

[0049] According to one embodiment, the present invention relates to a method for the diagnosis, in particular the early diagnosis, of nephropathy in a subject, comprising detecting the presence or absence of podocalyxin or measuring the level of podocalyxin in a sample of biological fluid from said subject. According to a particular embodiment, the method comprises measuring the ratio of podocalyxin to a standardized marker, in particular annexin A5 or β-actin. More particularly, the method according to the invention may comprise detecting the presence or absence of a marker, a cell microvesicle comprising podocalyxin, in particular according to the above-mentioned embodiment comprising the use of a support according to the invention. The present invention also relates to assessing the risk of developing nephropathy, prognosis of nephropathy, monitoring the progression of nephropathy or monitoring the effectiveness of a therapeutic treatment for nephropathy, comprising detecting the presence or absence of podocalyxin in a sample of biological fluid from a subject, in particular podocalyxin contained in a cell microvesicle as characterized by the above-mentioned embodiment, or measuring the level of podocalyxin. According to a particular embodiment, the method according to the invention comprises a step of quantifying the podocalyxin / annexin A5 ratio or the podocalyxin / β-actin ratio, more particularly the podocalyxin / annexin A5 ratio, for detecting renal damage complications in microvesicles derived from a sample of biological fluid from a patient, more particularly a urine sample. If this ratio is statistically higher than the ratio calculated in healthy subjects, it indicates nephropathy. Moreover, if this ratio increases or decreases with respect to the ratio previously calculated for the same patient, it is evidence of progression or remission of nephropathy, respectively.

[0050] Furthermore, the present invention relates to a method for the diagnosis of Parkinson's disease in a subject, comprising the detection of the presence or absence of α-synuclein or the measurement of the level of α-synuclein in a sample of biological fluid from said subject. According to a particular embodiment, the method comprises the measurement of the ratio of α-synuclein to a standardized marker, in particular Annexin A5 or β-actin. More particularly, the method according to the invention may comprise the detection of the presence or absence of a marker, a cell microvesicle comprising α-synuclein, in particular according to the above-mentioned embodiment comprising the use of a support according to the invention. The present invention also relates to the assessment of the risk of developing Parkinson's disease, the prognosis of Parkinson's disease, the monitoring of the progression of Parkinson's disease or the monitoring of the effectiveness of a therapeutic treatment of Parkinson's disease, comprising the detection of the presence or absence of α-synuclein, in particular contained in a cell microvesicle or in the membrane of a cell microvesicle as characterized according to the above-disclosed embodiment, or the measurement of the level of α-synuclein, in a sample of biological fluid from a subject. According to a particular embodiment, the method according to the invention comprises a step of quantifying the α-synuclein / annexin A5 ratio or the α-synuclein / β-actin ratio, more particularly the α-synuclein / annexin A5 ratio, for detecting these neuropathies (e.g. Parkinson's disease) in microvesicles derived from a sample of biological fluid from a patient. If this ratio is statistically higher than the ratio calculated for a healthy subject, it is indicative of Parkinson's disease. Moreover, if this ratio is increased or decreased with respect to a ratio previously calculated for the same patient, it is evidence of progression or remission, respectively, of Parkinson's disease. The invention further relates to a method for the diagnosis, in particular the early diagnosis, of diabetic nephropathy in a subject. According to a particular embodiment, the method according to the invention comprises the detection of the presence or absence of the platelet marker CD41 or the measurement of the level of CD41 in a sample of biological fluid, in particular a urine or plasma sample, more particularly a plasma sample, from said subject. According to a particular embodiment, the method comprises the measurement of the ratio of CD41 to a standardized marker, in particular Annexin A5 or β-actin.More particularly, the method according to the invention may comprise the detection of the presence or absence of cell microvesicles containing the marker CD41, in particular according to the above-mentioned embodiment comprising the use of a support according to the invention. According to a particular embodiment, the method according to the invention comprises the step of quantifying the CD41 / Annexin A5 ratio or the CD41 / β-actin ratio, more particularly the CD41 / Annexin A5 ratio, for detecting diabetic nephropathy complications in microvesicles derived from a sample of biological fluid from a patient, in particular a urine or plasma sample. If this ratio is statistically higher than the ratio calculated for a healthy subject, it is indicative of diabetic nephropathy. Moreover, if this ratio increases or decreases with respect to the ratio previously calculated for the same patient, it is evidence of the progression or remission, respectively, of diabetic nephropathy. Detection of the stage of nephropathy may in particular comprise a step of comparing the characterization results obtained from the test sample of biological fluid with the characterization results of one or more control samples characteristic of different stages of the disease (in particular control samples obtained from diabetic patients with nephropathy in the normoalbuminuric stage (Normo), in the microalbuminuric stage (Micro) or in the macroalbuminuric stage (Macro), these stages corresponding to the severity of the disease, the Macro stage being the most advanced). Thus, according to a particular embodiment, the method according to the invention allows the monitoring and early detection of diabetes and its potential complications, in particular its renal or neuropathic complications.

[0051] The invention will now be illustrated by the following non-limiting examples. EXAMPLES

[0052] Abbreviation: DCM: dichloromethane DMSO: Dimethyl sulfoxide Eq: equivalent EtOAc: Ethyl acetate MES: 2-(N-morpholino)ethanesulfonic acid MilliQ: Water purified by the Milli-Q water purification system MV: Microvesicle NHS: N-hydroxysuccinimide NOS: N-oxysuccinimide Cplx: Complex PVC: Poly(vinyl chloride) PS: Phosphatidylserine Pht: Phthalimide MeCN: Acetonitrile XPS: X-ray photoelectron spectroscopy EDA: Ethylenediamine HUVEC: Human umbilical vein endothelial cells

[0053] The complexes used in this experimental section were synthesised according to the embodiments presented in International Application No. PCT / FR2012 / 050610, in particular the complexes C1, C2 and C4 of the following formulae:

[0054] [ka]

[0055] [ka]

[0056] Example 1 Functionalization of PET supports and grafting of complexes onto the surface. Polyethylene terephthalate (PET) was chosen for the covalent grafting of the complex, which occurs by reaction of the primary amine functional groups of the complex with carboxylic acids present on the surface.

