Fluorescent reporters for the detection of target molecules and their uses

Abstract

The present invention relates to a device for detecting a target molecule (2) and / or measuring the concentration of the target molecule (2), comprising a substrate having covalently bound graft molecules on its surface, at least one receptor (11) covalently bound to a polypeptide (12), and two fluorescent dyes F. a and F b and at least one fluorescent probe (1) containing a fluorescent dye F a binds to the receptor (11) and the fluorescent dye F b is bound to a polypeptide (12); and the fluorescent dye F a and F b form a FRET donor / acceptor pair; and a polypeptide (12) is covalently attached to the graft molecule. The present invention also relates to fluorescent probes and methods for detecting and / or measuring the concentration of target molecules.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to fluorescent reporters, or fluorescent probes, for detecting and / or measuring the concentration of target molecules in a sample. [Background technology]

[0002] prior art The detection of target molecules in samples has become essential in medical research, such as for the detection of contaminants in agricultural products and wastewater, or for the diagnosis of many disease states, including cancer, infectious diseases, autoimmune diseases, and allergies. Detection of target molecules using FRET technology, based on non-radiative energy transfer between two fluorescent dyes, traditionally requires a FRET donor / acceptor pair, each of which possesses a recognition molecule such as an antibody. This fluorescent reporter, or fluorescent probe, has certain drawbacks: two parts must recognize the target molecule, generate a FRET effect, and thus detect the target molecule. The detection process is lengthy, and its sensitivity is fundamentally limited by the concentration of the target molecule and the affinity of the antibody. The development of fluorescent probes based on intramolecular FRET makes it possible to circumvent some of these limitations.

[0003] Such fluorescent probes are known from the prior art. In particular, Grant et al. (Grant et al., "Effects of immobilization on a FRET immunosensor for the detection of myocardial infarction," Anal Bioanal Chem (2005), 381:1012-1018) and Ko et al. (Ko et al., "A novel FRET-based optical fiber biosensor for rapid detection of Salmonella typhimurium," Biosensors and Bioelectronics (2006), 21:1283-1290) describe fluorescent probes containing an antibody and protein A bound to two fluorescent dyes that form a FRET donor / acceptor pair. Since the antibody and protein A are not covalently bound, there is a risk that the fluorescent probe will dissociate during detection of the target molecule, and therefore the probe cannot be used for in vivo detection. A second consequence of weak antibody-Protein A binding is a high detection threshold.

[0004] Document FR3040789 further describes a fluorescent probe comprising two antibodies each labeled with a fluorochrome member of a FRET donor / acceptor pair, the two antibodies not binding to each other and thus not allowing the achievement of an intramolecular FRET effect.

[0005] In parallel, the in vivo application of fluorescent probes remains very limited. In this context, the methods used are generally based on the injection of nonspecific tracers or antibodies coupled to fluorescent dyes. Detection is then carried out by measuring the intensity of the signal present at the tissue surface. Although sometimes used, these approaches have many drawbacks. In particular, the obtained signal is strongly influenced by the probe's stability in vivo, its biodistribution, or its specificity. Furthermore, the safety of these injectables needs to be systematically demonstrated. The development of fluorescent probes composed of single molecules that react to the presence of target molecules by a change in their optical properties would enable the detection of markers of interest by simple contact, without the constraints associated with injections. Summary of the Invention [Problem to be solved by the invention]

[0006] During in vivo use, for example, during surgery or exploration of parts of the human body by endoscopy, it is important for surgeons to be able to reliably and quickly identify tumor cells present in the tissue, so fluorescence-assisted surgery is currently a booming field, but is limited by the inherent deficiencies of marker injection.

[0007] There is therefore a real need for fluorescent probes that can be used during surgery or endoscopy, for example at the end of optical fibers, to make it possible to establish a diagnosis without the risk of biological contamination. [Means for solving the problem]

[0008] As with previously developed fluorescent probes, during in vitro use, e.g., during rapid analysis of solutions for medical diagnostic purposes or for targeting molecules in environmental samples, it is necessary to avoid biological contamination of the sample under study by the fluorescent probe. The present invention proposes a solution to this problem by providing a fluorescent probe in which different elements are tightly bound to each other, particularly to the receptor moiety, to assist in the detection of the target molecule, without separation from the other elements that form the fluorescent probe and without inducing biological contamination of the sample. Furthermore, the fluorescent probe according to the present invention has the advantage that it does not require additional steps of handling, washing, secondary labeling, or any other kind. It is sufficient to simply contact it with the sample to be analyzed, significantly reducing the number of sample handlings and the time to obtain a result. [Effects of the Invention]

[0009] The present invention provides an apparatus for detecting and / or measuring the concentration of a target molecule, comprising: - a substrate to the surface of which graft molecules are covalently attached; at least one fluorescent probe, ■ at least one receptor covalently bound to the polypeptide; ■Two types of fluorescent dyes F a and F b At least one fluorescent probe comprising Including, Fluorescent dye F a binds to the receptor and the fluorescent dye F b binds to the polypeptide; and Fluorescent dye F a and F b forms a FRET donor / acceptor pair; The polypeptide is covalently attached to a graft molecule.

[0010] According to an embodiment, the receptor is selected from an antibody, an antibody fragment, an aptamer, a protein, a peptide, or a derivative thereof. According to an embodiment, the polypeptide is a binding protein selected from Protein G, Protein L, Protein A, Protein Z, Protein M, an immunoglobulin, a complete or partial immunoglobulin, or a derivative thereof. According to an embodiment, the polypeptide comprises 2 to 100 amino acids, preferably 4 to 50 amino acids. According to an embodiment, the fluorescent dye F a and / or F b is selected from fluorescent molecules or fluorescent proteins. According to embodiments, the substrate is selected from a cell culture plate, a well plate, a film, a strip, an agarose gel, a cellulose gel, nanoparticles or microparticles, preferably spherical, preferably silica or polymer, a microscope slide, a glass slide, a substrate configured to be attached to the periphery of an optical fiber or the head of an optical fiber. According to embodiments, the substrate is a polymer film. According to embodiments, the polymer film is selected from polyethylene terephthalate, fluorinated polyethylene-co-propylene, polymethyl methacrylate, polytetrafluoroethylene, polymethylpentene, polyvinyl chloride, styrene methyl methacrylate, polyethylene naphthalate, derivatives thereof, or mixtures thereof. According to embodiments, the grafted molecule comprises at least two reactive groups selected from maleimide, N-hydroxysuccinimide (NHS) ester, sulfo-N-hydroxysuccinimide ester, sulfo-NHS, azide, alkyne, epoxide, carboxylic acid, aldehyde, aziridine, alkene, or derivatives thereof. According to an embodiment, the device further comprises an optical fiber and a probe head, said probe head comprising a body and a light emitting surface, at least a part of which is transparent and forms a port, said substrate being said port.

[0011] The present invention also provides - at least one receptor covalently bound to the polypeptide; -Two types of fluorescent dyes F a and F b Including, Fluorescent dye F a binds to the receptor and the fluorescent dye F b binds to the polypeptide; and Fluorescent dye F a and F b relates to fluorescent probes that form a FRET donor / acceptor pair.

[0012] According to embodiments, the receptor is selected from an antibody, an antibody fragment, an aptamer, a protein, a peptide, or a derivative thereof. According to embodiments, the polypeptide is a binding protein selected from Protein G, Protein L, Protein A, Protein Z, Protein M, an immunoglobulin, a full or partial immunoglobulin, or a derivative thereof. According to embodiments, the polypeptide comprises 2 to 100 amino acids, preferably 4 to 50 amino acids.

[0013] The present invention also provides a method for detecting a target molecule and / or measuring the concentration of a target molecule, comprising the steps of: at least one fluorescent probe, ■ at least one receptor covalently bound to the polypeptide; ■Two types of fluorescent dyes F a and F b Including, Fluorescent dye F a binds to the receptor, and the fluorescent dye F b is bound to the polypeptide; and Fluorescent dye F a and F b form a FRET donor / acceptor pair; contacting the sample with at least one fluorescent probe, the receptor of which has affinity for said target molecule; - exciting the fluorescent probe at a predetermined wavelength such that it excites the donor fluorescent dye; - measuring the ratio between the intensity of the fluorescence emitted by the donor fluorescent dye and the intensity of the fluorescence emitted by the acceptor fluorescent dye; and - determining the presence or absence of said target molecule in the sample and / or calculating the concentration of said target molecule in the sample The present invention relates to a method comprising:

[0014] definition For purposes of the present invention, the following terms are defined as follows:

[0015] An "antibody" (also known as an immunoglobulin and abbreviated Ig) refers to a gamma globulin protein found in the blood or other bodily fluids of vertebrates and used by the immune system to recognize and neutralize foreign substances such as bacteria and viruses. Antibodies consist of two pairs of polypeptide chains, called heavy and light chains, arranged in a Y-shape. At both ends of the Y are regions that bind to antigens and inactivate them. As used herein, the term "antibody" (Ab) includes monoclonal, polyclonal, and multispecific antibodies (e.g., bispecific antibodies). The term "immunoglobulin" (Ig) is used interchangeably with "antibody."

