Method for detecting peptide biomarkers

Abstract

The invention relates in particular to a method for detecting a peptide biomarker in a biological fluid comprising the steps of: providing a biological fluid sample; adding the fluid to a sensor system comprising, in a 2 to 5 M solution of alkali metal halide, alkaline earth halide or mixtures thereof having a pH of between 3 and10, at least one aerolysin nanopore inserted into a lipid or polymer membrane, the fluid being added on the cis side of the membrane; applying a difference in potential of between -300 and +300 mv to the system comprising the fluid, so as to promote the passage of the at least one biomarker; recording the electrical signal generated at a frequency of 100 kHz to 1 MHz; analysing the signal, the analysis of the generated electrical signal making it possible to detect the presence and / or to determine the concentration or absence of the biomarker.

Description

[0001]Method for detecting peptide biomarkers The present invention relates to the biotechnology sector. More particularly, the invention relates to the field of medical or animal diagnosis by the detection of peptide biomarkers. Prior art More than 200 blood protein biomarkers are already currently used in medical biology, but the needs to improve diagnosis and patient monitoring are still significant. While techniques based on mass spectrometry allow the identification of numerous peptide biomarkers, their routine uses in the clinic still pose difficulties (https: / / www.inserm.fr / dossier / proteomique / ). Furthermore, methods based on antibodies pose problems of cross-reactions and do not allow, for example, the identification of the presence of peptides differing for example, only by an amino acid, or even more difficultly different conformations. Thus, for example,The detection of fibrinopeptide A (FPA), although a proven biomarker of coagulation or even certain cancers, is not implemented in the clinic, particularly due to cross-reactions when using techniques based on recognition by antibodies. The principle of electrical detection of the transport of molecules through a nanopore consisting of a protein channel inserted in a lipid membrane separating two compartments is well known: the membrane is subjected to a potential difference which induces an ionic current through the nanopore in the presence of an electrolyte solution. The passage of a molecule through the nanopore, or the interaction of the molecule with the nanopore, induces a measurable drop in current. In this field,the use of the aerolysin nanopore is known and it has been shown that aerolysin forms a channel sensitive to electrical current in a lipid bilayer. Patent EP3270139 describes the electrical detection of peptides differing in their amino acid sequence by an aerolysin nanopore and this at the scale of an amino acid. Nevertheless, methods that would allow the detection, within complex biological media such as biological fluids (serum, urine, cerebrospinal fluid), of peptide biomarkers by analyzing the electrical signal of an aerolysin nanopore are not known in the state of the art. Indeed, directly identifying and quantifying the target biomarker from a sample of biological fluid containing by definition a mixture of a large number of molecules may require varying the experimental conditions (pH, temperature,etc.) to ensure specific identification of the target biomarker. The inventors have developed a method for analyzing peptide biomarkers using aerolysin nanopores in biological fluids that are complex mixtures. Compared, for example, to mass spectrometry methods, the invention allows for the reliable and sensitive detection of these biomarkers, and thus a rapid diagnostic test "at the patient's bedside". This method also has the particular advantage of gains in sensitivity and specificity for biomarkers with a short half-life, and / or not achieved by methods based on the use of antibodies for the reasons mentioned above. These methods are of particular interest for implementation in diagnostic methods. Thus,an object of the invention is to propose a diagnostic method comprising the detection of at least one peptide biomarker in a complex mixture such as a biological fluid, by analyzing the electrical signal associated with aerolysin in the presence of the biological fluid, in response to the application of a potential difference. Brief description of the invention As mentioned above, the inventors have developed a method for characterizing and quantifying peptides present in a biological fluid. The inventors have notably realized that the components or characteristics of the electrical signal generated during the passage of peptides through the nanopore were characteristics of the latter and made it possible to differentiate the same peptide with different conformations,of post-translational modifications and only slightly different in their sequence. Such a method is of interest both in the field of biotechnology and in the medical field. Therefore, a first subject of the invention is a method for detecting at least one peptide biomarker in a biological fluid comprising the following steps: - providing a sample of biological fluid, - adding the biological fluid to a sensor system comprising, in a solution of alkali metal halide, alkaline earth halide or mixtures thereof, from 2 M to 5 M and a pH of 3 to 10, at least one aerolysin nanopore inserted into a lipid, polymeric or solid membrane defining a cis side and a trans side, said biological fluid being added to the cis side of the membrane, - applying to said sensor system comprising the biological fluid a potential difference of between -300 mV and +300 mV, preferably between -200 mV and +200 mV,of a nature to promote the passage of said at least one peptide biomarker, - recording the electrical signal generated at a frequency of 100 kHz to 1 MHz, and - analyzing the electrical signal generated: o by determining a mean, median, minimum and / or maximum occupancy time of the at least one aerolysin nanopore of the sensor system, o by determining a mean, minimum, median and / or maximum blocking rate of the at least one aerolysin nanopore of the sensor system, o by analyzing the shape of the electrical signal generated, o by determining the standard deviation of the signal measured during the passage of said peptide biomarker through said nanopore, and o by determining the number of events, said analysis of the electrical signal generated making it possible to detect the presence and / or determine the concentration or absence of the at least one peptide biomarker in said biological fluid. Indeed,it has been discovered that the analysis of the characteristics of the electrical signal alone makes it possible to differentiate identical peptides but with different conformations (such as a secondary structure, a statistical conformation or a particular enantiomerism), or carrying post-translational modifications or even peptides not differing by sequences of only a few amino acids, or even just one. Thus this analysis of these characteristics makes it possible to envisage the characterization in complex biological samples of biomarkers differing by their conformation of post-translational modifications or their amino acid sequence by using the aforementioned characteristics of the electrical signals generated by these biomarkers in the sensor system mentioned above. According to other optional characteristics of the method, the latter may optionally include one or more of the following characteristics,alone or in combination: - the at least one peptide biomarker corresponds, for a given peptide, to the detection of the presence and / or absence of at least one post-translational modification and / or at least one conformation of said peptide; in fact, the method of the invention allows the efficient characterization of the presence in a mixture of peptides which only present such differences, without heavy equipment and complicated handling, - the at least one peptide biomarker has a size of between 3 and 100 amino acids; - the at least one peptide biomarker is selected from: histones H1, H2A, H2B, H3 and / or H4, B-type natriuretic peptide, chemokines, interleukins, or one of their receptors or its subunit, adiponectin, NLRP3, insulin chain A, insulin chain B, a cytochrome C, fibrinopeptide A (FPA), phosphorylated FPA (FPA-P), FPA-3, FPA-6, bradykinin, des-arginine, 9 bradykinin, Aβ1-15 , Aβ 1-16 , Aβ 1-17 , Aβ 1-40 , Aβ 1-42, Aβ11-40, Aβ17-40, Aβ17-42, ADA, ARTN, AXIN1, Beta-NGF, CASP- 8, CD40, CDCP1, CST5, DNER, CCL11, 4E-BP1, FGF-21, LAP TGF- beta-1, LIF, LIF-R, CSF-1, MMP-1, MMP-10, MCP-1, MCP-2, MCP-3, MCP-4, CD244, NT-3, NRTN, OSM, OPG, FGF-23, FGF-5, FGF-19, Flt3L, GDNF, HGF, IFN-gamma, PD-L1, EN-RAGE, SLAMF1, SIRT2, STAMBP, SCF, ST1A1, CD6, CD5, CD8A, TSLP, TNFB, TRANCE, TRAIL, TGF-alpha, TWEAK, TNF, TNFSF14, TNFRSF9, uPA, and VEGF-A; these biomarkers are of particular interest in medicine and biotechnology, - the electrical signal is collected over a period of time of at least 1 minute; in fact, a sufficiently long acquisition time increases the precision and the probability of detection of the biomarker, - the sensor system comprises at least two sensor systems; multiplying the sensor systems increases sensitivity, - the biological fluid is selected from: blood, plasma, serum, cerebrospinal fluid, bronchoalveolar lavage,saliva, synovial fluid, amniotic fluid, urine, a cell lysate, a culture medium of eukaryotic or bacterial cells; in fact the method of the invention is little or not sensitive