[0057] General Protocol: First, a functionalization step (Figure 1) was used to i) cleave the ester bond, ii) increase the density of COOH functional groups, and iii) make the carbonyl group more electrophilic with an electron-withdrawing group and good leaving group, N-hydroxysuccinimide, to react with the complex more quickly and in better yield. 1) Cut untreated PET film (obtained from Goodfellow) into a 1 cm 2 The PET was pre-cut into a rectangle measuring 5 cm wide by 2 cm long to obtain 10 small squares of PET. The rectangles were cleaned in a tube containing 96% ethanol (Falcon) for 1 hour with sonication. The PET was dried on Kimtech paper for a few minutes. 2) Dip the cleaned rectangle of untreated PET into a solution of sodium hydroxide (10% NaOH in 40 mL MilliQ / MeCN v / v 1 / 1). -3 M) at 60°C for 18 hours. 3) Wash with MilliQ water (3 times), then for 48 hours. 4) Oxidation of PET rectangles in a solution of potassium permanganate (2 g KMnO4 in 38.4 mL MilliQ water and 1.6 mL H2SO4) at 60 °C for 1 h. 5) Wash with acid (HCl 6M), then wash with MilliQ water (twice), and cut the rectangle into small squares measuring 1 cm wide and 1 cm long. 6) The squares were placed into a pill container containing 2 mL of solution (76.7 mg EDC, 23 mg NHS, 39 mg MES in 2 mL MilliQ water per square). 7) Add 2 mL of complex solution (0.2 mL DMSO and 1.8 mL 10 in MilliQ) to each square. -3 M) at room temperature for 16 hours. 8) Rinse with MilliQ water (3 times with sonication) and wash for 48 hours.

[0058] "Control" samples were prepared as required for characterization of the materials. These were samples of untreated PET without hydrolysis, oxidation, and EDC / NHS activation. Steps 1), 7), and 8) were performed, but steps 2), 3), 4), 5), and 6) were not included.

[0059] Material Characterization Quantification of the density of carboxylic acids on the surface for all functionalization steps: A method used to quantify the density of COOH groups present on the surface of a polymer is the use of Toluidine Blue O, a cationic chemical reagent that quantifies the COOH groups present on the surface. - In fact, a basic pH (e.g., pH=10) is required to deprotonate the carboxylic acid groups so that they can react with TBO and form non-covalent bonds. To resorb TBO immobilized on the surface of the material, acetic acid is added to reprotonate the COO- groups and thus form the ionic bond [COO - …TBO] is disconnected.

[0060] A calibration curve is previously constructed using different concentrations of TBO. The optical density readings at 630 nm are reported for the samples, and the surface concentration of COOH can be found by the calibration curve. From the calibration curve, for untreated PET (PET as delivered by the supplier, washed with ethanol, and then dried), we found a 1.5 pmol / mm 2 The density of COOH functional groups is obtained. Similarly, the surface density of COOH functional groups can be evaluated on the PET surface at various processing steps. We obtained the following: - 28 pmol / mm for hydrolyzed PET 2 - 210 pmol / mm for oxidized PET 2 - PET-NHS: 136 pmol / mm 2 - 50 pmol / mm for PET+C1 2

[0061] Determination of atomic percentages on the surface of PET at different stages of processing by photoelectron spectroscopy. These analyses were carried out using a VG ESCALAB 220i-XL spectrometer. Atomic quantification of Si, S, C, N, O, or Zn atoms was carried out by X-ray photoelectron spectroscopy (XPS) in the solvent DMSO / H2O 10 / 90.

[0062] [Table 1]

[0063] [Table 2]

[0064] Quantification of the atomic percentages allows us to state that the complexes are indeed immobilized on the surface of PET. Indeed, PET+C1 contains an atomic percentage of Zn, in contrast to activated PET, which does not contain any metal. Furthermore, note the increase in the theoretically predicted percentage of nitrogen, since the complexes contain six nitrogen atoms at the level of the ligands. This result is supported by the ratios COO / CO, which change from 0.93 (PET-NHS) to 0.65-0.68 (PET+C1), and NCO / COO, etc., which change from 0.06 (PET-NHS) to 0.17-0.25 (PET+C1).

[0065] XPS analysis of functionalized versus non-functionalized supports and / or supports grafted with complexes C1, C2, or C4 These analyses were carried out using a "VG ESCALAB 220i-XL" spectrometer. Nine samples were prepared for X-ray photoelectron spectroscopy analysis: cleaned virgin PET (Smp1), oxidized PET (Smp2), PET-NHS or activated PET (i.e., PET that was hydrolyzed, oxidized, and then activated with EDC / NHS; Smp3), activated PET+C1 (Smp4), activated PET+C2 (Smp5), activated PET+C4 (Smp6), virgin PET+C1 (Smp7), virgin PET+C2 (Smp8), and virgin PET+C4 (Smp9). The high-resolution spectra O1s obtained by XPS show that the complexes are grafted onto the surface of the materials, based on the appearance of peaks for "Zn-O" and "Cu-O" at 530.5-530.7 eV starting from sample 4. At the level of the area ratio Zn-O (or Cu-O) / C=O, the values ​​are 0.15 for Smp4, 0.25 for Smp5, 0.11 for Smp6, and the values ​​decrease later without the activation step to 0.08 for Smp7, 0.15 for Smp8, and 0.14 for Smp9. These results can be explained by the fact that without activation (Smp7, 8, and 9), the complexes are adsorbed on the surface of the support and are not grafted by covalent bonds. Thus, after several rinsings, the complexes are removed and are hardly detectable anymore.

[0066] [Table 3]

[0067] If covalent grafting occurs, a peak at 400 eV (high-resolution spectrum N1s) corresponding to the amide bond NC=O is easily observed by XPS, which means that a bond between the NH2 of the complex and the carbonyl group of the material has indeed occurred. Furthermore, when EDC / NHS activation is not performed and the complex is in contact with the surface (Smp7, 8, and 9), a peak different from the peak at 400 eV is observed. This peak corresponds to the free NH2 generated by the X group of the complex, which can be correlated with adsorption and not with a covalent bond between the complex and the material.

[0068] Optimization of the density of complexes grafted onto surfaces As a variant, the support may be prepared according to the general protocol in which steps 2 and 3 are not performed, or steps 2, 3, 4, and 5 are not performed, in order to facilitate better accessibility of the complex by the phosphatidylserine present on the microvesicles with only the required amount of complex on the surface. Indeed, by omitting the steps of hydrolysis and / or oxidation of PET, it is possible to control the amount of COOH groups on the surface of PET and therefore the amount of complex immobilized on the surface of PET. In the proposed variant, virgin PET is cleaned and then functionalized without prior oxidation or without prior oxidation and hydrolysis. With the proposed variant, only the -COOH functions present on the surface of the support are activated / functionalized for the purpose of grafting the complex. Advantageously, the PET that has not undergone an oxidation step contains a lower amount of carboxylic acid groups, and therefore the resulting grafting density will be lower (d1). - 1.5 pmol / mm for untreated PET 2 COOH - 28 pmol / mm for hydrolyzed PET 2 COOH - 210 pmol / mm for oxidized PET 2 COOH