[0016] "Antigen" refers to a molecule that induces an immune response. This immune response may involve either antibody production or activation of cells with specific immunological capabilities, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or any peptide, can serve as an antigen.

[0017] "Aptamer" refers to a synthetic oligonucleotide, generally RNA, that is capable of binding to a specific ligand.

[0018] The "active configuration" (denoted "on") is the fluorescent dye F a and F b This refers to the configuration of a fluorescent probe in the presence of energy transfer (FRET effect) between the two.

[0019] The "inactive configuration" (denoted "off") is the fluorescent dye F a and F b This refers to the configuration of a fluorescent probe in the absence of energy transfer (FRET effect) between the two.

[0020] "Fluorescent dye" (or fluorophore) refers to a chemical substance that can emit fluorescent light after excitation.

[0021] "Antibody fragments" include portions of intact antibodies, particularly the variable regions responsible for specific antigen recognition. Examples of antibody fragments include Fab, Fab', (Fab')2, and Fv, scFv, and scFv-Fc fragments; dimeric antibody fragments; linear antibodies (see U.S. Patent 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The expression "antibody fragment" (or functional fragment) refers to a compound that shares qualitative biological activity with a full-length antibody. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, and a remaining "Fc" fragment, a term reflecting the ability to readily crystallize. The Fab fragment consists of the entire light chain, along with the variable region domain of the heavy chain (VH) and the first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Treatment of an antibody with pepsin yields a single large fragment (Fab')2 fragment, roughly corresponding to two Fab fragments linked by disulfide bridges with divalent antigen-binding activity and always capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines in the antibody hinge region. Fab'-SH is the term used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced in the form of pairs of Fab' fragments with hinge cysteines between them. Chemical coupling of additional antibody fragments is also known.

[0022] "Ligand" refers to a specific target molecule that can reversibly bind to a receptor. The ligand interacts specifically with said receptor non-covalently. The bond is formed by intermolecular forces such as ionic bonds, hydrogen bonds, and van der Waals forces. An antibody / antigen pair is an example of a receptor / ligand pair. As used herein, the terms "ligand," "antigen," and "target molecule" are interchangeable.

[0023] "Optical transparent" refers to a material that absorbs less than 50%, preferably less than 20%, and more preferably less than 10% of light at wavelengths between 350 nm and 1100 nm.

[0024] "Polypeptide" refers to a chain of amino acids linked by peptide bonds. This definition includes amino acid chains containing 1 to 100 amino acids and those containing more than 100 amino acids, more commonly referred to as proteins.

[0025] "Protein" refers to a functional entity formed from one or more peptides. It consists of a polypeptide containing more than 100 amino acids.

[0026] "Protein G" refers to a surface protein expressed by certain streptococcal strains. It binds with high affinity to the Fc fragments of different classes of immunoglobulins from many species. It specifically binds to all IgG subtypes of humans, mice, and rats, as well as IgG subtypes from many other mammalian species. It binds preferably to Fc fragments, but can also bind to Fab fragments. Due to its affinity for the Fc region of immunoglobulins from many mammalian species, Protein G is now considered a standard reagent in biochemistry and immunology.

[0027] "Fluorescent reporter" refers to an entity that has fluorescent properties for the detection of a specific target molecule (or ligand), i.e., a fluorescent probe. Such entities can include, for example, receptor-polypeptide pairs as described hereinbelow. The terms "fluorescent reporter," "fluorescent biosensor," and "fluorescent probe" are used interchangeably hereinbelow.

[0028] A "receptor" refers to a biological molecule that can recognize and / or reversibly bind to a specific target molecule (or ligand). The receptor interacts specifically with the target molecule non-covalently. The bond is formed by intermolecular forces such as ionic bonds, hydrogen bonds, and van der Waals forces. An antibody / antigen pair is an example of a receptor / ligand pair.

[0029] Detailed Description Fluorescent probes The present invention provides - at least one receptor covalently bound to the polypeptide; -Two types of fluorescent dyes F a and F b The present invention relates to a fluorescent probe (also called a fluorescent reporter) comprising:

[0030] Fluorescent dye F a binds to the receptor and the fluorescent dye F b binds to the polypeptide. a and F b form a FRET donor / acceptor pair.

[0031] Therefore, fluorescent probes consist of a part that is responsible for the specific binding of the target molecule (receptor) to be detected, and two types of fluorescent dyes (F) that can convert the recognition of the target molecule into a measurable fluorescent signal. a and F b ) and a support system for one of two fluorescent dyes that can also function as a fastener to allow controlled binding on the substrate (polypeptide).

[0032] During target molecule recognition by a fluorescent probe, a conformational change occurs in the receptor. This conformational change affects the relative positions of the variable fragments, VH (variable heavy chain) and VL (variable light chain), as well as the constant fragment, which alters the distance between the two fluorescent dyes. This alters the emission levels of the donor and acceptor fluorescent dyes through non-radiative energy transfer, i.e., the Förster resonance energy transfer (FRET) effect. This non-radiative energy transfer allows the optical signature of the fluorescent probe to change when the distance between the two fluorescent dyes changes. This changes the fluorescence intensity emitted by each of the two fluorescent dyes, thereby converting the change in receptor conformation induced by target molecule recognition into a measurable fluorescent signal. Therefore, the fluorescent signal emitted by the probe makes it possible to detect target molecules in vitro or in vivo.

[0033] Two fluorescent dyes, F, are used to form a FRET donor / acceptor pair. a and F b They must have compatible spectral properties, in particular an overlap between the emission spectrum of the so-called "donor" fluorochrome and the excitation spectrum of the so-called "acceptor" fluorochrome. When the donor fluorochrome is excited, its fluorescence excites the acceptor fluorochrome. The efficiency of this energy transfer essentially depends on the distance between the two fluorochromes, their extinction coefficients and quantum yields, as well as the degree of overlap between their emission and excitation spectra.

[0034] The specific locations of the fluorescent dyes on the receptor and polypeptide are optimized to promote a change in their optical signature in fluorescence upon recognition and / or binding of the target molecule. Preferably, the fluorescent dye F b is grafted onto the free amines of the polypeptide.

[0035] The polypeptide has a particular affinity for a receptor, for example, the receptor is an antibody and the binding protein is protein G.

[0036] The covalent bond between the receptor and the polypeptide is stronger than the receptor-target molecule bond formed upon recognition of the target molecule by the receptor. This ensures that the receptor does not separate from the polypeptide upon target molecule recognition, thereby avoiding separation of the fluorescent probe into two parts. Preventing separation of the receptor and the polypeptide is particularly important when the fluorescent probe is used for in vivo detection of a target molecule, especially when it is grafted onto the distal end of an optical fiber for intracellular probing, because it limits the risk of leaving a portion of the fluorescent probe (with the receptor) in the patient's body upon fiber removal. Furthermore, without being bound by any theory, the applicant has observed that the covalent bond between the receptor and the polypeptide improves the effectiveness of the FRET effect, and that the detection threshold for the target molecule is lower, particularly in the case of a fluorescent probe comprising a covalently linked receptor and polypeptide, indicating a significant improvement in the sensitivity of the fluorescent probe.

[0037] According to embodiments, the receptor is linked to the polypeptide via a hetero- or monobifunctional linking molecule ("crosslinker"). Preferably, the linking molecule has two or more reactive groups selected from: a carboxyl-to-amine reactive group, e.g., carbodiimide; an amine-reactive group, e.g., NHS ester, imidoester, pentafluorophenyl ester, hydroxymethylphosphine; a sulfhydryl-reactive group, e.g., maleimide, haloacetyl (bromo or iodo), pyridyl disulfide, thiosulfonate, vinyl sulfone; an aldehyde-reactive group, e.g., hydrazide, alkoxyamine; a photoreactive group, e.g., diazirine, aryl azide; or a hydroxyl (non-aqueous)-reactive group, e.g., isocyanate.

[0038] In a preferred configuration of this embodiment, the binding molecule is selected from glutaraldehyde, formaldehyde, disuccinimidyl tartrate, tris(hydroxymethyl)phosphine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, bis(sulfosuccinimidyl)suberate, 1,3-butadiene diepoxide, succinimidyl iodoacetate, succinimidyl (4-iodoacetyl)aminobenzoate, sulfosuccinimidyl (4-iodoacetyl)aminobenzoate, mixtures thereof, or derivatives thereof. Preferably, the binding molecule is selected from glutaraldehyde, succinimidyl iodoacetate, succinimidyl (4-iodoacetyl)aminobenzoate, or sulfosuccinimidyl (4-iodoacetyl)aminobenzoate.

[0039] According to embodiments, the covalent bond between the antibody and the binding protein can be obtained by photoactivation of a modified amino acid containing a photoinducible reactive group. In a preferred configuration of this embodiment, the modified amino acid can be photoleucine or photomethionine and the reactive group is diazirine or aryl azide.