to the biological fluid comprising the at least one biomarker; - the ratio of the volume of biological fluid to the volume of the electrolyte in the compartment on the cis side of the membrane of the at least one sensor system is less than or equal to 1 / 50; in fact the method according to the invention is particularly sensitive and therefore makes it possible to detect the at least one biomarker at dilutions of the biological fluid, which limits, if necessary,the risks of unexpected interference of certain compounds of the biological fluid with the function of the aerolysin nanopore. A second subject of the invention relates to a method for diagnosing a pathology associated with a coagulation disorder in a subject suspected of suffering or suffering from such a pathology comprising the detection of FPA or its derivatives such as phosphorylated FPA (FPA-P), FPA-3, FPA-6, or mixtures thereof, in a biological fluid originating from said subject, said method comprising the following steps: - providing a sample of biological fluid from said subject, - introducing the biological fluid into a sensor system comprising, in a solution of alkali metal halide, alkaline earth halide or mixtures thereof, from 2M to 5M and with a pH of 4.1 to 10, at least one aerolysin nanopore inserted into a lipid or polymeric membrane defining a cis side and a trans side,said biological fluid being added to the cis side of the membrane, - applying to said sensor system comprising the biological fluid a potential difference of between +1 mV and +300 mV, preferably between +10 mV and +100 mV, preferably +50 mV such as to promote the passage of FPA and its derivatives, - recording the electrical signal generated at a frequency of 100 kHz to 1 MHz, and - analyzing the electrical signal generated, said analysis of the electrical signal generated making it possible to detect the presence and / or determine the concentration or absence of FPA or its derivatives in said biological fluid, a number of events detected, in said biological fluid, for one or more of the biomarkers selected from FPA, FPA-P FPA-3, and FPA-6, greater than that of a control or a threshold value determined for subjects not having a thrombotic event,being a sign of increased thrombotic activity in said subject. Such a method is effective in the characterization and quantification of biomarkers such as FPA, FPA-P FPA-3, and FPA-6 of FPA and their different conformations which are related to the coagulation processes taking place in the body. A third subject of the invention relates to a method for diagnosing bradykinin angioedema in a subject suspected of suffering or suffering from such a pathology comprising the following steps: - providing a sample of biological fluid from said subject, - introducing the biological fluid into a sensor system comprising, in a 2M to 5M alkali or alkaline earth metal halide solution and a pH of 3.1 to 10, an aerolysin nanopore inserted into a lipid or polymeric membrane, said biological fluid being added to the cis side of the membrane,- applying to said sensor system comprising the biological fluid a potential difference of between -300 mV and -1 mV, preferably between -100 mV and -10 mV, preferably -50 mV, such as to promote the passage of bradykinin and / or des-arginine, 9 bradykinin, - record the electrical signal generated at a frequency of 100 kHz to 1 MHz, - analyze the generated electrical signal, said generated electrical signal making it possible to detect the presence and / or determine the concentration or absence of bradykinin and / or des-arginine 9 bradykinin or their different conformations in biological fluid, characterization of the presence of levels of bradykinin and / or des-arginine 9bradykinin levels higher than the control or a threshold value determined for subjects not suffering from bradykinin angioedema or angioedema being the sign of the presence of bradykinin angioedema in said subject. Such a method is of significant interest, because at present there is no rapid method for diagnosing these angioedemas which have a specific therapeutic solution and different from histamine angioedemas and for which the wrong diagnosis can have vital consequences for the subject.Brief description of the drawings Other characteristics, advantages and details of the invention will be better understood on reading the additional description which follows of embodiments by way of example in relation to the appended drawings in which: Figure 1 represents the different fragments resulting from the fibrinolysis of fibrinogen, which results in the release of fibrinopeptide A (FPA), which can be phosphorylated (FPA-P) in the organism or not, or cleaved in its terminal part thus generating the loss of 3 (FPA-3) or 6 amino acids (FPA-6). The P circled with a dotted line indicates the location of the phosphorylation present in FPA-P. Figure 2 represents a part of a sensor system used in the method according to the invention comprising the lipid bilayer in which an aerolysin nanopore is embedded, in an electrolyte mixture (4M KCl, 25 mM Tris HCl, pH7.5).The membrane defines a cis compartment and a trans compartment, the cis compartment being the one where the part of the nanopore outside the membrane is located. The potential difference applied to the system generates a force to attract the peptides included in the electrolyte mixture into the cis compartment relative to the orientation of the nanopore. The electrical signal is detected and analyzed according to different parameters that allow the presence of peptides in the cis compartment to be identified, following the addition of the biological fluid. Figure 3 is a representation of an electrical signal generated when a peptide biomarker passes through the aerolysin nanopore, in the sensor system used in the method. I0 is the base ionic current when no analyte passes through the nanopore. The passage of the biomarker through the nanopore causes a drop in the measured current Ib.ΔI therefore represents the blockage of the nanopore caused by the passage of the analyte. The blocking time Dt is the time taken by the analyte to disrupt the current. This time therefore corresponds to the time taken for the analyte (the peptide biomarker) to pass through the nanopore or to the time taken for the analyte to interact with the nanopore. Figure 4 represents the analysis of the electrical signals generated by FPA (left panel), FPA-P (middle) and their mixture (right panel). The representation, for each event, of the blocking current as a function of the blocking time reveals the different populations, and makes it possible to distinguish the two species in their mixture (right panel). The concentration in the cis compartment of the sensor system of FPA and FPA-P, added alone, is 40 µM. The concentration of each of these species in the mixture of FPA and FPA-P is 10 µM. Data is filtered at 5 kHz and recorded at 250 kHz intervals.In Figure 5, the upper panel represents the analysis of the electrical signal generated by a mixture of FPA (10 µM), FPA-P (20 µM), FPA-3 (10 µM) and FPA-6 (2.5 µM). The representation of the signal of each event as a function of ΔInorm as a function of Dt clearly allows to differentiate and detect each of the species and their conformation. The lower panel is a representation of the ΔInorm as a function of the number of events for the same mixture. This representation allows to estimate for each species the average normalized blocking time. Figure 6 presents for each species of FPA and its derivatives, the determination of the average blocking time. A log-normal distribution is used. Figure 7 represents a combinatorial analysis of the ΔI. norm and Dt of an electrical signal recording of a mixture comprising FPA (10 µM), FPA-P (20 µM), FPA-3 (10 µM) and FPA-6 (2.5 µM), at 50 mV. Figure 8 represents the determination of the different ΔInorm for des-arginine 9bradykinin (2 µM, left panel) and bradykinin (2 µM, middle panel) and their equimolar mixture (1.3 µM, right panel). For both bradykinin and des-arginine 9 bradykinin, two conformations each characterized by a specific ΔInorm are detected. A Gaussian distribution is used. Acquisitions are carried out at a potential difference of –50 mV and the electrolyte of the sensor system is 4M KCL 25 mM Tris pH 7.5. Figure 9 represents a combinatorial analysis of the ΔInorm and Dt of a recording of the electrical signal of a mixture comprising their equimolar mixture 1.3 µM bradykinin and 1.3 µM des-arginine 9 bradykinin. This representation shows the two conformations of bradykinin (BK1 and BK2) and the two conformations of des-arginine 9bradykinin (DAB1 and DAB2). Acquisitions are performed at a potential difference of –50 mV and the electrolyte of the sensor system is 4M KCL 25 mM Tris pH 7.5. Figure 10 represents the analysis of a serum containing 100 nM des-arginine 9 bradykinin. The serum was diluted to one hundredth in the cis compartment of the sensor system. The representation of the number of events as a function of their ΔI normallows the identification of the two forms DAB1 and DAB2. The electrical signals were recorded over 27 minutes. The average ΔInorm determined by assuming a Gaussian distribution of the data correspond to those determined in Figure 9. The acquisitions are carried out at a potential difference of –50 mV and the electrolyte of the sensor system is 4M KCL 25 mM Tris pH 7.5. Figure 11 represents the possible classification of different peptides and different conformations of these peptides according to the shape of the electrical signal generated by the