[0069] Example 2 Functionalization of PVC and grafting of complexes. General Protocol: The protocol used is described with reference to FIG. 1) 1cm 2 A PVC plate with 10 squares was cleaned in a 50 mL tube by sonication in 40 mL MilliQ water for 1 h. The plate was then immersed in a solution containing 2 mL of 97% EDA and 38 mL MilliQ water for 2 h at 30° C. Finally, the 10 square plate was rinsed three times in 10 mL MilliQ water, with sonication performed at each rinse step. 2) The plate was immersed in a solution containing 4 mL of 50% glutaraldehyde and 30 mL of MilliQ water at pH 9.5 for 2 hours at 50° C. The plate was then rinsed three times with 10 mL of MilliQ water, with ultrasonic treatment being performed at each rinsing step. 3) Finally, the squares from each plate treated according to the above steps were-3 The complexes are grafted by immersion in a solution containing 1.9 mL of the complex, 0.1 mL of DMSO, and 1.9 mL of citrate phosphate buffer (0.5 M citric acid, 0.5 M disodium phosphate) at pH 4 at room temperature with stirring for 16 hours. The PVC squares are then rinsed three times in 2 mL of MilliQ water, with ultrasonic treatment being carried out during these rinsing steps. The treated PVC squares are then rinsed three times in 2 mL of MilliQ water over a period of two days.

[0070] Surface characterization by photoelectron spectroscopy (XPS) To characterize the surfaces of the materials, XPS spectroscopy analyses were carried out on the PVC at each step of their functionalization with complex C1. The materials are each shown diagrammatically in FIG. 3. Five surface points were investigated for each material, four corners and one in the center. These analyses were carried out using a VG ESCALAB 220i-XL spectrometer.

[0071] result The spectra and deconvolution of the spectra, as well as the atomic percentages of the various elements present on the surface of the material (Table 3), show that after functionalization with complex C1, zinc (Zn) is present on the surface of the material at a level of 0.25% for PVC. As zinc is the metal in the composition of complex C1, its presence tells us that complex C1 is fixed on the surface of the material. Thus, the functionalization is effective on PVC. Moreover, the atomic percentages determined at five different points on the surface (four corners + one in the center) show low standard deviations (Table 3), demonstrating the uniformity of the treatment and the reproducibility of the results. From this, we assume that the functionalization is uniform on the PVC.

[0072] Example 3 Grafting of Complex C1 on DNA-BIND® Plates General Protocol The complexes were immobilized on a 96-well plate available from Costar as DNA-BIND®, which is made of polystyrene pre-functionalized with N-oxysuccinimide (NOS) functional groups. 14 NOS / cm 2 The surface density of this NOS is approximately the same as the density of COOH functional groups (8.18 × 10) that the inventors have incorporated onto polyethylene terephthalate (PET). 15 COOH / cm 2 ) 1) A solution (total volume = 2 mL) was prepared with 1.78 g of complex, 0.2 mL DMSO, 1.8 mL MilliQ water. 200 μL of this solution was deposited in each well (10 wells). The grafting reaction was carried out for 16 hours at room temperature in the dark. 2) After this reaction time, the solution is removed from the wells and the wells are rinsed with three streams of MilliQ water. Each well is then incubated with fresh MilliQ water and rinsed three times per day for 48 hours.

[0073] Surface characterization by photoelectron spectroscopy To characterize the surfaces of the materials, XPS spectroscopy analyses were carried out on DNA-BIND® at each step of their functionalization with complex C1. The materials are each shown diagrammatically in FIG. 4. Five surface points were investigated for each material, four corners and one in the center. These analyses were carried out using a VG ESCALAB 220i-XL spectrometer.

[0074] result The spectra and deconvolution of the spectra, as well as the atomic percentages of the various elements present on the surface of the material (Table 4), show that after functionalization with complex C1, zinc (Zn) is present on the surface of the material at a level of 0.41% for "DNA-BIND®". As zinc is the metal in the composition of complex C1, its presence tells us that complex C1 is immobilized on the surface of the material. Thus, the functionalization is effective for "DNA-BIND®". Moreover, the atomic percentages determined at five different points of the surface (four corners + one in the center) show a low standard deviation (Table 4), demonstrating the uniformity of the process and the reproducibility of the results. From this, it is deduced that the functionalization is uniform in the two materials: PVC and "DNA-BIND®".

[0075] [Table 4]

[0076] Example 4 Probing the markers carried by the microvesicles after their capture by the kit DNA-BIND® polystyrene grafted with complex C1 Materials and Methods a / Collection of biological fluid samples from diabetic patients Urine and plasma samples were obtained from diabetic patients at the Diabetology Department of Pr Vincent Rigalleau. All patients signed a free and informed consent, explained according to the Declaration of Helsinki and approved by the Ethics Committee. A clinical profile, including the level of albuminuria in the urine, was established for each diabetic patient. Patients were divided into three groups: normoalbuminuric patients or Normo (stage 1 of the complications of the disease), microalbuminuric patients or Micro (stage 2), and macroalbuminuric patients or Macro (stage 3). Urine samples were also collected from healthy donors without known renal complications. All samples were stored at -80°C until used.

[0077] b / Cell model: Human umbilical vein endothelial cells, HUVEC Endothelial cells, human umbilical vein endothelial cells (HUVEC) (Promocell, C-12208), are cultured in Endothelial Cell Growth Medium 2 Kit (Promocell, C-22111), which consists of basal medium and growth factor mixture, without antibiotics.

[0078] c / Isolation of model microvesicles from cell cultures (human umbilical vein endothelial cells or HUVECs) For activation of model microvesicle production, HUVECs are rinsed twice with PBS buffer and stimulated for 24 hours in culture medium containing 100 ng / mL TNF-α (Peprotech, ref. 300-01A). The cell culture medium is collected after 24 hours of stimulation and centrifuged in a 50 mL tube at 12000 g for 2 minutes at 4 °C to remove cells, cell debris and apoptotic bodies. The supernatant is transferred to a clean tube and centrifuged at 20000 g for 90 minutes at 4 °C to sediment the microvesicles. The resulting pellet of model microvesicles is washed by resuspension in 1.5 mL of cold PBS buffer, transferred to a 1.5 mL tube and then centrifuged at 20000 g for 90 minutes at 4 °C. This washing step is repeated once. The pellet of model microvesicles is then resuspended in cold PBS (100-500 μL) depending on the size of the pellet. The corresponding amount of protein is determined by absorbance at 280 nm using a nanodrop spectrophotometer. Samples of the model microvesicles are stored at -80°C until use.