[0040] According to an embodiment, the receptor is selected from an antibody, an antibody fragment, an aptamer, a peptide, or a derivative thereof.

[0041] According to embodiments, receptors are capable of recognizing and / or reversibly binding to ligands, i.e. target molecules. A ligand corresponds to any molecule that can reversibly bind to a given receptor with affinity and high specificity.

[0042] In a particular configuration of this embodiment, the target molecule (or ligand) is an antigen.

[0043] In a particular configuration of this embodiment, the antibody or antibody fragment is selected from Fab, Fab', (Fab')2, scFv, or scFv-Fc.

[0044] According to embodiments, the polypeptide comprises a terminal group selected from a thiol, an amine, an azide, an alkyne, an epoxide, a carboxylic acid, an aldehyde, an aziridine, an alkene, or a derivative thereof.

[0045] According to an embodiment, the polypeptide is a binding protein, which allows precise control of the grafting position of the fluorescent dye in the peptide chain, and the distance between the polypeptide and the fluorescent dye can be adjusted by inserting a linker.

[0046] According to a first particular configuration of this embodiment, the binding protein is preferably an immunoglobulin-binding protein.

[0047] According to a second particular configuration of this embodiment, the binding protein is selected from Protein A, Protein G, Protein L, Protein M, Protein Z, an immunoglobulin, a complete or partial immunoglobulin, or a derivative thereof.

[0048] According to an embodiment, the polypeptide comprises 2 to 100 amino acids, preferably 4 to 50 amino acids, preferably 5 to 20 amino acids, preferably 5 to 10 amino acids, even more preferably 8 amino acids. The use of such polypeptides has many advantages: -Fluorescent labeling is better controlled; Indeed, such polypeptides can be used to bind a single antibody or a small number of antibodies to a polypeptide by controlling the number of antibodies bound to the polypeptide, i.e., by adjusting the number of binding sites that can accommodate receptors, thereby allowing for fine control of the donor / acceptor fluorochrome ratio. -Improved sensitivity of fluorescent probes; In fact, by adjusting the number of amino acids constituting the peptide chain or by changing the position of the receptor binding site on the peptide chain, the fluorescent dye F a and F b The distance between them can be adjusted. -Fluorescent probes have great flexibility; In fact, the size of the solid is limited in this case.

[0049] According to a first particular configuration of this embodiment, the polypeptide is a linear or cyclic polypeptide. Preferably, the polypeptide comprises the amino acid sequence (RRGW) set forth in SEQ ID NO: 1. These amino acids form the Ig-binding unit.

[0050] According to a second particular configuration of this embodiment, the polypeptide comprises 8 amino acids comprising the amino acid sequence (RRGW) set forth in SEQ ID NO: 1. More preferably, the polypeptide comprises the amino acid sequence (CCGGRRGW) set forth in SEQ ID NO: 2. Even more preferably, the polypeptide comprises the sequence of 8 amino acids (CCGGRRGW) set forth in SEQ ID NO: 2.

[0051] According to an embodiment, the polypeptide can be replaced by an aptamer, which has affinity for the antibody, i.e., is capable of specifically binding to the constant part of said antibody in order to immobilize the latter.

[0052] According to a preferred embodiment, the fluorescent dye F a is the donor and fluorescent dye F b is the acceptor of the FRET donor / acceptor pair.

[0053] According to a further embodiment, the fluorescent dye F a is the acceptor and the fluorescent dye F b is the donor of the FRET donor / acceptor pair.

[0054] According to an embodiment, the fluorescent dye F a and / or F bhas a fluorescence emission peak within the range of 350 nm to 399 nm (UV range), 400 nm to 499 nm (blue range of the visible spectrum), 500 nm to 599 nm (green range of the visible spectrum), or 600 nm to 719 nm (red range of the visible spectrum), 720 nm to 850 nm (near infrared range).

[0055] According to an embodiment, the fluorescent dye F a and F b The fluorescence emission peaks of the two fluorochromes have an overlapping region. Advantageously, this overlap allows for non-radiative energy transfer between the two fluorochromes.

[0056] According to an embodiment, the fluorescent dye F a and / or F b is selected from a fluorescent molecule or a fluorescent protein.

[0057] In a particular configuration of this embodiment, the fluorescent molecule is selected from rhodamine, coumarin, evoblue, oxazine, carbopyronine, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole, xanthene, cyanine, fluorescein, squaraine, squaraine rotaxane, oxadiazole, acridine, arylmethine, tetrapyrrole, dipyrromethene, or any other fluorescent derivative thereof.