nanopore in contact with one of the peptides BK1, BK2, DAB1 and DAB2. A) shape of the identified signals; B) the representation for each event of ΔInorm as a function of Dt clearly allows the differentiation of species.C) Principal component analysis diagram after classification of the blockages observed in an equimolar mixture of 100 nM or 50 nM peptides under serum-free conditions (diamond) shows that the signal shape allows classification of the events. Figure 12 represents a graph showing the proportionality of the frequency of current blockage events as a function of the peptide concentration, equimolar mixture of BK1 and DAB1, for DAB2 and BK2 for concentrations varying from 10 nM to 1 µM. Figure 13 represents the representative electrical signals generated during the transition from the oxidized (A) or reduced (B) form of the L-AVP or D-AVP peptides which are differentiated by the presence of the enantiomeric form of asparagine present at residue 8 of these 9 amino acid peptides.Figure 14 is another representation of an electrical signal generated during the passage of a peptide biomarker through the aerolysin nanopore, in the sensor system used in the method. Characteristics of the signal are indicated therein which can be used to characterize each blockage such as the minimum, average, maximum blocking rate (ΔIbmin, ΔIb, ΔIbmax), the standard deviation (σ) and occupation time (Dt). Description of embodiments of the invention As is apparent from the above, it is now possible, according to the method according to the invention, to detect from an electrical measurement and the analysis of its different components, to distinguish and characterize directly in a biological liquid the presence of peptide biomarkers, which are distinguished, for example, only by at least one post-translational modification or their conformation, and this for the same primary sequence, and / or a difference as small as a single amino acid.This is illustrated by the experimental data accumulated by the researchers. For example, the method according to the invention makes it possible to reliably discriminate between FPA in its phosphorylated or non-phosphorylated form and also between different conformations of FPA, which is not possible using methods based on epitope / antibody recognition due to the proximity of the sequences or very close or even identical molecular masses. According to another example, the method according to the invention makes it possible to discriminate between bradykinin and des-arginine. 9bradykinin, and to identify the presence of different conformations of these molecules, directly in human serum without any particular treatment or purification. According to yet another example, the invention makes it possible to differentiate peptides distinguished only by their conformation, for example a secondary structure or an enantiomerism, as is currently demonstrated for vasopressin and desmopressin. In the remainder of the description below, the implementation of the method according to the invention for the detection of biomarkers according to their conformation, their post-transcriptional modifications or their sequence will be described more particularly for illustrative purposes. The normalized blocking current ΔI norm , or blocking rate, is defined according to the following formula: ΔI norm =(I0-I b) / I0In which - I0 is the current passing through the nanopore when the nanopore is free, - Ib is the blocking current which passes through the nanopore when it is transiently blocked during the passage of a peptide biomarker molecule through the nanopore. The blocking time Dt is the duration of occupation of the nanopore by the biomarker and corresponds to the duration of the current drop generated by the passage of the peptide biomarker or its interaction time with the nanopore. The number of events corresponds to the number of current drops observed over a given duration in the presence of the or a mixture of peptide biomarkers, for a sensor system. An event therefore corresponds to the passage of a species through the nanopore or a prolonged interaction with it: events with a duration greater than or equal to 75 µs are considered for the purposes of the invention. The number of events is therefore associated with the concentration of the peptide biomarker.A sensor system is for example described in Figure 2. For the purposes of the method according to the invention, several sensor systems can be used independently and separately. Although the method of the invention is particularly sensitive and makes it possible to detect peptide biomarker concentrations in the nanomolar range, using several sensor systems in parallel has the advantage of increasing the number of events analyzed and consequently of further lowering the detection threshold of the method according to the invention. Also, using a sensor system comprising several aerolysin nanopores is possible, however this configuration is less preferred. Any functional aerolysin nanopore can be used for the purposes of the present invention. The so-called "wild" aerolysin nanopore from Aeromonas hydrophila (uniprot reference P09167; https: / / www.uniprot.org / uniprotkb / P09167 / entry) can be used.A peptide biomarker is defined as a peptide whose presence, concentration or absence in a biological sample is associated with a pathological, physiological or metabolic state. Detected in a subject's organism, the presence, concentration or absence in a biological sample is associated with the presence or an increased risk of the occurrence of a pathological, metabolic or physiological state in said subject. According to a particular embodiment, a peptide biomarker that can be detected with the method of the invention may comprise 3 to 100 amino acids, or even 3 to 25 amino acids. Also, the method may comprise subjecting the biological sample to enzymatic digestion of the peptides and proteins it contains.The method according to the invention is particularly suitable for detecting the presence and quantifying biomarkers characterized by a significant polymorphism in the population or a specific polymorphism linked to a pathological, physiological or metabolic state (this polymorphism may be linked to a conformation, a post-translational modification, a sequence polymorphism, etc. as described previously).The peptide biomarkers detected by the method according to the invention can be selected from the following group of peptide biomarkers: histones H1, H2A, H2B, H3 and / or H4, B-type natriuretic peptide, chemokines such as those listed in Tables 1, 2 and 3, adiponectin, NLRP3, insulin chain A, insulin chain B, a cytochrome C, an interleukin or one of its receptors or one of the subunits of this receptor, fibrinopeptide A (FPA), phosphorylated FPA (FPA-P), FPA3, FPA6, bradykinin (BK), des-arginine. 9bradykinin (DAB), Aβ1-15, Aβ1-16, Aβ1-17, Aβ1-40, Aβ1-42, Aβ11-40, Aβ17-40, Aβ17-42,ADA, ARTN, AXIN1, Beta-NGF, CASP-8, CD40, CDCP1, CST5, DNER, CCL11, 4E-BP1, FGF-21, LAP TGF- beta-1, LIF, LIF-R, CSF-1, MMP-1, MMP-10, MCP-1, MCP-2, MCP-3, MCP-4, CD244, NT-3, NRTN, OSM, OPG, FGF-23, FGF-5, FGF-19, Flt3L, GDNF, HGF, IFN-gamma, PD-L1, EN-RAGE, SLAMF1, SIRT2, STAMBP, SCF, ST1A1, CD6, CD5, CD8A, TSLP, TNFB, TRANCE, TRAIL, TGF-alpha, TWEAK, TNF, TNFSF14, TNFRSF9, uPA, VEGF-A. Chemokines are a family of small, soluble proteins, 8-12 kilodaltons in size. Chemokines are involved in various biological processes such as hematopoiesis and angiogenesis. As such, they can be markers of tumor proliferation or inflammation, for example. Most of them are characterized by the presence of four cysteine ​​residues in conserved positions that are essential to their three-dimensional structure.There are four families of chemokines, depending in particular on the positioning of the two proximal cysteines of their N-terminal end. CXC chemokines (Table 1) have one amino acid between the first two cysteines, so-called CC chemokines (see Table 2 specifying, but not limited to, human CC chemokines) have the first two adjacent cysteines, so-called CX3C chemokines (see Table 3, specifying, but not limited to, human CX3C chemokine), whose only member (fractalkine) has three amino acids between the first two cysteines, C chemokines (Lymphotactin alpha and beta) have only two cysteines in total (see Table 3 specifying, but not limited to, human C chemokines).The method according to the invention is particularly suitable for characterizing and quantifying the presence in a biological fluid of such proteins of close structure and / or sequences whose presence is linked to a pathological, physiological or metabolic state. Table 1 CXC N chemokines. om Référence Nom Reference U niprot Uniprot CXCL1 P09341 CXCL10 P02778 CXCL2 P19875 CXCL11 O14625 CXCL3 P19876 CXCL12 P48061 CXCL4 P02776 CXCL13 O43927 CXCL5 P42830 CXCL14 O95715 CXCL6 P80162 CXCL15 Q9WVL7 CXCL7 P02775 CXCL16 Q9H2A7 CXCL8 P10145 CXCL17 Q6UXB2 CXCL9 Q07325 Table 2 Chemokines CC N om Référence Nom Reference U niprotUniprot CCL1 P22362 CCL16 O15467 CCL2 P13500 CCL17 Q92583 CCL3 P10147 CCL18 P55774 CCL4 P13236 CCL19 Q99731 CCL5 P13501 CCL20 P78556 CCL6 P27784 CCL21 O00585 CCL7 P80098 CCL22 O00626 CCL8 P80075 CCL23 P55773 CCL9 / CCL10 P51670 CCL24 O00175 CCL11 P51671 CCL25 O15444 CCL12 Q62401 CCL26 Q9Y258 CCL13 Q99616 CCL27 Q9Y4X3 CCL14 Q16627 CCL28 Q9NRJ3 C CL15 Q16663Table 3 Chemokines C Reference name Uniprot XCL1 P47992 XCL2 Q9UBD3 Chemokines CX3C Reference name Uniprot CX3CL1 P78423 Also, interleukins (and some of their receptors) are proteins experiencing significant polymorphism in