[0079] d / Purification of microvesicles from human urine samples Urine from healthy donors and patients is thawed and microvesicles are purified by differential centrifugation: an initial centrifugation is performed at 12000g for 2 min at 4°C to remove cells, cell debris and apoptotic bodies. The supernatant is centrifuged at 20000g for 90 min at 4°C to sediment the microvesicles. The resulting pellet of microvesicles is washed by resuspension in 1.5 mL of cold PBS buffer, transferred to a 1.5 mL tube and then centrifuged at 20000g for 90 min at 4°C. This washing step is repeated once. Samples are stored at -80°C until use.

[0080] e / Purification of microvesicles from human blood samples The blood collected in the sodium citrate tube is subjected to a first centrifugation at 1500 g. The upper phase, corresponding to the plasma, is carefully removed and centrifuged at 12000 g for 2 min to remove the platelets. The supernatant, corresponding to the platelet-free plasma, PFP, is then centrifuged at 20000 g at 4° C. to sediment the microvesicles. The resulting pellet of microvesicles is washed by resuspension in 1.5 mL of cold PBS buffer, transferred to a 1.5 mL tube and then centrifuged at 20000 g for 90 min at 4° C. This washing step is repeated once. The samples are stored at −80° C. until use.

[0081] f / Protein biomarker detection Enzymatic method: Model microvesicles produced by activation of HUVEC cells have plasminogen activator on their surface, which activates plasminogen (present on the surface of the microvesicles) to plasmin; this activity of plasmin can be detected using a chromogenic substrate, methylmalonyl-hydroxypropyl-arginyl-paranitroanilide, CBS0065. The chromophore is cleaved in the presence of plasmin to a yellow product, the absorbance of which can be measured using a spectrophotometer at a wavelength of 450 nm. Microvesicles resuspended in PBS buffer after isolation from HUVEC are deposited in the wells of a 96-well polystyrene plate (DNA-BIND® Costar) grafted with a binuclear metal complex 1, called complex 1. A total of 2.5 μg of microvesicles (determined by A280 nm nanodrop technique) in 25 μL are incubated for 1 hour at room temperature. After 3 rinses, the enzyme activity of each well is determined. Certain wells are not rinsed and represent 100% of the enzyme activity of the control. First, a standard range is obtained by serial dilution at the following concentrations of plasminogen activator in IU / mL: 0, 0.00078125, 0.0015625, 0.003125, 0.00625, 0.0125, 0.025, and 0.05. Second, a solution of plasminogen at 4 μM and a solution of the chromogenic substrate CBS0065 at 3 mM are prepared and then mixed volume-to-volume. A volume of 25 μL of this mixture is added to each well. The plate is covered with cling film to simultaneously read the absorbance at 405 nm and 450 nm in each well. Place in the spectrophotometer at 37° C. for 18 hours. This kinetic study makes it possible to calculate the average rate of appearance of product, which reflects the enzymatic activity in each well; it is expressed in mOD / min.

[0082] g / Western blot The first step consists of lysis of the microvesicles with RIPA buffer + protease and phosphatase inhibitors (990 μL RIPA buffer + 10 μL (100x) of a cocktail of protease and phosphatase inhibitors). Before adding PBS, add RIPA buffer + inhibitors in a volume as a function of the size of the pellet of microvesicles obtained in the last step of isolation (approximately 4 times the volume of RIPA as the volume of the pellet). The pellet is resuspended by pipetting up and down and left for 15 minutes in ice. The lysate is then centrifuged at 10000xg for 10 minutes at 4°C to remove debris. The supernatant is transferred to a clean tube. The amount of protein present in the lysate of microvesicles is measured by the bicinchoninic acid assay (BCA) technique. The proteins are then denatured using buffer Laemmli 4x + reducing agent (DTT, dithiothreitol). The denatured lysate can be stored at -80°C before use. The denatured lysate of microvesicles is placed on an acrylamide gel under SDS-PAGE denaturing conditions with a gradient of 4-12%. The run is carried out at a constant voltage of 120V for approximately 1.5 hours (until the migration front reaches the bottom edge of the gel). The gel is transferred onto a PVDF membrane. After saturation of the membrane for 1 hour at room temperature with a 5% milk solution in TBS-Tween 0.1% buffer, the three primary antibodies diluted in a 5% milk solution in TBS-Tween 0.1% buffer are incubated overnight at 4°C. The following dilutions are used: antibody against podocalyxin (Santacruz, Sc-23904) 1 / 2000 or antibody against CD41 (Novus, MAB 7616) 1 / 1000; and antibody against β-actin (SIGMA, A1978-100UL) 1 / 10000; and antibody against annexin A5 (SIGMA, A8604-100UL) 1 / 2000. After two washing steps in TBS-Tween 0.1% buffer, the secondary antibody coupled with the enzyme horseradish peroxidase, HRP, at a dilution of 1 / 5000 is incubated in a 5% milk solution in TBS-Tween 0.1% for 1 hour at room temperature.After two washing steps in TBS-tween 0.1% buffer, chemiluminescence detection is performed. The signal is captured by a CCD camera, and the signal intensity is then analyzed using the free software ImageJ.

[0083] h / Statistical analysis For validation of biomarkers in microvesicles obtained from urine of diabetic patients, statistical analysis of the Western blot results was performed using GraphPad Prism software version 7.

[0084] i / ELISA After isolation from urine or plasma from patients or healthy individuals, microvesicles resuspended in PBS buffer are deposited in the wells of a 96-well polystyrene plate (DNA-BIND®, Costar) grafted with a binuclear metal complex 1, called complex 1. An overnight incubation was carried out at 4° C. with gentle horizontal shaking. After two washes in PBS, a saturated solution consisting of 5% “bovine serum albumin” (BSA) protein was deposited in all wells of the plate and incubated for 2 hours at room temperature with gentle agitation. Then, either a primary antibody against podocalyxin at 1 / 500 (Santacruz, Sc-23904) or a primary antibody against the CD41 platelet marker at 1 / 500 (Novus, MAB 7616) is contacted with the microvesicles captured in the wells for 2 hours at room temperature with gentle horizontal shaking. After two washing steps, the secondary antibody linked to the enzyme HRP at a dilution of 1 / 5000 is incubated for 1 hour at room temperature with gentle horizontal rocking. After six washing steps, 100 μL of the chromogen TMB (3,3',5,5'-tetramethylbenzidine) is deposited in the wells for 30 minutes at room temperature with gentle horizontal rocking. The reaction is stopped by adding 100 μL of sulfuric acid and the absorbance of each well is measured at 450 nm using a spectrophotometer.