[0058] In particular configurations of this embodiment, the fluorescent protein is green fluorescent protein (GFP), 22G, aceGFP, amFP486 ("GFP-like fluorescent protein amFP486", "Anemonia manjano FP486"), amm2CP, avGFP, AvicFP1, cFP484 ("GFP-like fluorescent protein cFP484", "Clavularia cFP484"), dendFP, dfGFP ("green fluorescent protein"), DrCBD, DsRed, EosFP ("green-to-red photoconvertible GFP-like protein EosFP"), eqFP578 ("red fluorescent protein eqFP578", "Entacmaea quadricolor FP578"), eqFP611 ("red fluorescent protein eqFP611", "Entacmaea quadricolor FP611"), HcRed ("GFP-like non-fluorescent protein", "Heteractis crispa Red"), KikG, KO, LanYFPn, Montipora sp20 ("cytochrome c oxidase subunit 1"), mRed7 ("rod shape determining protein MreD", "mine drainage metagenome 7"), NpR3784g, RpBphP1 ("Rhodopseudomonas palustris BphP1"), RpBphP2 ("Rhodopseudomonas palustris BphP2"), RpBphP6 ("Rhodopseudomonas palustris BphP6"), TeAPCalpha, zFP538 ("GFP-like fluorescent protein FP538", "Zoanthus FP538")AausFP1, ​​vsfGFP-0, LanYFP, bfloGFPa1, RRvT, dLanYFP, dVFP, ccalYFP1, efasGFP, pcDronpa(green), aeurGFP, Skylan-S(On), mVenus-Q69M, tdTomato, PlamGFP, eechGFP1, mNeonGreen, Kaede(green), mClover3, Clover, VFP, pcDronpa2(green), moxNeonGreen, tdimer2(12), Dronpa(On), YPet, Skylan-NS(On), ffDronpa(On), eechGFP2, gfasGFP,Gamillus (On), sarcGFP, vsfGFP-9, mEos3.1 (green), pmeaGFP1, mVFP, pmimGFP1, pmimGFP2, mEos4a (green), pcDronpa2 (red), pdae1GFP, pmeaGFP2, mScarlet, mCitrine, ccalGFP3, phiYFP, SYFP2, Citrine2, mVFP1, Gamillus0.4, SHardonnay, aacuGFP2, mVenus, mEos4b (green), mKOI, fabdGFP, mGeos-C (On), rsKame (On), Tu rboRFP, afraGFP, stylGFP, phiYFPv, folding reporter GFP, citrine, dendFP (green), PSmOrange (orange), anobGFP, mRuby3, RFP611, Topaz, SEYFP, mScarlet-I, mWasabi, iq-mVenus, meffRFP, d2EosFP (green), eqFP578, EYFP-Q69K, mTFP1, ccalRFP1, eechGFP3, cgreGFP, superfolder GFP, meffGFP, mEos3.2 (green), pporG FP, muGFP, Venus, mGeos-E (On), Gamillus0.2, mEosFP-M159A (green), pporRFP, pcDronpa (red), D1EosFP (green), moxGFP, oxGFP, EosFP (green), amilFP513, KO, mGeos-S (On), tdKatushka2, mOrange, mEYFP, anm1GFP1, meffCFP, AzamiGreen, obeGFP, TagRFP, dimer2, dTomato, mEos2 (green), usGFP, M355NA, cgfTagRFP, mG eos-F(On), EYFP, Gamillus0.3, Kohinoor(On), amilFP593, ccalOFP1, obeYFP, mGeos-M(On), moxVenus, oxVenus, WasCFP, mEos4a(red), mUkG, NowGFP, mEosFP(green), mRuby2, ppluGFP2, mAvicFP1, dTFP0.2, AvicFP1, scubRFP, mKO2, UnaG, mEos4b(red), GFPmut2, mRuby, Emerald, mEmerald, ppluGFP1, meleRFP,mGeos-L(On), OFP, mmilCFP, KikGR1 (green), TurboGFP, mApple, CyRFP1 (CyRFP1), E2-Red / Green, ccalGFP1, mPapaya, moxCerulean3, GFP(S65T), eqFP611, meleCFP, GFPmut3, SGFP2(E222Q), mCerulean3, mOrange2, G3, Dreiklang(On), TagGFP2, mAzamiGreen, mKikGR (green), iq-mEmerald, cfSGFP2, Dendra2-M159A (orange), mEGFP, EGFP, TagRFP-T, TagGFP, anobCFP2, KCY, td-RFP611, TagBFP, smURFP, mTagBFP2, mLumin, SGFP2, SGFP2(T65G), PSmOrange2 (orange), eqFP611V124T, efasCFP, TagYFP, mKO, TDsmURFP, CyOFP1, MEos2 (red), SGFP2(206A), LanFP1, rsFastLime(On), mTFP0.7(On), cerFP505, psamCFP, Katushka2S, DsRed.T3, E2-Crimson, FR-1, DsRed2, mCerulean2, Dendra2-M159A (green), PATagRFP1314(On), mTurquoise2, Padron(On), aceGFP, AcGFP1, NijiFP (orange), SPOON(on), Cerulean, amilFP490, dTFP0.1, PATagRFP1297(On), mT-Sapphire, T-Sapphire, NijiFP (green), mAmetrine, mNectarine, mStrawberry, SGFP1, cgfmKate2, BrUSLEE, rsGreen1 (bright), mIrisFP (green), G2, mTurquoise, moxDendra2 (green), iq-mCerulean3, PATagRFP(On), mKate2, d1EosFP (red), Turquoise-GL, MiCy, mBlueberry2, FusionRed-M, dendFP (red), Dendra2-T69A (green), anobCFP1, I±GFP, mCerulean2.N, LSSmOrange, mCerulean2.D3,cpT-Sapphire174-173, Aquamarine, oxCerulean, mEosFP (red), mEosFP-F173S (green), KikGR1 (red), mNeptune2.5, EBFP1.5, Dendra2-T69A (orange), EosFP (red), aacuGFP1, bsDronpa (On), Dendra2 (green), mKateS158A, IrisFP (green), ZsGreen, mCerulean.B24, obeCFP, Katush ka, Padron0.9(On), mNeptune2, AausGFP, TagCFP, mCerulean.B, LanFP2, mKateS158C, mEos3.1(red), Dronpa-2(On), D10, shBFP-N1 58S / L173I, Kaede (red), sg25, d2EosFP (red), mCerulean2.N (T65S), avGFP, E2-Orange, BDFP1.6, DsRed-Max, mCerulean.B2, mIrisFP (red), CGFP, Dendra2 (red), Dronpa-3 (On), Sapphire, EBFP1.2, rsEGFP2 (On), FusionRed, EBFP2, CyPet, eforCP, mEos3.2 (red), mKikGR (red), mBeRFP, mCyRFP1, mKillerOrange, RFP630, mCherry2, moxBFP, oxBFP, KillerOrange, moxDendra2 (red), mClavGR2 (red), cFP484, rsEGFP (On), KCY-G4219, Gamillus0.1, mMaple (red), SCFP3A, IrisFP (orange), RFP637, mCardinal, mRFP1-Q66T, mKalama1, mCerulean, mKateM41GS158C, SBFP2, KCY-R1, mPapaya0.7, mCherry, iq-EBFP2, eqFP650, G1, sg12, mmiCy, mEos2-A69T (green), SCFP3b, DsRed-Express2, mKate, mScarlet-H, Dendra (green), mClavGR2 (green), td-RFP639, Azurite, Dendra (red), miRFP670-2, PA-GFP (On), iRFP713 / V256C, miRFP680, mECFP,SuperNovaRed, mNeptune, DsRed.T4, BFP.A5, mCRISPRed, ECFP, ZsYellow1, Neptune, mRaspberry, rsFolder(green), PAmCherry2(On), DsRed-Express, m oxMaple3 (red), iRFP670, mRFP1, mMaple3 (red), RFP639, iq-mApple, emiRFP670, miRFP670, shBFP, SiriusGFP, AvicFP4, W7, H9, SCFP2, mRFP1-Q66S, mTang erine, IrisFP-M159A (green), KillerRed, mMaple (green), dsFP483, GZnP3, PS-CFP2 (green), sg11, mRFP1-Q66C, miRFP670nano, mEosFP-F173S (red), miRFP682, LSSmCherry1, rsFolder2 (green), iRFP682, R3-2+PCB, amFP486, PSmOrange (far red), mGarnet2, miRFP, Jred, mMaroon1, PS-CFP2 (cyan), mGarnet, zFP538, PAmChe rry1(On), miRFP670v1, W1C, dTG, rsFusionRed1(On), P4-1, emiRFP703, miRFP703, mKelly2, mStable, dKeima, mEos2-A69T(orange), iRFP702, deGFP2, PSmOrange2 (far red), miRFP702, lanRFP-I“S83l, laRFP, W2, mKelly1, SCFP1, miRFP713, iFP2.0, pHuji, mIFP, iFP1.4, SuperNovaGreen, HcRed-Tandem, E BFP, iRFP713, SOPP3, SOPP2, miniSOG, HcRed7, miRFP720, RDSmCherry0.1, P4-3E, mTFP0.3, mMaple3 (green), mCarmine, iRFP720, SOPP, Maroon0.1, moxMap le3 (green), PS-CFP (Cyan), BFP, mBlueberry1, eechRFP, PS-CFP (green), deGFP3, PAmCherry3 (On), RDSmCherry1, deGFP1, PAmKate (On), LSS-mKate2, Wi-Phy,rsFusionRed2(On)、miRFP709、eqFP670、mBanana、KFP1(On)、sg50、mPlum、rsTagRF P(ON)、deGFP4、iq-mKate2、sg42、Pp2FbFPL30M、TagRFP675、mRojoB、AQ143、Sirius、 s, mKeima, TagRFP657, ECGFP, SNIFP, tKeima, Pp2FbFP, rsFusionRed3(On), AsRed2, LSS-mKate1, AvicFP2(before transformation), ECFPH148D, dKeima570, mHoneydew, cpCitrine, RsCherry(On), mNeptune681, mGrape1, mGinger2, mGrape3, mNeptune684, mGinger1, RDSmCherry0.2, mGrape2, mRojoA , SBFP1, mRouge, P4, RDSmCherry0.5, GZnP3 (GZnP3 (apostate)), rsCherryRev(On), Sandercyanin, HcRed, mRtms5, anm2CP, mTFP1-Y67W, Ultra marine, 10B, 11, 22G, (3-F)Tyr-EGFP, 5B, 6C, A1a, A44-KR, aacuCP, AausFP2, AausFP3, AausFP4(On), acanFP, aceGFP-G222E-Y220L, aceGFP -h, Achilles, AdRed, AdRed-C148S, ahyaCP, alajGFP1, alajGFP2, alajGFP3, amCyan1, amFP495, amFP506, amFP515, amilCP, amilCP580, am ilCP586, amilCP604, amilFP484, amilFP497, amilFP504, amilFP512, amilFP597, anm1GFP2, apulCP584, apulFP483, AQ14, asCP562, asFP49 9, asulCP, atenFP, avGFP454, avGFP480, avGFP509, avGFP510, avGFP514, avGFP523, AvicFP3 (before conversion), bfloGFPc1, BFP5, BFPsol, Blue102, BR 1, cEGFP, CFP, CFP4, cgigCP, cgigGFP, CheGFP1, CheGFP2, CheGFP3, CheGFP4, Clomeleon, Clover1.5, cpasCP, cp-mKate, Cy11.5, dClavGR1.6, dClover2, dClover2A206K, dfGFP, dhorGFP, dhorRFP, dimer1, dis2RFP, dis3GFP, dPapaya0.1, DrCBD, d-RFP618, Dronpa-C62S, DspR1, DsRed.M1, DsRed-Timer, DstC1, EaGFP, echFP, echiFP, eGFP203C, eGFP205C, EnhancedCyan-Emitting GFP, EYFP-F46L, fcFP, fcomFP, Flamindo2, FP586, Fpaagar, Fpag_frag, Fpcondchrom, FPmann, FPmcavgr7.7, FPrfl2.3, Gamillus0.5, GCaMP2, GCaMP6f(Ca. 2+ in the presence of), gdjiCP, gfasCP, GFP-151pyTyrCu, GFPhal, GFP-Tyr151pyz, GFPxm16, GFPxm161, GFPxm162, GFPxm163, GFPxm18, GFPxm181UV, GFPxm18UV, GFPxm19, GFPxm191uv, GFPxm19uv, gtenCP, HcRed1-Blue, hcriCP, hcriGFP, hfriFP, hmGFP, HriCFP, HriGFP, iLov, jRGECO1a(CA 2+ in the absence of), jRGECO1a(Ca 2+(in the presence of aG1ea, McaG2, mcavFP, mcavGFP, mcavRFP, mcCFP, mcFP497, mcFP503, mcFP506, mCherry1.5, mClavGR1, mClavGR1.1, mClavGR1.8, mClover1.5, mcRFP, meffCP, mEos2-N A, meruFP, MfaG1, miniSOG2, miniSOGQ103V, mKate2.5, mKG, mK-GO (late), mK-GO (early), mMaple2 (green), mMaple2 (red), mmGFP, mOFP.T.12, mOFP.T.8, montFP, Montiporasp.#20-9115, moxEos3.2, mPA-GFP, mPapaya0.3, mPapaya0.6, mPlum-E16P, mRed7, mRed7Q1, mRed7Q1S1, mRed7Q1S1BM, mRFP1.1, mRFP1.2, mRFP1.3, mRFP1.4, mRFP1.5, mTFP *, mTFP0.4, mTFP0.5, mTFP0.6, mTFP0.8, mTFP0.9, mTFP1-Y67H, mTurquoise-146G, mTurquoise-146S, mTurquoise2- G, mTurquoise-DR, mTurquoise-GL, mTurquoise-GV, mTurquoise-RA, NpR3784g, OFPxm, P11, P9, Padron(STAR)(On) , PdaC1, PDM1-4, pHluorin2 (acidic), pHluorin2 (alkaline), pHluorin, psupFP, ptilGFP, Q80R, RCaMP, R-FlincA, rfloGFP, rfloGFP2, rfloRFP, RFP618, roGFP1, roGFP1-R1, roGFP1-R8, roGFP2, RpBphP1, RpBphP2, RpBphP6, rrenGFP, rrGFP , rsCherryRev1.4(On), RSGFP1, RSGFP2, RSGFP3, RSGFP4, RSGFP6, RSGFP7, Rtms5, SAASoti(red), SAASoti(green), scleFP1, scleFP2, scubGFP1, scubGFP2, secBFP2, sfCherry, sfCherry2, sfCherry3C, SH3, ShG24, spisCP, stylCP, SuperfoldermTurquoise2, SuperfoldermTurquoise2ox, sympFP, TeAPCI±, tPapaya0.01, Trp-lessGFP, TurboGFP-V197L, V127TSAASoti(red), V127TSAASoti(green), vsGFP, Xpa, yEGFP, YFP3, zGFP, zoan2RFP, zRFP, or any other fluorescent derivative thereof. Fluorescent proteins are genetically encoded.