the population and of particular interest in the study of inflammatory states of individuals, the diagnosis of pathologies and the understanding of the inflammatory mechanisms underlying certain pathologies (such as for example COVID-19). Interleukins, or one of their receptors, different forms can be detected by the method of the invention are listed in Table 4 below. Table 4 N om Référenceuniprot Interleukin-1 alpha (IL-1 alpha) P01583 Interleukin-2 (IL-2) P60568 Interleukin-2 (receptor subunit beta, IL-2RB) P14784 Interleukin-4 (IL-4) P05112 Interleukin-5 (IL5) P05113 Interleukin-6 (IL6) P05231 Interleukin-7 (IL-7) P13232 Interleukin-8 (IL-8) P10145 Interleukin-10 (IL10) P22301 Interleukin-10 (receptor subunit alpha, IL-10RA) Q13651 Interleukin-10 (receptor subunit beta, IL-10RB) Q08334 Interleukin 11 (IL-11) A8K3F7 Interleukin-12 (beta subunit, IL-12B) P29460 Interleukin-13 (IL-13) P35225 Interleukin-15 (alpha subunit of the receptor, IL-15RA) Q13261 Interleukin-17A (IL-17A) Q16552 Interleukin-17C (IL-17C) Q9P0M4 Interleukin-18 (IL-18) Q14116 Interleukin-18 receptor 1 (IL-18R1) Q13478 Interleukin-20 (IL-20) Q9NYY1 Interleukin-20 (alpha subunit of the receptor, IL-20RA) Q9UHF4 Interleukin-22 (alpha subunit 1 of the receptor, IL-22 RA1) Q8N6P7 Interleukin-24 (IL-24) Q13007 Interleukin-33 (IL-33) O95760 More generally,The method is also particularly suitable for the identification of biomarkers related to the other inflammation proteins listed in Table 5 below. Table 5 N, om Référence Reference u niprot Nom uniprot Adenosine Deaminase Fibroblast growth ( ADA) P00813 factor 23 (FGF-23) Q9GZV9 Artemin (ARTN) Q5T4W7Fibroblast growthfactor 5 (FGF-5)Q8NF90 Axin-1 (AXIN1) O15169Fibroblast growthfactor 19 (FGF-19)O95750 Fms-related Beta-nerve growth factor (Beta-NGF) P01138 tyrosine kinase 3 P49771 ligand (Flt3L) Caspase-8 (CASP-8) Q14790Glial cell line- P39905 derived neurotrophic factor (GDNF) CD40L receptor Hepatocyte growth (CD40) P25942 factor (HGF) P14210 CUB domain-containing protein 1 Q9H5V8 Interferon gamma P01579 (IFN-gamma)(CDCP1) Programmed cell Cystatin D (CST5) P28325 death 1 ligand 1 Q9NZQ7 (PD-L1) Delta and Notch-like epidermal growth Q 8NFT8 Protein S100-A12P80511 factor-related (EN-RAGE) receptor (DNER) Signaling lymphocytic Eotaxin (CCL11) P51671 Q13291 activation molecule (SLAMF1) Eukaryotic translation initiation factor Q13541 SIR2-like protein Q8IXJ6 2 (SIRT2) 4E-binding protein 1 (4E-BP1) Fibroblast growth STAM-bindingfactor 21 (FGF-21) Q9NSA1 protein (STAMBP) O95630 Latency-associated peptide transforming growth P01137 Stem cell factor P21583 (SCF) factor beta-1 (LAP TGF-beta-1) Leukemia inhibitory Sulfotransferasefactor (LIF) P15018 1A1 (ST1A1) P50225Leukemia inhibitory T cell surface factor receptor P42702 glycoprotein CD6 Q8WWJ7 (LIF-R) isoform (CD6) Macrophage colony- T-cell surface stimulating factor 1 P09603 glycoprotein CD5 P06127 (CSF-1) (CD5) Matrix T-cell surface metalloproteinase-1 P03956 glycoprotein CD8 P01732 (MMP-1) alpha chain (CD8A) Matrix Thymic stromal metalloproteinase- P09238 lymphopoietin Q969D9 10 (MMP-10) (TSLP) Monocyte chemotactic protein P13500 TNF-beta (TNFB) P01374 1 (MCP-1) TNF-related Monocyte activation- chemotactic protein P80075 O14788 induced cytokine 2 (MCP-2) (TRANCE) TNF-related Monocyte apoptosis- chemotactic protein P80098 P50591 inducing ligand 3 (MCP-3) (TRAIL) Monocyte Transforming chemotactic protein Q99616 growth factor P01135 4 (MCP-4) alpha (TGF-alpha) Tumor necrosis Natural killer cell factor (Ligand) receptor 2B4 (CD244) Q9BZW8 O43508 superfamily, member 12 (TWEAK) Neurotrophin-3 (NT- Tumor necrosis 3 ) P20783 factor (TNF) P01375Tumor necrosis factor ligand Neurturin (NRTN) Q99748 superfamily O43557 member 14 (TNFSF14) Tumor necrosis factor receptor Oncostatin-M (OSM) P13725 Q07011 superfamily member 9 (TNFRSF9) Urokinase-type Osteoprotegerin ( OPG) O00300plasminogen P00749 activator (uPA) Vascular endothelial P15692 growth factor A (VEGF-A) B-type natriuretic peptide (BNP) is a well-known marker of heart failure. The precursor of BNP is secreted by cardiac muscle cells of the atria and ventricles. Its synthesis is increased in cases of distension of this tissue. BNP is notably a biomarker of acute heart failure. BNP has 32 amino acids. Histones are elements of the nucleosome, which comprises 2 copies of each of the four histones and a 147-base pair stretch of DNA. Post-translational modifications of histones are numerous, such as, for example, acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP-ribosylation, deamination, and proline isomerization or citrullination.These modifications are strongly involved in the epigenetic mechanisms of gene regulation, and constitute biomarkers of certain pathologies such as multiple sclerosis, Alzheimer's disease, sepsis. The method according to the invention is particularly suitable for characterizing and quantifying the presence in a biological fluid of such structural proteins and / or close sequences presenting post-translational modifications, the presence of which is linked to a pathological, physiological or metabolic state. Adiponectin (Uniprot reference: Q15848) is a protein whose plasma levels are inversely correlated with the risk of metabolic pathologies such as insulin resistance, diabetes or metabolic syndrome. The level of adiponectin is notably used in the diagnostic orientation and therapeutic management of lipodystrophy and severe insulin resistance syndromes.NLRP3 (Uniprot reference: Q96P20) is a protein that is part of one of the inflammasome complexes and has been identified as a biomarker of the inflammatory state of an organism. High levels of inflammasome proteins are detected in the biofluids of patients suffering from chronic inflammatory pathologies such as diabetes, HIV, chronic obstructive pulmonary disease (COPD), pneumonia, psoriasis, psoriatic arthritis, macular degeneration, and neurodegenerative pathologies. More specifically, NRLP3 is described as a prognostic biomarker in sepsis, or for the diagnosis of neuroinflammatory pathologies such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis. For example, higher levels of plasma NLRP3 are detected in subjects suffering from Parkinson's disease.The cited Uniprot references are commonly accessible on the website https: / / www.uniprot.org / which lists sequence and functional data from international research. Aβ1-15, Aβ1-16, Aβ1-17, Aβ1-40, Aβ1-42, Aβ11-40, Aβ17-40, Aβ17-42 correspond to the different peptides derived from the amyloid peptide found in particular in the cerebrospinal fluid of subjects suffering from Alzheimer's disease. A biological fluid is understood to be any liquid sample secreted, produced or isolated from a living organism. More particularly, such biological fluid may include: blood, plasma, serum, cerebrospinal fluid, bronchoalveolar lavage, saliva, synovial fluid, amniotic fluid, urine, cell lysate, eukaryotic or bacterial cell culture medium.Although not necessary, said biological fluid may be treated before its application to the sensor system, the pretreatment aiming to facilitate the detection, or the identification of the peptide biomarker(s) present in the biological fluid. FPA is a 16 amino acid peptide, originating from the proteolysis of the amino-terminal end of the Aα chain of fibrinogen. It can undergo various degradations at its N-terminal end, thus generating several peptides of a size less than 16 amino acids, in particular for example the peptides FPA-3 and FPA-6. An increase in the phosphorylation of FPA is reported in patients who have undergone surgical procedures. Thus, “FPA and its derivatives” includes all peptides resulting from a degradation, cleavage or post-translational modification of FPA. “FPA and its derivatives” includes in particular the peptides selected from the group consisting of FPA, FPA-3, FPA-6, FPA-P illustrated in Figure 1.Subject, within the framework of the methods for identifying at least one peptide biomarker and the diagnostic methods of the invention, means a mammal, preferably a human. Method for detecting at least one peptide biomarker in a biological fluid. As mentioned previously, the method for detecting at least one peptide biomarker in a biological fluid according to the invention makes it possible to detect the presence or absence of said at least one peptide biomarker, and that it varies slightly in size, or in conformation or in post-translational modification. This method is particularly advantageous with regard to the usual methods of recognition by dedicated antibodies which do not allow, or do so unsatisfactorily, to specifically identify within a sample biomarkers of close sequences due to cross-reactions.Furthermore, this method is particularly easy to implement and does not require pretreatment of the biological fluid as may be the case for other methods such as, for example, spectrometry. Furthermore, mass spectrometry alone does not allow the characterization of peptides that differ only by their enantiomeric conformation. The aforementioned methods also do not allow the statistical conformations of a peptide (the transient secondary structures of peptides) to be differentiated and quantified, even less so within a biological sample. Furthermore, this method can easily and quickly be implemented as close as possible to the patient and the care. In the method for identifying at least one peptide biomarker in a biological fluid according to the invention, the electrolyte is a solution of alkali metal or alkaline earth metal halide or their mixture.Said electrolyte may be selected from