[0085] result a / Identification of protein markers carried by microvesicles Validation of podocalyxin as a biomarker in microvesicles obtained from urine of diabetic patients according to the stage of renal complications by Western blot technique Podocalyxin is a transmembrane protein expressed by podocytes, glomerular cells found exclusively in the kidney; it has been described in the literature as a marker of podocyte cellular pathology. The protein β-actin is a cytoskeletal protein expressed in all cells; its stable and ubiquitous expression means that it is often used in Western blots as a reference protein when it is necessary to quantify the levels of proteins whose expression may vary as a function of disease.

[0086] The protein annexin A5 is localized near the plasma membrane in all cells; like β-actin, it is also used as a reference protein in Western blots. Thus, the analysis of the expression of the protein podocalyxin in microvesicles obtained from the urine of healthy donors or diabetic patients by Western blot is expressed rather in the form of the ratios podocalyxin / β-actin and podocalyxin / annexin A5. FIG. 5 shows the podocalyxin / β-actin ratio. Each point represents the ratio obtained after Western blot analysis of urinary microvesicle samples obtained from healthy donors or diabetic patients. It can be seen that healthy donors express a low podocalyxin / β-actin ratio of 0.70 on average, which corresponds to the baseline level of natural production of podocyte-derived microvesicles in a non-pathological state. This ratio gradually increases according to the stage of nephropathy associated with diabetes. Thus, it can be seen that the podocalyxin / β-actin ratio reaches, on average, 1.09 in normoalbuminuric patients, then 1.70 in microalbuminuric patients and finally 3.91 in macroalbuminuric patients. ANOVA statistical analysis combined with Tukey's between-group comparison test using GraphPad Prism software revealed significant differences between groups: p-values: p<0.001 for Healthy vs. Macro, p<0.01 for Normo vs. Macro, and p<0.05 for Micro vs. Macro. FIG. 6 shows the podocalyxin / annexin A5 ratio. Each point represents the ratio obtained after Western blot analysis of urinary microvesicle samples obtained from healthy donors or diabetic patients. As before, it can be seen that healthy donors exhibit a low podocalyxin / annexin A5 ratio of 0.50 on average; this corresponds to the baseline level of natural production of podocyte-derived microvesicles in a non-pathological state. Again, the ratio increases gradually according to the stage of nephropathy associated with diabetes. Thus, it can be seen that the podocalyxin / annexin A5 ratio changes, on average, to 0.93 in normoalbuminuric patients, then to 2.15 in microalbuminuric patients, and finally to 4.33 in macroalbuminuric patients. ANOVA statistical analysis combined with Tukey's between-group comparison test using GraphPad Prism software revealed significant differences between groups: Healthy vs. Normo, p<0.05, Healthy vs. Macro, p<0.0001, Normo vs. Macro, p<0.0001, and Micro vs. Macro, p<0.01. In conclusion, it can be envisioned that the podocalyxin / annexin A5 ratio can be used in the context of a kit for detecting renal damage complications in microvesicles derived from patient urine. If this ratio is increased, this is evidence of podocyte cytopathies.

[0087] Detection of platelet marker, CD41, in microvesicles derived from plasma of diabetic patients according to the stage of renal complications by Western blot technique The aim of our experiments in this part is to show that markers can be detected in microvesicles obtained from human plasma and purified by centrifugation. For this purpose, we developed the detection of a protein localized on the surface of the platelet membrane, CD41, by Western blot technique. Microvesicles resulting from the budding of the platelet membrane express CD41. We therefore analyzed the CD41 / Annexin A5 expression ratio in microvesicles derived from the plasma of normoalbuminuric and monadic diabetic patients (Figure 7 below). By Western blot technique, we are able to observe the specific detection of the CD41 platelet marker in protein extracts from microvesicles derived from plasma. The CD41 / Annexin A5 ratio varies from individual to individual. This experiment shows that markers carried by circulating microvesicles can be quantified in plasma.

[0088] conclusion The inventors show that protein biomarkers can be quantified in microvesicles isolated from different biological fluids: urine and plasma.

[0089] Furthermore, the podocalyxin / annexin A5 ratio could be used in a diagnostic kit to detect renal damage complications.

[0090] b / Identification of the surface of DNA-BIND® polystyrene grafted with complex C1 Capture of model microvesicles and enzymatic detection of biomarkers carried by these microvesicles The microvesicles carry biological materials such as enzymes, which are proteins with biological activity. The inventors used the enzymatic technique described in International Application No. PCT / FR2012 / 050610 to verify the grafting of complex C1 on the surface of DNA-BIND® polystyrene. Model microvesicles with a total microvesicle amount of 2.5 μg (protein equivalent) in 25 μL are captured by the material for 1 hour at room temperature. The enzymatic activity present in each experimental condition is analyzed by enzyme kinetics: the rate of appearance of the chromogenic product, CBS0065, catalyzed by the enzyme is monitored for 18 hours. The experimental conditions are as follows: No rinsing condition 100%: This is the control condition, the model microvesicles are left in the wells for 1 hour and the enzyme activity is determined directly, without rinsing the wells. The enzyme activity detected is the maximum activity of the extract of the model microvesicles. Therefore, this condition represents the positive control, 100%. Condition without complex and with rinsing: model microvesicles are incubated for 1 hour in the wells of the plate that are not grafted with complex C1, then rinsed three times and analyzed for enzyme activity. This condition represents a negative control; the enzyme activity obtained is evidence of a non-specific interaction of the model microvesicles. Condition with complex and rinsing: model microvesicles are incubated for 1 hour in the wells of the plate grafted with complex C1, then rinsed three times and analyzed for enzyme activity. This condition represents the capture of the microvesicles by the complex; the enzyme activity obtained is evidence of the specific interaction of the model microvesicles. FIG. 8 shows the mean values ​​of Vi (mean values ​​of calculated initial velocities) obtained for each of the above conditions. In the control condition of microvesicles without rinsing, a mean value of Vi of 0.226 mOD / min is observed, which represents the maximum mean value of Vi obtained for 2.5 μg microvesicles, which is therefore 100%. In the wells in which model microvesicles are incubated on a support not grafted with metal complexes, a mean value of initial velocities of 0.026 mOD / min is measured. The degree of nonspecific interactions is 0.026 / 0.226=0.115, i.e. 11.5%. In the wells in which microvesicles are incubated on a support grafted with metal complex C1 and then rinsed, a mean value of initial velocities of 0.152 mOD / min is measured. The specific capture rate is therefore 0.152 / 0.226=0.672, i.e. 67.2%. In conclusion, this experiment shows that (1) the surface of DNA-BIND® polystyrene grafted with complex C1 allows the capture of HUVEC cell model microvesicles, and (2) the surface of DNA-BIND® polystyrene grafted with complex C1 allows the quantification of the enzyme carried by the model microvesicles. Furthermore, a specific capture of 67.2% of the microvesicles can be evaluated.