[0059] According to an embodiment, the fluorescent dye F a The bond between the receptor and the NHS-NH2, maleimide-SH or derivatives thereof is a covalent bond. In a particular configuration of this embodiment, the bond is of the NHS-NH2, maleimide-SH or derivatives thereof, and the NHS or maleimide group is linked to the fluorescent dye F. a The amine or thiol is supported on the receptor.

[0060] According to an embodiment, the fluorescent dye F a is a fluorescent protein, and the receptor and F a In this embodiment, the receptor and F are linked in the form of a fusion protein. a are encoded by the same gene.

[0061] According to an embodiment, the fluorescent dye F b The bond between the polypeptide is a covalent bond. In a particular configuration of this embodiment, this bond is of the type NHS-NH, maleimide-SH or derivatives thereof, and the NHS group or the maleimide group is linked to the fluorescent dye F b The amine or thiol is carried on the polypeptide.

[0062] According to an embodiment, in the inactive configuration (off configuration) of the fluorescent probe, the fluorescent dye F a and F b The distance between the fluorescent dye F and the fluorescent dye F does not enable the FRET effect. a and F b The distance between the fluorescent dye F and the fluorescent dye F is greater or smaller than the distance that allows for the FRET effect; a and F b In other words, the distance between the fluorescent dye F and the fluorescent probe in the inactive configuration (i.e., the fluorescent probe at rest, in the off configuration) a and F b is the distance between the fluorescent dye F in the active (ON) configuration. a and F bThe distance between the receptor and the target molecule is either greater or smaller than the distance between the receptor and the target molecule. The fluorescent probe is at rest when the receptor is not bound to the target molecule. In the case of an antibody, the fluorescent probe is at rest when the antibody recognition site is free. The fluorescent probe is in the active configuration when the receptor is bound to the target molecule; in the case of an antibody, the fluorescent probe is in the active configuration when the antibody recognition site is free, i.e., when the antigen is recognized and bound to the antibody. There is also a scenario in which the ON configuration occurs when the antibody does not detect its target molecule and switches to the OFF configuration when the receptor binds to the target molecule (FRET in the absence of the target molecule and cessation of FRET by the target molecule). This results in a change in the fluorescent signal in both cases when the target molecule is recognized.

[0063] Apparatus for detecting and / or measuring the concentration of a target molecule - Patent Application 20070122997 The present invention also relates to an apparatus for detecting a target molecule and / or for measuring the concentration of a target molecule.

[0064] The device comprises: - a substrate to the surface of which graft molecules are covalently attached; at least one fluorescent probe, ■ at least one receptor covalently bound to the polypeptide; ■Two types of fluorescent dyes F a and F b Including, Fluorescent dye F a binds to the receptor and the fluorescent dye F b binds to the polypeptide; and Fluorescent dye F a and F b comprises at least one fluorescent probe that forms a FRET donor / acceptor pair.

[0065] The polypeptide is covalently attached to the graft molecule.

[0066] The fluorescent probes are as described above and the embodiments relating to the fluorescent probes or the different components of said probes (receptor, polypeptide, fluorescent dye) apply to the device of the invention.

[0067] The covalent bond between the receptor and the polypeptide, and the covalent bond between the polypeptide and the graft molecule, which is stronger than the receptor-target molecule bond, can ensure that the receptor does not leave the polypeptide or the fluorescent probe does not leave the substrate once the target molecule is recognized, thus avoiding separation of the fluorescent probe into two parts or probe-substrate separation.

[0068] During in vitro target molecule detection, avoiding separation between receptor and polypeptide is particularly important so as not to induce contamination of the sample.

[0069] During in vivo target molecule detection, avoiding separation between the receptor and polypeptide or between the fluorescent probe and substrate is particularly important when the fluorescent probe is used at the distal end of an optical fiber for in vivo probing, as it limits the risk of leaving part (receptor-bearing) or all of the fluorescent probe inside the patient's body upon fiber removal. Such biological contamination can cause a variety of adverse effects, similar to those observed during direct injection of antibodies, including symptoms of discomfort such as headache, nausea, or asthenia; reactions such as fever or chills; allergy-like dermatotropism (itching, rash, hives); respiratory (bronchospasm, cough, dyspnea) or cardiovascular (low blood pressure) symptoms; or tumor lysis syndrome (in cases of high tumor burden). These adverse effects depend on both the properties of the ligand and the receptor.

[0070] The role of the graft molecule is to ensure the correct orientation of the receptor so that its recognition site is accessible to the target molecule.

[0071] The use of grafting molecules on the surface of a substrate also allows for fine control over the functionalization of said surface, in particular by adjusting the number of fluorescent probes grafted to the surface of the substrate, i.e. by varying the coverage of said substrate with grafting molecules, the density (or coverage) of fluorescent probes per unit area of ​​the surface of the substrate, which advantageously allows for the provision of devices designed for the specific needs of the user.

[0072] In an alternative embodiment of the invention, the polypeptide could be attached to the graft molecule by a non-covalent bond.

[0073] According to an embodiment, the target molecule (or ligand) is a molecule for which the receptor has affinity and high specificity. Preferably, the target molecule (or ligand) is an antigen.

[0074] According to an embodiment, the substrate is selected from a cell culture plate, a well plate, a film, a strip, an agarose gel, a cellulose gel, nanoparticles or microparticles, preferably spherical, preferably silica or polymer, a microscope slide or a glass slide. The purpose of the device according to this embodiment is the detection of target molecules and / or the measurement of their concentration in vitro, i.e. in solution or on a substrate.

[0075] Preferably, the substrate is a polymer film selected from polyethylene terephthalate, fluorinated polyethylene-co-propylene, polymethyl methacrylate, polytetrafluoroethylene, polymethylpentene, polyvinyl chloride, styrene methyl methacrylate, polyethylene naphthalate, derivatives thereof, or mixtures thereof. Advantageously, such polymer films are chemically inert, transparent, and / or resistant to high temperatures, i.e., resistant to temperatures of at least 90°C, preferably at least 110°C, preferably at least 130°C.

[0076] According to an embodiment, the device further comprises an optical fiber. Preferably, the device comprises an optical fiber and a probing head. The head is detachably attached or firmly connected (via a ferrule) to the optical fiber. In a known manner, an optical fiber comprises a cladding surrounding one or more fiber cores, and its distal end for probing is presented in the form of a rigid ferrule, the outer surface of which transversely to the fiber axis is transparent, firmly connected to the end of the fiber cladding. The purpose of the device according to this embodiment is the detection of target molecules and / or the measurement of their concentration in vivo, i.e., in the body of a patient, for example by endoscopy or during surgery.

[0077] A "probing head" is a part of the fiber that acts as a probe or, in general, has any technical function that uses the light emitted at the end of the optical fiber to cooperate or interact with the medium into which the end of the fiber is introduced. The ferrule of the optical fiber only has the function of mechanical attachment of the probing head.

[0078] In the case of a detachable probe head, the ferrule and probe head are either firmly connected by mechanical assembly (crimping, fitting, screwing, clipping, quarter-turn locking) or each include a cooperating attachment means (male-female coupling means). The attachment of the head to the ferrule is designed so that the head and ferrule cannot separate during use, especially when the fiber is introduced into the patient's body.

[0079] In a particular configuration of this embodiment, the substrate is selected from a substrate configured to be attached around the optical fiber or to the head of the optical fiber, preferably an optically transparent substrate configured to be attached to the distal end (i.e., probing end) of the optical fiber, more particularly to the distal end of the probing head.