the group consisting of LiCl, NaCl, KCl or KBr, RbCl, CsCl, KF, ammonium chloride, tetramethylammonium chloride, BeCl2, CaCl2, MgCl2, SrCl2, BrCl2, BaCl2 or any combination thereof. KCl is particularly preferred. The concentration of the electrolyte is between 2M and 5M, inclusive. The pH of the electrolyte solution may vary, for example depending on the biomarker that one wishes to characterize and / or detect. This pH must not, however, be such as to call into question the structure or the activity of the aerolysin nanopore of the sensor system. Thus, the pH of the electrolyte solution is between 3 and 10. Advantageously, the pH can be chosen to increase the driving force via changing the selectivity of the nanopore, as well as the net charge of the analyzed peptide biomarker.The temperature of the at least one sensor system is such that there is no formation of ice crystals and / or that the aerolysin nanopore does not denature. In certain instances, placing the sensor system at a low temperature, for example below 15°C, or even below 10°C, has the advantage of slowing down events and therefore making it easier to characterize components of the electrical signal associated with the passage of a peptide biomarker. Conversely, placing the at least one sensor system at a high temperature, for example a temperature above 40°C, above 45°C, or even above 55°C, can make it possible to denature at least partially the peptide biomarker and thus facilitate its passage through the aerolysin nanopore. Thus, the at least one sensor system, in the method of the invention, is at a temperature of 5°C to 55°C.In the sensor system used in the method of the invention, the membrane in which the aerolysin nanopore is inserted is: - either of lipid nature, consisting, for example, of diphytanoylphosphatidylcholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine DLPC; 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine DDPPC; 1,2-dieicosenoyl-sn-glycero-3- phosphocholine DEPC or their mixture, - or of polymeric nature, based on diblock polymers (poly(1,2-butadiene)-b-poly(ethylene oxide) or triblock PDMS- PMOXA-PDMS which has the advantage of more easily controlling the number of nanopores formed in the system, but also having better stability than a lipid membrane over time and subjected to high electrical potential differences (> 200 mV). In the method of the invention, the acquisition frequency varies from 100 kHz to 1 MHz, preferably from 250 kHz and 400 KHz.The acquisition frequency can in particular be adjusted according to the size of the biomarker; in fact, the smaller the peptides, the higher the acquisition frequency required for adequate detection of events. Advantageously, a Bessel filter is used. The Bessel filter used will also be adjusted according to the size of the biomarkers (it can have a frequency value varying from 1 kHz to 100 kHz). This Bessel filter makes it possible to reduce the noise without modifying the information contained in the electrical signal. In the method of the invention, the analytes (peptide biomarkers) are directly added to the cis compartment of the membrane of the at least one censor system. Such a sensor system is illustrated in Figure 2. Said at least one peptide biomarker added in cis can be included in the biological fluid without treatment of the latter aimed at facilitating the detection of said at least one biomarker.Said biological fluid may also have been subjected to a prior treatment aimed at facilitating the detection of the at least one biomarker such as, for example, filtration, fractionation, precipitation, the addition of reagent aimed at adding at least one group on at least one of the amino acids of said biomarker. As mentioned previously, however, pretreatments such as filtration, fractionation, precipitation or any other treatment aimed at purification or concentration are less desirable because they often induce a loss of sensitivity. This prior treatment may for example aim at modifying the charge of the at least one peptide biomarker so as to facilitate its translocation from the cis side to the trans side of the membrane of the sensor system upon application of the potential difference.This is advantageous in cases where the at least one peptide biomarker that one wishes to characterize and / or detect has a neutral or insufficient charge. This treatment may also comprise an enzymatic treatment aimed at producing peptides from a large biomarker protein, with the aim of detecting and characterizing at least one of these peptides by the sensor system of the invention. This treatment may also comprise the use of sufficiently denaturing conditions so as to dissociate complexes involving the peptide biomarkers whose presence one wishes to detect, and / or unfold at least in part secondary or tertiary structures.The biological fluid may be included in the group comprising blood, plasma, serum, cerebrospinal fluid, bronchoalveolar lavage, saliva, synovial fluid, amniotic fluid, urine, cell lysate, eukaryotic or bacterial cell culture medium. As shown in Figure 9, the method of the invention allows the identification of peptide biomarkers in a biological fluid such as serum, which is a complex biological fluid without prior treatment. Thus, this can avoid a loss in sensitivity, due to the loss of biomarkers due to these treatments, in particular filtration, fractionation, precipitation and also contributes to the speed of implementation of the method.Enzymatic digestion of peptides in biological fluid is less subject to a loss of sensitivity related to the loss of biomarkers, and has the advantage of being able to focus on the characterization of the presence of large molecules (e.g. peptides of 25 amino acids or more (i.e. approximately more than 2.75 kDa). In Figure 4, FPA and FPA-P were used in a sensor system according to the method of the invention. The representation for each event of the ΔInorm as a function of the blocking duration makes it possible to identify a specific signature for FPA and FPA-P. The method of the invention makes it possible to reveal two signals for FPA-P, FPA-P1 and FPA-P2. Without wishing to be bound by any theory, it is believed that these two signals correspond to two different confirmations of the FPA-P peptide, induced by the phosphorylation of the peptide.As illustrated in the right panel of Figure 3, the method makes it possible to identify the presence of FPA and FPA-P in a mixture. Thus, the method according to the invention makes it possible to distinguish up to a single post-translational modification of a peptide biomarker, such as, for example, phosphorylation. In addition, the distinction of several conformations for the same species makes it possible to infer a gain in sensitivity and specificity for said method. Indeed, such additional information increases the probability of detection and correct identification of the peptide biomarker. The representation of ΔInorm as a function of the number of events makes it possible to arrive, with regard to FPA, at the determination of an average blocking rate of 0.64±0.01, 0.70±0.01 for FPA-P1 and 0.61±0.01 for FPA-P2.The method of the invention is thus sufficiently powerful to allow the detection of unique post-translational modifications and / or different conformations of the same peptide biomarker. These performances are confirmed by the data presented in Figure 5, where it is seen that the analysis of the electrical signal of a mixture of FPA, FPA-P, FPA-3 and FPA-6, makes it possible to identify the presence of these species unambiguously in this mixture. The upper panel of Figure 5 shows the specific signatures of these peptide biomarkers as determined by the representation of the blocking rate as a function of the nanopore blocking duration. The lower panel of Figure 5 shows the determination of the average blocking rate for each of the deduced species as a function of the number of events. An average blocking rate of 0.64±0.01, 0.70±0.01 for FPA-P1, 0.62±0.01 for FPA-P2, 0.56±0.01 for FPA-3 and 0.44±0.01 for FPA-6.The method according to the invention therefore makes it possible to discriminate between peptides differing by N-terminal cleavages, confirmation and / or post-translational modification, in a mixture, using for example ΔInorm or ΔI. normaverage as defined above. Figure 6 shows the determination for each of the species FPA, FPA-3, FPA-6 and FPA-P, that it is possible to determine an average occupancy time for each of these, which is specific to them, despite the similarity of these peptides. As illustrated, this is obtained by plotting the blocking time of each event as a function of the number of events and thus estimating the average occupancy time by fitting to a Gaussian distribution. Average occupancy times of 5.74±0.22 ms for FPA, 0.62±0.04 ms for FPA-P1, 4.41±0.33 ms for FPA-P2, 2.50±0.12 ms for FPA-3, and 0.95±0.04 ms for FPA-6 are thus determined.Thus, it is determined that the electrical signal can be used to determine characteristics such as: - the average, median, minimum and / or maximum occupancy time, - the average, minimum, median and / or maximum blocking rate of the at least one aerolysin nanopore of the sensor system, - the shape of the electrical signal which, being specific to the peptide biomarkers, can be used to differentiate them even if these differences consist only of a single post-translational modification or N-terminal cleavages, - the number of events which make it possible to determine the concentration of the identified peptide biomarker, or - the standard deviation of the