[0091] Capture of microvesicles isolated from human urine and ELISA detection of podocyte markers carried by these microvesicles We tested the presence and detection of podocalyxin by an ELISA immunological method to verify the specific capture of microvesicles isolated from urine and to demonstrate the capacity for quantification of the biomarker after capture on our material. To do this, microvesicles isolated by centrifugation from urine were incubated overnight at 4° C. with gentle rocking on DNA-BIND® Costar 96-well polystyrene plates grafted entirely with complex C1. A maximum amount of microvesicles equivalent to 20 μg of protein (i.e. 100 ng / μL in a final reaction of 200 μL) assayed spectrophotometrically was deposited. After several washing procedures, the presence of the protein podocalyxin was detected with an antibody specifically directed against it. A secondary antibody coupled to the enzyme horseradish peroxidase is added to amplify and detect the signal spectrophotometrically at 450 nm in the presence of the chromogenic substrate TMB. The results are reported in FIG. 9. Thus, a specific signal can be observed in the condition: microvesicles captured by the material + primary antibody against podocalyxin + secondary antibody. A signal equivalent to the background noise is observed in all other conditions corresponding to the various controls. It can be concluded that the microvesicles isolated from human urine are specifically captured and that podocalyxin on the surface of the microvesicles is specifically detected. To demonstrate that the ELISA detection of the biomarker protein podocalyxin in microvesicles derived from human urine after capture on the material can be considered semi-quantitative, a range of deposits of microvesicles was carried out, contained at concentrations comprised between 0.5 μg and 20 μg, i.e. between 2.5 ng / μL and 100 ng / μL in a final reaction of 200 μL. The results of the absorbance measurements as a function of the different amounts of microvesicles are presented in FIG. 10. A linear relationship can be observed between the concentration of microvesicles deposited and captured by the material and the signal detected by the ELISA technique for the protein podocalyxin. Thus, the material (complex C1 grafted onto DNA-BIND® polystyrene plates from Costar) not only allows the specific capture of microvesicles isolated from urine, but also makes it possible to quantify the presence of podocalyxin by ELISA techniques, with a detection threshold of 2.5 ng / μL of deposited microvesicles.

[0092] Capture of microvesicles isolated from human plasma and ELISA detection of the CD41 platelet marker carried by these circulating microvesicles We wanted to show that the material (complex C1 grafted onto DNA-BIND® polystyrene plates from Costar) is also capable of capturing circulating microvesicles previously isolated by centrifugation (i.e. isolated from human plasma) and that it is possible to quantify the presence of proteins on the captured microvesicles by ELISA techniques. For this, we chose the protein CD41, which is present on the surface of platelets and is therefore found on the surface of microvesicles derived from their membrane budding. Circulating microvesicles previously isolated by centrifugation from plasma from normoalbuminuric diabetic patients were deposited in polystyrene plates functionalized with complex C1 and incubated overnight at 4° C. with gentle rocking. An amount of microvesicles isolated from 1.5 mL of plasma was deposited per well. After several washing operations, the presence of the protein CD41 was detected with an antibody specifically directed against it. A secondary antibody linked to the enzyme horseradish peroxidase is added to amplify and detect the signal spectrophotometrically at 450 nm in the presence of the chromogenic substrate TMB. The results are reported in FIG. 11. A specific signal is observed in the wells where the plasma microvesicles from the patients (P1 and P2) were deposited. These results show not only the specific capture of circulating microvesicles but also the possibility to quantify by ELISA techniques the presence of a protein on their surface, in this case the platelet protein CD41.

[0093] Capture of microvesicles in human plasma and detection of platelet protein biomarker, CD41, by ELISA Finally, we wanted to show that the material (complex C1 grafted on DNA-BIND® polystyrene plates from Costar) is capable of capturing non-isolated microvesicles. In this case, the sample is therefore obtained directly from human plasma containing microvesicles, and not from microvesicles previously isolated by centrifugation. We tried to quantify the presence of the protein CD41 carried by the microvesicles in the plasma samples using the ELISA technique. For this, 300 μL of plasma from a normoalbuminuric diabetic patient was incubated overnight at 4° C. with gentle rocking on a DNA-BIND® polystyrene plate grafted with complex C1. After several washing operations, the presence of the protein CD41 was detected with an antibody specifically directed against it. A secondary antibody linked to the enzyme horseradish peroxidase is added in order to amplify and detect the signal spectrophotometrically at 450 nm in the presence of the chromogenic substrate TMB. The results are reported in FIG. 12. Although its levels are low, we observe a signal for the detection of CD41 in wells in which plasma from patients (P3, P4, and P5) has been deposited in wells grafted with complex C1. These results show not only the specific capture of microvesicles (not previously purified) present in plasma, but also the possibility of quantifying the presence of a protein on their surface, in this case the platelet protein CD41, by ELISA techniques.

[0094] Example 5 Early diagnosis of diabetic nephropathy by capturing cellular microvesicles on solid supports Materials and Methods Human biofluid samples: All samples were stored at -80°C until use. Human urine samples from healthy subjects: Human urine samples were collected from healthy subjects during the day in normal urine pots without any additives at the Institute of Chemistry and Biology of Membranes and Nano-objects (CBMN) in Pessac. No initial urination urine was collected and samples were stored at -80°C until use. Human urine samples from diabetic patients: Donors were diabetic patients with nephropathy at the normoalbuminuric stage (Normo), microalbuminuric stage (Micro), or macroalbuminuric stage (Macro). These stages correspond to the severity of the disease, with the Macro stage being the most advanced. Urine samples were collected during the day in a normal urine pot without the addition of any additives. Furthermore, no urine was collected at the beginning of urination. Purification of microvesicles Microvesicles derived from cell models: Human umbilical vein endothelial cells, HUVEC (Promocell; ref. C-12208), were cultured in complete medium (Promocell; endothelial cell growth medium 2 kit; ref. C-22111) according to the supplier's recommendations. Between passages 3 and 6, the cells were rinsed twice in PBS×1 and then activated with a solution of TNF-α (Peprotech; ref. 300-01A) prepared at 100 ng / mL in complete medium for 24 hours at 37° C. to produce microvesicles. The supernatant containing the microvesicles was then transferred to a 50 mL tube. First, cell debris and apoptotic bodies were sedimented by centrifugation at 12000 g for 2 hours at 4° C. (SIGMA; thermostatically controlled centrifuge 3-18KHS) to remove them. Secondly, the supernatant containing the microvesicles was transferred to a new 50 mL tube and the microvesicles were sedimented by centrifugation at 20000 g for 1.5 hours at 4° C. The supernatant was removed and the pellet was rinsed with 1 mL of buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4). The solution of microvesicles was transferred to a new 1.5 mL microtube and centrifuged at 20000 g for 1.5 hours at 4° C. The supernatant was removed and the pellet of microvesicles was rinsed again and then centrifuged at 20000 g for 1.5 hours at 4° C. The final pellet of microvesicles was finally resuspended in buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) and this solution was stored at −80° C. until use. Microvesicles from human urine samples: To isolate microvesicles, urine was thawed overnight at 4° C. and 40 mL was centrifuged at 1500 g for 15 min at 4° C. The supernatant was transferred to a new tube and centrifuged at 12000 g for 2 min at 4° C. to remove cell debris and apoptotic bodies. The supernatant was then transferred to a new tube and centrifuged at 20000 g for 1.5 h at 4° C. to sediment the microvesicles. The supernatant was removed and the pellet was rinsed in 1 mL of buffer (HEPES 10 mM; NaCl 0.15 M; pH 7.4). The solution of microvesicles was transferred to a 1.5 mL microtube and centrifuged at 20000 g for 1.5 h at 4° C. The supernatant was removed and the pellet of microvesicles was rinsed again and then centrifuged at 20000 g for 1.5 h at 4° C. The final pellet of microvesicles was finally resuspended in a buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) and the solution was stored at -80°C until use.