[0080] Preferably, the substrate is a polymer film located and / or attached to the distal end of the probing head, in this embodiment the polymer film may be glued to the distal end of the probing head using, for example, an epoxy resin, or may be mechanically attached to the distal end of the probing head.

[0081] In a specific configuration of this embodiment, the probe head has a body and an outer surface at its tip, called the emission surface, at least a portion of which is transparent and forms a port, intended to face the core(s) of the optical fiber for light passage. Preferably, a polymer film is used as the transparent part so that it is functionalized with a fluorescent probe, i.e., the substrate of the device is the port of the probe head. This conveniently allows for internal probing and in vivo target molecule detection to be performed by contacting the distal end of the probe head with a member. Preferably, the probe head is detachably attached to the optical fiber, conveniently allowing for changing the probe head and therefore the fluorescent probe depending on the target molecule detection while maintaining the same optical fiber. Preferably, the body is made of a polymer material, glass, ceramic, stainless steel, composite material, or a combination of these materials, and the outer emission surface is made of a polymer, glass, ceramic, silica, composite material, or hybrid material.

[0082] Advantageously, the optical fiber is not subjected to any processing such as tapering. Preferably, the fluorescent probe is grafted to the port of the probing head, thus leaving the fiber intact. This allows for improved sensitivity of the device and reproducibility of the grafting surface. The role of the optical fiber is then to transport the optical signal to the target.

[0083] According to an embodiment, the optical fiber comprises a core partially covered by a metal cladding, a part of said core is not covered by the cladding, and the fluorescent probe is grafted to the surface of the part of the core that is not covered. This embodiment makes it possible to obtain surface waves on a longitudinal part of the optical fiber.

[0084] According to an embodiment, the grafting molecule comprises at least two reactive groups selected from maleimide, N-hydroxysuccinimide (NHS) ester, sulfo-N-hydroxysuccinimide (NHS) ester, sulfo-NHS, azide, alkyne, epoxide, carboxylic acid, aldehyde, aziridine, alkene, or derivatives thereof. The grafting molecule allows for covalent bonding between the substrate and the fluorescent probe via a grafting molecule-polypeptide bond. Preferably, two reactive groups are located at each of its ends. The bond between the polypeptide and the grafting molecule occurs between an end group of the polypeptide, preferably a thiol or amine, and one of the two reactive groups of the grafting molecule described herein.

[0085] In a preferred configuration of this embodiment, the grafting molecule contains a maleimide group that can react with the thiol terminal group of the polypeptide to form a covalent bond. The thiol group is terminal and singular, and this configuration has the advantage that the orientation of the polypeptide on the substrate can be controlled, and the orientation of the fluorescent probe can ensure optimal accessibility of the receptor to the target molecule.

[0086] In another configuration of this embodiment, the graft molecule comprises an N-hydroxysuccinimide group that can react with an amine group of a polypeptide to form a covalent bond.

[0087] According to an embodiment, the substrate is coated with a layer of an organic or inorganic material selected from zirconia, titanium dioxide, epoxy, and organosilanes such as amine organosilanes, thiol organosilanes, azide organosilanes, alkyne organosilanes, carbonyl organosilanes, or organosilanes having carbon-carbon double bonds. Preferably, the substrate is coated with a layer of amine organosilanes or thiol organosilanes. Typically, a substrate, such as a polymer film, is coated with an amine organosilanes and then immersed in a (3-aminopropyl)triethoxysilane (APTES) solution, resulting in a substrate coated with a layer of graft molecules bearing maleimide groups. Alternatively, a substrate, such as a polymer film, is coated with a thiol organosilanes and then immersed in an amine solution, resulting in a substrate coated with a layer of graft molecules bearing maleimide groups.

[0088] Method for detecting a target molecule and / or for measuring the concentration of a target molecule The present invention also relates to methods for detecting and / or measuring the concentration of a target molecule.

[0089] The method comprises the following steps: at least one fluorescent probe, ■ at least one receptor covalently bound to the polypeptide; ■Two types of fluorescent dyes F a and F b containing the fluorescent dye F a binds to the receptor, and the fluorescent dye F b is bound to the polypeptide; and Fluorescent dye F a and F b form a FRET donor / acceptor pair, and contacting the sample with at least one fluorescent probe, the receptor of which has affinity for said target molecule; - exciting the fluorescent probe at a predetermined wavelength such that the donor fluorescent dye is excited; - measuring the ratio between the intensity of the fluorescence emitted by the donor fluorescent dye and the intensity of the fluorescence emitted by the acceptor fluorescent dye; - determining the presence or absence of said target molecule in the sample and / or calculating the concentration of said target molecule in the sample Includes.

[0090] Upon recognition of the target molecule by the receptor, a conformational change occurs in the receptor, which allows the fluorescent dye F a and F b When the distance between the donor and acceptor fluorochromes changes, non-radiative transfer (FRET effect) occurs from the donor fluorochrome to the acceptor fluorochrome. Therefore, fluctuations in the intensity of the fluorescence peaks of the donor and acceptor fluorochromes are a direct result of the concentration of the target molecule in the sample. Therefore, if the target molecule is present in the sample, an increase or decrease in the fluorescence intensity emitted by the donor fluorochrome is expected, which increases or decreases the ratio between the intensity of the fluorescence emitted by the donor fluorochrome and the intensity of the fluorescence emitted by the acceptor fluorochrome (hereinafter referred to as the FRET index). If the intensity of the fluorescence peak does not change (zero FRET index), this indicates the absence of the target molecule in the sample. Therefore, determining the presence or absence of the target molecule in the sample and / or calculating the concentration of the target molecule in the sample is a result of interpreting the fluctuations in the FRET index. The reverse is also possible.

[0091] Advantageously, the detection of target molecules is fast.The use of the above-mentioned fluorescent probe, especially the presence of receptor-polypeptide covalent bond, significantly increases the sensitivity of target molecule detection.For example, the detection threshold is observed to be less than 25nM.Preferably, the method according to the present invention has a detection threshold of less than 10nM, preferably less than 5nM, more preferably less than 1nM, even more preferably less than 0.1nM.

[0092] The embodiments relating to fluorescent probes, different elements of said probes (receptors, polypeptides, fluorescent dyes), devices or different elements of said devices are applied to the implementation of the method according to the invention. In particular, the method according to the invention is implemented by the device according to the invention.

[0093] According to embodiments, the fluorescent reporter is contacted with the sample by any contacting means. Preferably, the contacting occurs in solution or by direct contact.

[0094] In certain configurations of this embodiment, the direct contact lasts for a few seconds. In other specific configurations of this embodiment, for example in solution, the contact in solution lasts for less than 1 hour, preferably less than 30 minutes, and more preferably less than 5 minutes. The longer the contact time, the sharper the optical signal.

[0095] According to an embodiment, the ratio between the intensity of the fluorescence emitted by the donor fluorochrome and the intensity of the fluorescence emitted by the acceptor fluorochrome is measured by spectroscopy.

[0096] According to an embodiment, the method also includes a pre-calibration step in which the device is contacted with a healthy sample. Advantageously, this step allows the selected F a / F b A reference FRET index measurement for the pair can be determined, and subsequent comparison of this reference measurement with the FRET index measured upon contact with a sample suspected of harboring the target molecule allows conclusions to be drawn about the presence or absence of the target molecule and its concentration in the test sample.

[0097] Detection of the presence, absence, or amount above or below a certain threshold of a target molecule allows one to characterize a sample and determine whether it is healthy or not.

[0098] According to the embodiment, the sample may be any sample that may contain a target molecule to be detected or measured, and may be a liquid sample or a solid sample.

[0099] According to embodiments, the sample is selected from a solution, a cell culture (e.g., eukaryotic or prokaryotic), whole blood, plasma, serum, sweat, or any biological liquid or fluid, organic tissue, or organ.

[0100] In a particular configuration of this embodiment, the liquid sample can be used directly as the detection or measurement target, or can be diluted with, for example, a buffer solution or saline, and then used as the detection or measurement target. Examples of liquid samples include, but are not limited to, cell cultures (e.g., eukaryotic or prokaryotic), culture supernatants, cell extracts, bacterial extracts, body fluids such as serum, plasma, saliva, sweat, cerebrospinal fluid, or urine, industrial wastewater, or agricultural food liquids such as milk.

[0101] In a particular configuration of this embodiment, the solid sample is selected from organic tissues and organs. The solid sample can be dissolved, suspended, or immersed in a liquid, such as a buffer or saline, in a state where it can be contacted with the free fluorescent probe, and then used as a sample. Preferably, the solid sample does not undergo any treatment before contacting with the fluorescent probe.

[0102] According to an embodiment, the detection threshold of a target molecule depends on the affinity between the receptor and the target molecule, and the detection threshold is on the order of several pmol / L (picomolar concentration), preferably on the order of several fmol / L (femtomolar concentration).

[0103] According to an embodiment, the method comprises: in vitro, for example in suspension, in solution, or on a substrate (e.g., a multiwell plate, a microscope slide, or a strip); or In vivo, for example by endoscopy or during surgery It can be implemented.