signal measured during the passage of said peptide biomarker.These electrical signal data, being found specific to peptide biomarkers, can be used to differentiate them, and characterize their presence and concentration, even if these differences consist only of a single post-translational modification or N-terminal cleavages. As explained later, these data also make it possible to differentiate different conformations of the same peptide biomarker, in relation to secondary structure (for example due to a disulfide bridge, but also a particular statistical conformation) or even one or more enantiomers.The inventors have identified that the analysis of the aforementioned characteristics of the electrical signal is sufficient in itself to detect the presence and / or determine the concentration or absence of peptide biomarkers distinguished by such subtle characteristics as the presence of one or more post-translational modifications or one or more N-terminal cleavages, a conformation. The determination of the concentration of peptides in a detection system with a nanopore is known in the art, it is based on the linear relationship between the frequency of events and the concentration (Pastoriza-Gallego M, Rabah L, Gibrat G, Thiebot B, van der Goot FG, Auvray L, Betton JM, Pelta J. Dynamics of unfolded protein transport through an aerolysin pore. J Am Chem Soc. 2011 Mar 9;133(9):2923-31).In a particular embodiment, the peptide biomarkers of the method according to the invention are identified and characterized by the coupled analysis of at least two parameters of the electrical signal such as those described above, this makes it possible to increase the resolution of the method and to improve the discrimination and identification of the peptide biomarkers.In a particular embodiment, the peptide biomarkers of the method according to the invention are identified and characterized by the coupled analysis of at least two parameters of the electrical signal selected from: - the average, median, minimum and / or maximum occupancy time, - the average, minimum, median and / or maximum blocking rate of the at least one aerolysin nanopore of the sensor system, - the shape of the electrical signal which, being specific to the peptide biomarkers, can be used to differentiate them even if these differences consist only of a single post-translational modification or N-terminal cleavages, - the number of events which make it possible to determine the concentration of the identified peptide biomarker, or - the standard deviation of the signal measured during the passage of said peptide biomarker.In a particular embodiment, the peptide biomarkers of the method according to the invention are identified and characterized by the coupled analysis of: - the mean, median, minimum and / or maximum occupancy time, with the mean, minimum, median and / or maximum blocking rate, or - the mean, median, minimum and / or maximum occupancy time, with the shape of the electrical signal, or - the mean, median, minimum and / or maximum occupancy time, with the standard deviation of the electrical signal, or - the mean, minimum, median and / or maximum blocking rate, with the standard deviation of the electrical signal, or - the mean, minimum, median and / or maximum blocking rate, with the shape of the electrical signal. In an even more particular embodiment of the method according to the invention, analyzing the generated electrical signal comprises: - determining the mean blocking rate, and - determining the mean blocking time as described above.Thus, these data can be used in two-dimensional representations as illustrated in Figure 7 which show that each population of events, and therefore each peptide biomarker can clearly be characterized and identified within a mixture of peptide biomarkers. In an even more particular embodiment of the method according to the invention, analyzing the generated electrical signal comprises: - determining a mean, median, minimum and / or maximum occupancy time of the at least one aerolysin nanopore of the sensor system, - determining a mean, minimum, median and / or maximum blocking rate of the at least one aerolysin nanopore of the sensor system, - analyzing the shape of the generated electrical signal, - determining the standard deviation of the signal measured during the passage of said peptide biomarker through said nanopore, and - determining the number of events.As shown in the experimental part, the combination of these characteristics makes it possible to differentiate the biomarkers according to variations in their primary sequence, the presence and / or absence of at least one post-translational modification and / or at least one conformation of said peptide. Figure 8 illustrates the implementation of the method of the invention for bradykinin. Bradykinin and des-arginine. 9 bradykinin are each characterized by two conformations detected by the sensor system of the method according to the invention. Blocking rates of 0.47 and 0.75 are determined for bradykinin (BK1 and BK2 forms), and blocking rates of 0.69 and 0.32 for des-arginine 9bradykinin (DAB1 and DAB2 forms). Similarly, occupancy times corresponding to each of the species are determined (not shown). The two-dimensional representation in Figure 9 confirms that the peptide biomarkers are effectively identified and discriminated by a coupled analysis of two parameters of the electrical signal, in this case the blocking time and the blocking rate. Figure 10 confirms the characterization in human serum of the presence of des-arginine 9bradykinin, by identifying two conformations characterized by an average blocking rate of 0.32±0.004 and 0.72±0.002. This is consistent with the blocking rates determined for the purified peptides (Figure 8). Figure 11 shows that it is possible to assign characteristics such as the general shape of the signal to the BK1, BK2, DAB1, DAB2 forms in a biological fluid such as serum, this characteristic allowing assignment of the signal to the peptide even at low concentration (50 nM). Figure 12 shows that the signal analysis allows determining the concentration of the biomarker, the frequency of which is linear with the concentrations of the biomarker peptides tested. In another case, the statistical analysis of the electrical signals allowed the inventors to surprisingly identify that the standard deviation (Figure 14, σ) of the signal allows the detection,the identification and differentiation of peptides such as vasopressin (L-AVP) and a synthetic enantiomer (D-AVP, precursor of desmopressin) differing by the presence of the enantiomer of arginine 8 present in the peptide. Specific electrical signals for these proteins in their reduced or oxidized form are observed, but also depending on the enantiomer of arginine 8 present in the peptide have been identified (Figure 13 A and B). In addition to the simple observation of a signal difference, it has also been determined by principal component analysis and statistical analysis of the data that the standard deviation (characteristic noted σ Figure 14) allows the detection and differentiation of D-AVP and L-AVP vasopressin. Thus, quite unexpectedly,the analysis of the different characteristics of the electrical signal of the nanopore is sufficient to allow in itself the characterization of biological samples by allowing rapid and direct detection of biomarkers in a sample of biological fluid, differentiated by their sequence, their conformation, and / or their post-translational modification. In the method according to the invention the duration of recording of the events is at least one minute. The longer the recording duration, the greater the probability of detecting a biomarker weakly represented in the biological fluid; the sensitivity and specificity of the method are also improved. Thus the duration of recording of the events is at least 2, 3, 4, 5, 10, 20, 30, 45, 60, 90, or even at least 120 min. Also,the method for detecting at least one peptide biomarker in a biological fluid according to the invention may comprise the use of several independent sensor systems, thus increasing the probability of detecting a peptide biomarker poorly represented in the biological fluid. Alternatively, a membrane comprising several nanopores may be used. Thus, in this embodiment, a sensor system comprises several nanopores. Also, the greater the potential difference applied, the greater the sensitivity of the method. Thus, the potential difference applied is less than or equal, in absolute value, to 300 mV, less than or equal to 250 mV, less than or equal to 200 mV, less than or equal to 150 mV, less than or equal to 100 mV, or even less than or equal to 50 mV. Obviously, when the at least one peptide biomarker sought is negatively charged, the potential difference applied is positive,and conversely when the at least one peptide biomarker sought is positively charged, the potential difference applied is negative. In the method according to the invention, the ratio of the volume of the electrolyte to the volume of the biological fluid in the cis compartment is greater than 10, greater than 50, greater than 75, greater than 100, greater than 500, or even greater than 1000. In a particular embodiment of the method according to the invention, the values ​​of the parameters of the electrical signal characterizing a peptide biomarker sought are previously established. For example, by analyzing the peptide biomarker purified by the sensor system of the invention. Thus, the method according to the invention comprises a step of comparing the