[0095] CRYOMEB Observation Capture of microvesicles: Buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) containing microvesicles (derived from cell activation of HUVEC cells or from human urine samples from healthy subjects) was incubated overnight at room temperature at a rate of 300 μL per material and then removed by 10 successive rinses with buffer solution. The materials used were PVC+C1 and 1 cm of "DNA-BIND®" plate wells+C1. 2 It is a rectangle. CRYOMEB OBSERVATION: The samples were observed using a "Quanta 250 FEG FEI" field effect scanning electron microscope (SEM) equipped with a "Quorum PP3000T" cryomodule. First, the samples were glued to a support using a Tissue-Tek® / carbon mixture and then rapidly cooled in pasty nitrogen. They were then placed in the first chamber of the microscope and sublimated at -95°C for 20 min, and then metallized in the second chamber by spraying platinum at 10 mA for 1 min under argon. Finally, the metallized samples were placed in the observation chamber for image acquisition. Capture of Microvesicles on DNA-BIND® Supports Protein assay: To determine the amount of microvesicles present in each solution, the protein was assayed by NanoDrop (ND1000; Thermo Fisher Scientific). Capture of microvesicles: The device used, also called "kit" in this application, is a DNA-BIND® 96-well plate (COSTAR; reference 2525) functionalized with complex C1. The experiment is divided into two parts: one related to the capture of microvesicles by the kit, and one related to control microvesicles that leave the plate and are therefore not captured by the kit. The "control" part conveys information about the initial composition of the microvesicles without capture. The buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) containing the microvesicles is then separated into two equal parts: one for capture and one for control. Part for capturing microvesicles: Buffer solution containing microvesicles (HEPES 10 mM; NaCl 0.15 M; pH 7.4) was incubated on the material at 37° C. for 1 hour or at 4° C. overnight at 50 μL per well. The plate was covered with a film to prevent any evaporation. Then, the buffer solution was removed and the wells were rinsed three times for 10 minutes with the same buffer preparation at 200 μL / well to isolate the captured microvesicles from other objects present in the solution and to remove those non-specifically adsorbed on the material. Then, the microvesicles were dissolved in 30 μL of lysis buffer (99% RIPA buffer, 1% protease inhibitor cocktail) per well for 30 minutes on ice to recover all proteins individually from the microvesicles. After homogenization, the solution was transferred to a 1.5 mL microtube. Membrane debris was sedimented to remove it by centrifugation at 10000 g for 10 min at 4° C. The protein-containing supernatant was collected in a new microtube. Part for control microvesicles: Buffer solution containing microvesicles (HEPES 10 mM; NaCl 0.15 M; pH 7.4) was incubated in 1.5 mL microtubes at 50 μL per microtube for 1 hour at 37° C. or overnight at 4° C. After homogenization of the solution, the microvesicles were sedimented by centrifugation at 20000 g for 1.5 hours at 4° C. The supernatant was removed by aspiration. The microvesicles were then resuspended and dissolved in 30 μL of lysis buffer per microtube for 30 minutes on ice in order to individually recover all proteins from the microvesicles. After homogenization of the solution, membrane debris was sedimented by centrifugation at 10000 g for 10 minutes at 4° C. in order to remove it. The supernatant containing the proteins was collected in a new microtube.

[0096] Western Blot Analysis: Each sample (protein extract) was prepared in a microtube with loading buffer (Bolt™ LDS sample buffer 4x + Bolt™ sample reducing agent 10x) in a ratio of 19.5 μL protein solution to 30 μL final sample in the ratio indicated by the supplier. Samples were vortexed and quickly centrifuged in a tabletop centrifuge before denaturing at 95° C. for 5 min. All samples (30 μL) were loaded onto an acrylamide gel in denaturing conditions. A size marker was always added as a reference. Samples were run at 50 V for 10 min and then at 120 V for 1.5 h. Proteins present in the gel were transferred onto a PVDF membrane by semi-fluid transfer at a constant 1.3 A for 15 min. Non-specific sites of the membrane were saturated in a milk buffer (5% skim milk in buffer Tris-buffered saline (TBS)-Tween 0.1%) bath for 1 h at room temperature with stirring. After two rinses for 10 min in TBS-Tween 0.1% buffer with stirring, the membrane was incubated overnight at 4° C. in a solution of primary antibodies prepared in milk buffer with stirring. The antibodies (Abs) used were Ab anti-podocalyxin (Santacruz, ref. Sc-23904; dilution 1 / 2000), Ab anti-β-actin (Sigma, ref. A1978; dilution 1 / 5000) and Ab anti-annexin A5 (Sigma, ref. A8604; dilution 1 / 2000). After two rinses for 10 min in TBS-Tween 0.1% buffer with stirring, the membrane was incubated for 1 h at room temperature in a solution of secondary antibodies prepared in milk buffer with stirring. Signals were detected by a gel imager (GeneGnome, SYNGENE) using a chemiluminescence reaction after incubation of the membrane with a chromogenic agent in Pico (ThermoFisher SCIENTIFIC, ref. 34580) for 5 min or with a chromogenic agent in Femto (ThermoFisher SCIENTIFIC, ref. 34094) for 2 min, depending on the signal intensity. Image acquisition was performed using GeneSys software. Both qualitative and quantitative analysis was performed using Image J.The qualitative aspect was evaluated by the presence of bands at molecular weights specific for each marker of interest, as well as the intensity of their signals. The quantitative aspect was determined by calculating the ratio of the intensity of the signal of the pathological marker, podocalyxin, to the intensity of the signal of the reference marker of microvesicles, annexin A5. This ratio makes it possible to determine the amount of the pathological marker relative to the number of microvesicles.