[0104] According to an embodiment, the device comprises excitation means configured to excite the sample and / or the fluorescent probe, and / or a fluorescent dye F a and F b and optical collection means configured to collect data from the fluorescence of the light.

[0105] In a particular configuration of this embodiment, the excitation means is a light source capable of irradiating at a given wavelength, such as for example a mercury lamp, a xenon lamp, an LED, a UV lamp or a laser light source.

[0106] In particular configurations of this embodiment, the optical data collection means is a microscope, a fluorometer, a cytometer, or a spectrophotometer.

[0107] Purpose The present invention also relates to the use of a fluorescent probe according to the invention and / or a device according to the invention for the detection of a target molecule and / or for determining the concentration of a target molecule in a sample.

[0108] According to an embodiment, the fluorescent probe according to the invention and / or the device according to the invention is used for detecting and / or measuring the concentration of a target molecule in a sample, for example in a solution, in vitro.

[0109] According to an embodiment, the fluorescent probe according to the invention and / or the device according to the invention is used for detecting a target molecule and / or measuring the concentration of a target molecule in a sample in vivo.

[0110] In a specific configuration of an in vivo embodiment, the fluorescent probe according to the present invention and / or the device according to the present invention is used to detect tumor cells, to screen for infections, or to detect markers that can assist in diagnosing or monitoring the progression of a pathological condition. In a specific configuration of an in vivo embodiment, the device is a probing head to which a fluorescent probe is grafted (in this case, the fluorescent probe is grafted to a port of the probing head, i.e., a transparent part of the light-emitting surface of the head), or an optical fiber with a catheter at its distal end to which a fluorescent probe is grafted. Preferably, the fluorescent probe according to the present invention and / or the device according to the present invention is used in endoscopy, which is used to examine the interior of a cavity by backscattered light detection techniques using optical fibers. The endoscope comprises a flexible casing that houses one or more optical fibers, the distal end of which is intended to be introduced into the cavity to be examined, and the opposite proximal end of which is intended to be connected to a light source aligned with the optical fiber(s) to transmit light to the distal end and into the cavity. A photodetector, aligned with the optical fiber and located at the proximal end, is also intended to receive the light emitted by the fluorescent probe and returned through the fiber.

[0111] According to an embodiment, the fluorescent probe according to the invention and / or the device according to the invention is used for detecting and / or measuring the concentration of a target molecule in industrial water, wastewater or agricultural food liquids, such as milk, etc. In a particular configuration of this embodiment, the fluorescent probe according to the invention and / or the device according to the invention is used for detecting drugs or pesticide residues or for detecting pathogens. [Brief explanation of the drawings]

[0112] [Figure 1] FIG. 1 is a diagram depicting the switching of a fluorescent probe from an inactive conformation (off) to an active conformation (on) upon recognition of a target molecule. [Figure 2] FIG. 2 depicts a detection device according to a particular embodiment. [Figure 3A] FIG. 3A is a diagram of optical spectra obtained by fluorimetric analysis of fluorescent probe responses to different antigen concentrations. [Figure 3B] FIG. 3B is an illustration of a dose-response curve obtained using a fluorescent probe by measuring the FRET index using the curve shown in FIG. 3A. [Figure 4] FIG. 4 shows the FRET response obtained when an antigen is added to a fluorescent probe (herein TrkB) and BSA, an unrelated molecule that does not undergo a conformational change. [Figure 5A] FIG. 5A shows the optical spectra measured after exposure of the fluorescent probe with a 488 nm laser in the presence of cell lines that do or do not express the target antigen. [Figure 5B] FIG. 5B is a plot of the FRET index obtained from the spectra shown in FIG. 5A. [Figure 6] FIG. 6 shows the optical spectra measured after excitation with a 488 nm laser for a fluorescent probe containing an antibody bound to protein G (gray curve) and a fluorescent probe containing an antibody bound to a polypeptide consisting of eight amino acids from the amino acid sequence set forth in SEQ ID NO: 1 (black curve). [Figure 7] Figure 7 shows the fluorescence intensity after excitation with a 488 nm laser for a fluorescent probe (Snc) containing an antibody noncovalently bound to a binding protein and a fluorescent probe (Sc) containing an antibody covalently bound to a binding protein before and after elution of the antibody at acidic pH (white column: solution containing the fluorescent probe before elution; gray column: solution containing the fluorescent probe after elution; black column: eluent). [Figure 8] FIG. 8 shows the magnitude of change in FRET index of a fluorescent probe in response to exposure to the target antigen EGFR for a fluorescent probe comprising an antibody non-covalently bound to a binding protein (Snc) and a fluorescent probe comprising an antibody covalently bound to a binding protein (Sc). DETAILED DESCRIPTION OF THE INVENTION

[0113] Exemplary Embodiments of the Invention The recognition of target molecule 2 by fluorescent probe 1 is illustrated in Figure 1, where fluorescent probe 1 is a receptor 11 bound to a polypeptide 12; and -Two types of fluorescent dyes F a and F b Includes.

[0114] Fluorescent dye F a binds to receptor 11 and fluorescent dye F b binds to polypeptide 12. Fluorescent dye F a and F b form a FRET donor / acceptor pair. Receptor 11 is covalently bound to polypeptide 12, i.e., stronger than the bond that can bind receptor 11 to recognition molecule 2.

[0115] Before recognition of the target molecule 2 by the fluorescent probe 1, the latter is in the so-called inactive configuration (“off”), i.e., two fluorescent dyes F a and F b In this configuration, the donor fluorochrome is excited and emits light by fluorescence, but the acceptor fluorochrome does not. When the target molecule 2 is recognized by the fluorescent probe 1, the latter then adopts the active configuration ("on"), which causes a conformational change in the acceptor 11, resulting in the two fluorescent dyes F a and F b This induces a non-radiative energy transfer (FRET effect) between the two fluorophores. This energy transfer occurs from the donor fluorophore to the acceptor fluorophore: the fluorescence intensity of the donor fluorophore decreases and the fluorescence intensity of the acceptor fluorophore increases; in this configuration, the fluorophores are expressed as F' a and F' b The change in the emission spectrum due to the FRET effect can be measured to detect the target molecule 2 and / or measure its concentration.

[0116] This embodiment is particularly advantageous because receptor 11-polypeptide 12 binding predominates over receptor 11-target molecule 2 binding, allowing for rapid detection of target molecule 2 while avoiding degradation of fluorescent probe 1.

[0117] In the embodiment shown in FIG. 2, the device for detecting a target molecule comprises: an optical fiber 4, ■ Cladding 46; ■ at least one core 45 of longitudinal axis XX'; ■Ferrule 44 ■ A probe head comprising a body 43 and an outer surface 42, called the emission surface, at least part of which is transparent, forming a port 41 and intended to face the core(s) 45 of the optical fiber 4 for the passage of light. an optical fiber 4; and - fluorescent probe 1, ■ Receptors bound to polypeptides; and ■Two types of fluorescent dyes F a and F b Fluorescent dye F a binds to receptor 11 and fluorescent dye F b is a fluorescent dye F that binds to polypeptide 12 and forms a FRET donor / acceptor pair. a and F b Fluorescent probe 1 containing Equipped with.

[0118] The optical fiber 4 has a proximal end, not shown here, which is intended to be connected in a known manner to a light source, and an opposite distal end which forms the probing end of the optical fiber 4, which emits the light necessary to illuminate the fluorescent probe. The same optical fiber is also used to collect the optical response of the probe, directing the light beam back to a box at the proximal end of the fiber.

[0119] The probing end of the optical fiber 4 is provided in known manner with a ferrule 44 forming a centrally drilled rigid end, in which the cladding 46 of the optical fiber 4 is mounted.

[0120] A port 41 on the outer surface 42 of the probe head is functionalized with a fluorescent probe 1 via a grafting molecule 3. The fluorescent probe 1 is thus functionalized with a fluorescent dye F a and F bIn order to detect cancer cells, by observing and / or measuring the variations in the emission spectrum of the target molecules present in the cavity probed by the optical fiber 4, in particular target molecules contained in cavities of the human body, i.e. on human tissue, that the probe head encounters during its use, can be recognized.

[0121] In a preferred method shown in FIG. 2, the probing head comprises a hollow cylindrical body 43 of longitudinal inner axis XX′ in the same position as the assembly of the head on the body of the optical fiber 4, and an outer distal end face 42 transverse to the axis of the cylinder and intended to emit light from the core 45 of the optical fiber 4.

[0122] This embodiment is particularly advantageous as it allows for the rapid detection of target molecules in body cavities (e.g. by endoscopy or during surgery) while avoiding degradation of the fluorescent probe 1 upon recognition of the target molecule. This in particular avoids parts of said fluorescent probe 1 being left behind in the cavity being probed, which would lead to contamination of the body being probed.

[0123] Example The invention will be better understood on reading the following examples, which illustrate the invention without limiting it.