values ​​of parameters of the electrical signal obtained during the analysis of the biological fluid with the predetermined parameters characterizing the peptide biomarker,such as for example for a preparation of pure or purified peptide biomarker. Said method for detecting at least one peptide biomarker in a biological fluid according to the invention can therefore be implemented in biotechnological applications, but also in diagnostic methods for the measurement and direct identification of very diverse biomarkers, in complex media, differing by characteristics which may be minute, not detectable for example by a simple mass analysis,as demonstrated above and illustrated in the figures. Thus, other objects of the present invention are diagnostic methods implementing the steps of the method for detecting at least one peptide biomarker in a biological fluid as described above. Methods for diagnosing at least one peptide biomarker in a biological fluid. Diagnosis of pathologies related to coagulation disorders. As shown by the experimental data illustrated in particular by the figures presented here,the method according to the invention is particularly suitable for the detection of FPA and its derivatives. FPA is a product of the cleavage of fibrinogen by thrombin. FPA and its derivatives are associated with the occurrence of coagulation in the body. FPA and its derivatives are therefore used in the diagnosis or monitoring of coagulation diseases or those associated with coagulation. Coagulation disorders are associated with many and diverse pathologies, such as venous thrombosis, disseminated intravascular coagulation which play a role in many cancers (lung, pancreatic, prostate, stomach, gastrointestinal, leukemia), ischemic heart disease, COVID-19, stroke, systemic lupus erythematosus. Thus the detection of FPA or its derivatives in a biological fluid from a subject reflects the activation of fibrin and therefore a thrombotic event. Also, for certain cancers,the activation of the thrombotic cascade can be associated with a poor prognostic factor in the patient suffering from cancer. Thus a second object of the invention relates to the detection of thrombogenesis in a subject comprising the detection of FPA or its derivatives such as phosphorylated FPA (FPA-P), FPA-3, FPA-6, or their mixtures, in a biological fluid originating from said subject. In a particular embodiment, this method allows the diagnosis of a pathology associated with a coagulation disorder in a subject suspected of suffering or suffering from such a pathology comprising the detection of FPA or its derivatives such as phosphorylated FPA (FPA-P), FPA-3, FPA-6, or their mixtures,in a biological fluid originating from said subject. This diagnostic method of a pathology associated with a coagulation disorder of the invention comprises the method for detecting at least one peptide biomarker in a biological fluid set out above and adapted in all its embodiments, to FPA and its derivatives. In a particular embodiment, a number of events detected, in a biological fluid, for one or more of the biomarkers selected from FPA, FPA-P FPA-3, and FPA-6, greater than that of a control, or a threshold value determined for subjects not having a thrombotic event is the sign of an increase in thrombotic activity in the subject. In a particular embodiment, this detection of thrombogenesis in a subject comprises the detection of FPA or its derivatives such as phosphorylated FPA (FPA-P), FPA-3, FPA-6, or mixtures thereof, in a biological fluid originating from said subject,said method comprising the following steps: - providing a sample of biological fluid from said subject, - introducing the biological fluid into a sensor system comprising, in a 2M to 5M alkali metal or alkaline earth metal halide solution and a pH of 4.1 to 10, at least one aerolysin nanopore inserted into a lipid or polymer membrane, said biological fluid being added to the cis side of the membrane, - applying to said sensor system comprising the biological fluid a potential difference of between +1 mV and +300 mV, between +10 mV and +100 mV, preferably +50 mV, such as to promote the passage of FPA and its derivatives, - recording the electrical signal generated at a frequency of 100 kHz to 1 MHz, and - analyzing the electrical signal generated, said analysis of the generated electrical signal making it possible to detect the presence and / or determine the concentration or absence of FPA or its derivatives in said biological fluid,a number of events detected, in said biological fluid, for one or more of the biomarkers selected from FPA, FPA-P FPA-3, and FPA-6, higher than that of a control or a threshold value determined for subjects not including a thrombotic event, being the sign of an increase in thrombotic activity in said subject. Diagnosis of bradykinin angioedema. Bradykinin angioedema (BA) is characterized by episodes of subcutaneous or submucosal edema that can be life-threatening in the case of pharyngeal localization. No emergency biological examination can confirm the diagnosis. Bradykinin dosage is not feasible in routine practice. This acute complication is therefore often confused with angioedema of allergic origin,which can lead to misdiagnosis and specific management and be life-threatening. Other serious attacks are abdominal pain crises responsible for subocclusive syndrome, or more rarely hypovolemic shock (javaud et al, 2016). The management of severe crises requires rapid diagnosis and treatment. There is therefore an unmet need for a diagnostic test that can: - characterize bradykinin in a biological fluid, and - detect and quantify the presence of bradykinin and des-arginine, 9 bradykinin As illustrated in figures 8 and 9, the method according to the invention makes it possible to identify and quantify bradykinin as well as des-arginine 9bradykinin in a biological fluid. Thus a third subject of the invention relates to a method for diagnosing bradykinin angioedema in a subject suspected of suffering or suffering from such a pathology comprising the detection of bradykinin and / or des-arginine 9 bradykinin or mixtures thereof, in a biological fluid originating from said subject. This diagnostic method for bradykinic angioedema comprises the method for detecting at least one peptide biomarker in a biological fluid set out above and adapted in all its embodiments, bradykinin and / or des-arginine 9 bradykinin or mixtures thereof. In a particular embodiment, a number of events detected, in a biological fluid, for bradykinin and / or for des-arginine 9bradykinin, higher than that detected in a control sample, or at a threshold value determined for subjects not suffering from bradykinin angioedema or angioedema, is a sign of the presence of bradykinin angioedema in the subject.A particular subject of the invention is a diagnostic method for bradykinin angioedema in a subject suspected of suffering or suffering from such a pathology comprising the following steps: - providing a sample of biological fluid from said subject, - introducing the biological fluid into a sensor system comprising, in a 2M to 5M alkali or alkaline earth metal halide solution and a pH of 3.1 to 10, an aerolysin nanopore inserted into a lipid or polymer membrane, said biological fluid being added to the cis side of the membrane, - applying to said sensor system comprising the biological fluid a potential difference of between - 300 mV and -1 mV, between -100 mV and -10 mV, preferably - 50 mV, such as to promote the passage of bradykinin and / or des-arginine. 9bradykinin, - record the electrical signal generated at a frequency of 100 kHz to 1 MHz, - analyze the generated electrical signal, said generated electrical signal making it possible to detect the presence and / or determine the concentration or absence of bradykinin and / or des-arginine 9bradykinin or their different conformations in the biological fluid, the characterization of the presence of levels of bradykinin and / or des-arginine9 bradykinin higher than the control or a threshold value determined for subjects not suffering from bradykinin angioedema or angioedema being the sign of the presence of bradykinin angioedema in said subject.Another subject of the invention is a method for treating bradykinin angioedema in the subject comprising the following steps: - providing a sample of biological fluid from said subject, - introducing the biological fluid into a sensor system comprising, in a 2M to 5M alkali or alkaline earth metal halide solution and a pH of 3.1 to 10, an aerolysin nanopore inserted into a lipid or polymer membrane, said biological fluid being added to the cis side of the membrane, - applying to said sensor system comprising the biological fluid a potential difference of between - 300 mV and -1 mV, between -100 mV and -10 mV, preferably -50 mV, such as to promote the passage of bradykinin and / or des-arginine. 9bradykinin, - record the electrical signal generated at a frequency of 100 kHz to 1 MHz, - analyze the generated electrical signal, said generated electrical signal making it possible to detect the presence and / or determine the concentration or absence of bradykinin and / or des-arginine 9 bradykinin or their different conformations in the biological fluid, and - administer to the subject a specific treatment for bradykinin angioedema in the event of the characterization of the presence of levels of bradykinin and / or des-arginine9 bradykinin higher than the control or a threshold value determined for subjects not suffering from bradykinin angioedema or angioedema.