[0097] result CRYOMEB analysis Images of capture of microvesicles by PVC+C1 are shown in Figure 13 (microvesicles derived from cellular activation of HUVEC cells) and by "DNA-BIND®"+C1 in Figure 14 (microvesicles derived from cellular activation of HUVEC cells) and Figure 15 (microvesicles derived from human urine samples from healthy subjects). These images show the presence of numerous spherical objects with diameters ranging from 40 nm to 100 nm for PVC+C1 (Figure 13) and an average of 150 nm for "DNA-BIND®"+C1 (Figures 14 and 15), and therefore the presence of phosphatidylserine-positive microvesicles captured by the different materials tested. The 96-well plate functionalized with C1 captures microvesicles derived from a cell model (HUVEC).

[0098] Capture of microvesicles and detection by Western blot First, the capture of microvesicles by the kit was demonstrated by the detection of a reference marker, Annexin A5, carried by model microvesicles after incubation on the kit at two different doses. The results are shown in FIG. 16. The presence of a prominent band at the molecular weight corresponding to Annexin A5 after capture provides evidence of specific and effective recruitment of microvesicles. Furthermore, the change in intensity of the signal as a function of the amount of deposited microvesicles shows a dose response that is also specific. Second, the capture of microvesicles by the kit was demonstrated by the detection of Annexin A5 on microvesicles derived from human urine samples from healthy subjects. The results are shown in FIG. 17. The presence of a prominent band at the molecular weight of Annexin A5 after incubation on the kit is evidence of specific capture of urinary microvesicles. After validation of capture, we tested the detection of pathological biomarkers on microvesicles captured by the kit. The biomarker studied is podocalyxin, which provides evidence of nephropathy at the podocyte level. The microvesicles used are derived from urine samples from healthy subjects and from diabetic patients with microalbuminuric stage (Micro) nephropathy. The results are shown in Figures 18 and 19. Donor-related data are shown in Table 5.

[0099] [Table 5]

[0100] The presence of specific bands of podocalyxin and annexin A5 after capture on the kit, shown in FIG. 18, indicates that Western blot makes it possible to detect pathological markers after capture on the kit. Furthermore, quantification of the signals, shown in FIG. 19, shows that the podocalyxin / annexin A5 ratio, which seems to reveal the disease, is of the same order of magnitude for microvesicles without the kit (shown in black) and for microvesicles captured by the kit (shown in gray). This result indicates that the population of captured microvesicles represents the population originally present in the biological fluid, allowing a relevant diagnosis. Finally, we compared the podocalyxin / annexin A5 ratio of microvesicles derived from urine samples from healthy subjects and from diabetic patients with nephropathy at various stages of the disease (Normo, Micro, Macro), without capture and after capture with the kit. The results are shown in FIG. 20, and the clinical information related to the donors is shown in Table 6.

[0101] [Table 6]

[0102] The schematic diagram in Figure 20 shows that the ratio calculated from the microvesicles after capture on the kit (grey) represents the ratio calculated from the microvesicles without capture (black), regardless of the stage of the disease (Normo, Micro, Macro). The Western Blot detection technique therefore makes it possible to verify that the kit can be used to perform an early diagnosis and to assess the severity of the disease.

Claims

1. A solid support having on its surface a compound of formula (I) or (II) covalently grafted: 【Chemistry 1】 During the ceremony, - M +i represents a metal ion, and i is 1, 2, or 3; - L represents an exchangeable ligand; - X is -(CH 2 ) m -NH 2 Group or -CH 2 -NHC(O)-R-NH 2 R represents a substituted or unsubstituted, linear or branched C 2 ~C 10 , especially C 5 ~C 10 is an alkyl group of the formula: - m=1 to 12; - n=1, 2, or 3; - Y is H or (CH 2 ) p -NH 2 where p=0 to 12.

2. 2. The solid support according to claim 1, characterized in that it is a solid support made of poly(vinyl chloride) (PVC), poly(ethylene terephthalate) (PET) or polystyrene (PS).

3. 3. A solid support according to claim 1 or 2, characterized in that it is a solid support of PET prefunctionalized with NHS functional groups, of PVC functionalized with glutaraldehyde, or of PS functionalized with N-oxysuccinimide functional groups.

4. 4. A solid support according to claim 1, characterized in that M is selected from Zn, Cu, Mn, Co, Ni and Fe, in particular from Zn and Cu, more in particular M is Zn.

5. A solid support according to any one of claims 1 to 4, characterized in that: - X is -(CH 2 ) m -NH 2 , m=1, n=1 and Y represents H; or - X is -(CH 2 ) m -NH 2 , m=1, n=2 and Y represents H; or -X-CH 2 -NHC(O)-R-NH 2 R stands for C 5 H 10 where n=1 or 2, and Y represents H.

6. A solid support according to any one of claims 1 to 5, characterized in that the grafted compound is a compound of formula (I) and in particular is selected from the following: 【Chemistry 2A】 【Chemistry 2B】

7. 7. A method for capturing microvesicles present in a sample of a biological fluid from a subject, the method comprising the step of contacting the sample with a support according to any one of claims 1 to 6.

8. 10. A method for characterizing microvesicles present in a sample of biological fluid from a subject, comprising the steps of contacting the sample with a support described in any one of claims 1 to 6, capturing the microvesicles, and characterizing the captured microvesicles.

9. A method for the diagnosis of a disease, comprising capturing microvesicles present in a sample of biological fluid from a subject on a support according to any one of claims 1 to 6, and detecting the presence or absence of a marker associated with said disease, or measuring the level of said marker specific to said disease.

10. Detecting the presence or absence of podocalyxin from the microvesicles captured on the support or measuring the level of podocalyxin for the diagnosis of nephropathy; or Detecting the presence or absence of alpha-synuclein from the microvesicles captured on the support or measuring the level of alpha-synuclein for the diagnosis of Parkinson's disease; or Detecting the presence or absence of CD41 from microvesicles captured on a support or measuring the level of CD41 for diagnosing diabetic nephropathy.

10. The method of claim 9, comprising:

11. A support according to any one of claims 1 to 6; and Means for detecting or quantifying disease-associated markers, in particular podocalyxin, alpha-synuclein, or CD41 Including the kit.

12. 12. The kit according to claim 11, further comprising means for detecting or quantifying a standardization marker, in particular Annexin A5 or β-actin.

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