Claims

CLAIMS 1. Method for detecting at least one peptide biomarker in a biological fluid comprising the following steps: - providing a sample of biological fluid, - adding the biological fluid to a sensor system comprising, in a solution of alkali metal halide, alkaline earth halide or mixtures thereof, from 2 M to 5 M and a pH of 3 to 10, at least one aerolysin nanopore inserted into a lipid or polymer membrane defining a cis side and a trans side, said biological fluid being added to the cis side of the membrane, - applying to said sensor system comprising the biological fluid a potential difference of between -300 mV and +300 mV, preferably between -200 mV and +200 mV, such as to promote the passage of said at least one peptide biomarker, - recording the electrical signal generated at a frequency of 100 kHz to 1 MHz, and - analyzing the electrical signal generated: o by determining a time average, median occupancy,minimum and / or maximum, of the at least one aerolysin nanopore of the sensor system, o by determining an average, minimum, median and / or maximum blocking rate of the at least one aerolysin nanopore of the sensor system, o by analyzing the shape of the electrical signal generated, o by determining the standard deviation of the signal measured during the passage of said peptide biomarker through said nanopore, and, o by determining the number of events, - said analysis of the generated electrical signal making it possible to detect the presence and / or determine the concentration or absence of the at least one peptide biomarker in said biological fluid.

2. Method according to any one of the preceding claims, in which the at least one peptide biomarker corresponds, for a given peptide, to the presence and / or absence of at least one post-translational modification and / or at least one conformation of said peptide.

3. Method according to any one of the preceding claims in which the at least one peptide biomarker has a size of between 3 and 100 amino acids. 4.Method according to any one of the preceding claims in which the at least one peptide biomarker is selected from: histones H1, H2A, H2B, H3 and / or H4, B-type natriuretic peptide, chemokines, interleukins or one of their receptors or its subunit, adiponectin, NLRP3, insulin A chain, insulin B chain, a cytochrome C, fibrinopeptide A (FPA), phosphorylated FPA (FPA-P), FPA-3, FPA-6, bradykinin, des-arginine. 9 bradykinin, Aβ 1-15 , Aβ 1-16 , Aβ 1-17 , Aβ 1-40 , Aβ 1-42 , Aβ 11-40 , Aβ17-40, Aβ17-42,ADA, ARTN, AXIN1, Beta-NGF, CASP-8, CD40, CDCP1, CST5, DNER, CCL11, 4E-BP1, FGF-21, LAP TGF-beta-1, LIF, LIF-R, CSF-1, MMP-1, MMP-10, MCP-1, MCP-2, MCP-3, MCP-4, CD244, NT-3, NRTN, OSM, OPG, FGF-23, FGF-5, FGF-19, Flt3L, GDNF, HGF, IFN-gamma, PD-L1, EN-RAGE, SLAMF1, SIRT2, STAMBP, SCF, ST1A1, CD6, CD5, CD8A, TSLP, TNFB, TRANCE, TRAIL, TGF-alpha, TWEAK, TNF, TNFSF14, TNFRSF9, uPA, VEGF-A.

5. Method according to any one of the preceding claims in which the electrical signal is collected over a period of time of at least 1 minute.

6. Method according to any one of the preceding claims in which the sensor system comprises at least two sensor systems.

7. Method according to one of the preceding claims in which the biological fluid is selected from: blood, plasma, serum, cerebrospinal fluid, bronchoalveolar lavage, saliva, synovial fluid, amniotic fluid, urine, a cell lysate, a culture medium of eukaryotic or bacterial cells.

8. Method according to one of the preceding claims in which the ratio of the volume of biological fluid to the volume of the electrolyte in the compartment on the cis side of the membrane of the at least one sensor system is less than or equal to 1 / 50. 9.A method for diagnosing a pathology associated with a coagulation disorder in a subject suspected of suffering from or suffering from such a pathology comprising the detection of FPA or its derivatives such as phosphorylated FPA (FPA-P), FPA-3, FPA-6, or mixtures thereof, in a biological fluid originating from said subject, said method comprising the following steps: - providing a sample of biological fluid from said subject, - introducing the biological fluid into a sensor system. comprising, in a solution of alkali metal halide, alkaline earth halide or their mixtures, from 2M to 5M and with a pH of 4.1 to 10, at least one aerolysin nanopore inserted into a lipid or polymer membrane defining a cis side and a trans side, said biological liquid being added to the cis side of the membrane, - applying to said sensor system comprising the biological liquid a potential difference of between +1 mV and +300 mV, preferably between +10 mV and +100 mV, preferably +50 mV, such as to promote the passage of FPA and its derivatives, - recording the electrical signal generated at a frequency of 100 kHz to 1 MHz, and - analyzing the electrical signal generated, said analysis of the electrical signal generated making it possible to detect the presence and / or determine the concentration or absence of FPA or its derivatives in said biological liquid, a number of events detected, in said biological liquid,for one or more of the biomarkers selected from FPA, FPA-P FPA-3, and FPA-6, higher than that of a control or a threshold value determined for subjects not having a thrombotic event, being a sign of an increase in thrombotic activity in said subject.

10. A method for diagnosing bradykinin angioedema in a subject suspected of suffering or suffering from such a pathology comprising the following steps: - providing a sample of biological fluid from said subject, - introducing the biological fluid into a sensor system, comprising, in a 2M to 5M alkali or alkaline earth metal halide solution and a pH of 3.1 to 10, an aerolysin nanopore inserted into a lipid or polymer membrane, said biological liquid being added to the cis side of the membrane, - applying to said sensor system comprising the biological liquid a potential difference of between - 300 mV and -1 mV, preferably between -100 mV and -10 mV, preferably -50 mV, such as to promote the passage of bradykinin and / or des-arginine 9 bradykinin, - record the electrical signal generated at a frequency of 100 kHz to 1 MHz, - analyze the generated electrical signal, said generated electrical signal making it possible to detect the presence and / or determine the concentration or absence of bradykinin and / or des-arginine 9bradykinin or their different conformations in biological fluid, characterization of the presence of levels of bradykinin and / or des-arginine 9 bradykinin levels above the control or a threshold value determined for subjects not suffering from bradykinin angioedema or angioedema being a sign of the presence of bradykinin angioedema in said subject.

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