[0124] Example 1a : In vitro detection of target molecules - detection in solution Preparation of fluorescent probes A solution of equimolar concentrations of anti-EGFR antibody (labeled with Alexa 488) and Protein G (labeled with Alexa 546) is incubated for 2 hours. The incubation should be long enough to ensure optimal binding protein-antibody binding.

[0125] Target molecule detection In parallel, antigen solutions of known concentrations were diluted with the same probe solution to generate a standard series ranging from 100 nM to 80 pM. To achieve the same concentrations as the standard series, the antigen-containing solution (recombinant EGFR) diluted in PBS was mixed with a solution containing a pre-prepared fluorescent probe. After 1 hour of incubation, the spectral characteristics were measured by fluorimetry. The FRET index was calculated for the standard series and the samples, allowing their concentrations to be calculated. The results obtained for the calibration curve are shown in Figures 5A and 5B.

[0126] The results shown in Figures 4, 5A, and 5B were obtained by depositing cell lines (HEK cells, A549 cells, and A431 cells) expressing or not expressing the antibody's target EGFR on microscope slides to which a fluorescent probe had been pre-adhered. For this purpose, 24 hours before deposition on the substrate, an organosilica (thiolsilica) / zirconia sol was prepared: 20 mL of a mixture of mercaptotriethoxysilane / zirconium chloride / ethanol / water (0.95 / 0.05 / 40 / 5) was stirred at ambient temperature. The sol was deposited on the microscope slide by dip coating (withdrawal speed 5.5 mm / s) and heat-treated at 80 °C for 12 hours. The first binding molecule, 1-(2-aminoethyl)-pyrrole-2,5-dione hydrochloride, containing maleimide and amine groups, was dissolved in DMSO at 0.1 mol / L, and the solution was then added to the slide for 1 hour with stirring at ambient temperature. After rinsing and washing with DMSO and distilled water, a second solution containing N-succinimidyl 4-maleimidobutyrate (at 0.1 mol / L), another conjugation molecule containing an NHS ester group and a maleimide group, in DMSO, is added to the slide. After rinsing and washing with DMSO and distilled water, a solution of Protein G (Alexa 546) at 10 μg / ml in PBS is incubated on the slide for 1 hour. The excess is washed off with PBS, and then a solution of anti-EGFR antibody (Alexa 488) in PBS is incubated for 1 hour. The excess is washed off with PBS, and then A431, HEK, and A549 cell suspensions are deposited on the surface and functionalized.

[0127] Fiber optics and a spectrophotometer are used for laser excitation and signal collection, allowing the determination of the amount of target molecule present on the cell surface (low to none for HEK cells, moderate for A549 cells, and high for A431 cells).

[0128] Example 1b : In vitro detection of target molecules Example 1a was reproduced, but the fluorescent probe was changed according to Table I.

[0129] [Table 1]

[0130] Example 2 : In vitro detection of target molecules Grafting on the substrate 24 hours before deposition onto the substrate, an organosilica (thiolsilica) / zirconia sol is prepared: a mixture of mercaptotriethoxysilane, zirconium chloride, ethanol, and water (0.95 / 0.05 / 40 / 5) is stirred at ambient temperature. The sol is deposited onto the substrate, typically the wells of a multiwell plate, and heat-treated at 80°C for 12 hours. The first binding molecule, 1-(2-aminoethyl)-pyrrole-2,5-dione hydrochloride, containing maleimide and amine groups, is dissolved in DMSO at 0.1 mol / L, and this solution is then added to the wells with stirring at ambient temperature for 1 hour. After rinsing and washing with water and DMSO, a second solution containing another binding molecule, N-succinimidyl 4-maleimidobutyrate, containing NHS ester and maleimide groups in DMSO (at 0.1 mol / L), is added to the wells at 100 μL per well with stirring for 1 hour. After rinsing and washing with DMSO and distilled water, the substrate is prepared for attachment of a first fluorescent binding protein (usually protein G terminated with a cysteine).

[0131] Preparation of fluorescent probes A solution of 10 μg / ml of Protein G (labeled with Alexa 546) in PBS is incubated on the functionalized surface for 1 hour. The excess is washed off with PBS. A 2 mM glutaraldehyde solution is incubated with the Protein G immobilized on the substrate for 15 minutes. The excess glutaraldehyde is washed off with PBS, and then a solution of anti-EGFR antibody (labeled with Alexa 488) is incubated for 2 hours. This time is long enough to ensure optimal Protein G-antibody binding. The excess antibody is then washed off with PBS.

[0132] Target molecule detection A solution containing the EGFR target antigen is added to the wells and detected in the same manner as in the test with the solution in Example 1.

[0133] Example 3a : Detection of target molecules in vivo Grafting onto the probe head or optical fiber The fluorescent probe prepared according to the method defined above is directly grafted onto the transparent port of a probe head (or capsule) designed to be assembled into an optical fiber. The port is typically made of PET (polyethylene terephthalate) or FEP (fluorinated polyethylene-co-propylene). Grafting is performed as in Example 2, first depositing a thin silica / zirconia layer on the port by dip-coating an organosilane / zirconia sol at a speed of 5.5 mm / s, followed by a surface functionalization step by grafting a molecule containing maleimide / NHS groups (1-(2-aminoethyl)-pyrrole-2,5-dione hydrochloride, followed by N-succinimidyl 4-maleimidobutyrate at 0.1 mol / L). The maleimide group can react with thiol groups of proteins, and the NHS group can react with amine groups of proteins, resulting in a covalent bond between the fluorescent probe and the substrate.

[0134] The method of grafting the fluorescent probe onto the end or port of the fiber corresponds to the method of grafting onto the substrate in Example 2.

[0135] EGFR detection on solid surfaces The port or optical fiber is brought into contact with the tissue or any other interface on the surface where the target molecule is likely to be present, and simultaneously illumination by a 488 nm laser beam is carried by the optical fiber to the fluorescent probe, and the reflected beam is collected by the same optical fiber into a spectrophotometer for analysis.

[0136] The FRET index is calculated to determine whether the interaction between the fiber (or port) and the tissue is positive (presence of the target molecule) or negative (absence of the target molecule), and this detection occurs within a few seconds.

[0137] Example 3b :Detection of target molecules Example 3a was reproduced, but the fluorescent probe was changed according to Table II.

[0138] Table II: Fluorescent Probe Composition [Table 2]

[0139] Example 4 : Fluorescent antibody-polypeptide probe Preparation of fluorescent probes 24 hours before deposition onto the substrate, an organosilica (thiolsilica) / zirconia sol is prepared: 20 mL of a mixture of mercaptotriethoxysilane / zirconium chloride / ethanol / water (0.95 / 0.05 / 40 / 5) is stirred at ambient temperature. The sol is deposited onto the substrate, typically a 50-micron-thick PET film, by dip coating (with a withdrawal speed of 5.5 mm / s) and heat-treated at 80°C for 12 hours. The first binding molecule, 1-(2-aminoethyl)-pyrrole-2,5-dione hydrochloride, containing maleimide and amine groups, is dissolved in DMSO at 0.1 mol / L, and the solution is then added to the polymer film with stirring at ambient temperature for 1 hour. After rinsing and washing with DMSO and distilled water, a second solution containing another conjugation molecule, N-succinimidyl 4-maleimidobutyrate, containing an NHS ester group and a maleimide group, in DMSO (at 0.1 mol / L) is added onto the polymer film with stirring for 1 hour. After rinsing and washing with DMSO and distilled water, the substrate is ready for conjugation with polypeptides.

[0140] An anti-EGFR antibody solution (labeled with Alexa 488) is incubated with a polypeptide (labeled with Alexa 546) having the amino acid sequence set forth in SEQ ID NO: 2 (CCGGRRGW) and immobilized on the substrate at ambient temperature for 60 minutes.

[0141] A similar process is followed to generate a fluorescent probe containing the same antibody conjugated to protein G. In this case, the protein G and the antibody carry the same fluorescent dye as in the previous case.

[0142] Target molecule detection After incubation, the spectral characteristics are measured using a fluorometer with excitation at 488 nm.

[0143] The two probes have different optical properties. In fact, as shown in Figure 6, the FRET effect is larger between the antibody and the peptide than between the antibody and Protein G. This difference in fluorescence intensity is due to the difference in the fluorescent dye F in the two probes. a and F bThis can be explained by the difference in the distance between the peptide chains. Since the peptide chains of the peptide are shorter than those of protein G, the distance between the fluorescent dyes is also shorter.

[0144] Example C1 Comparative Example 1 - Covalent binding of antibody to protein G Preparation of fluorescent probes containing antibodies non-covalently bound to Protein G-S nc Protein G (labeled with Alexa 546) is covalently grafted onto the bottom of the previously functionalized well plate as described above. To non-covalently attach the antibody to Protein G, a solution of anti-EGFR antibody (labeled with Alexa 488) is incubated at 10 μg / ml. Excess antibody is washed off with PBS.

[0145] Preparation of fluorescent probes containing antibodies covalently bound to Protein G-S c Protein G (labeled with Alexa 546) is covalently grafted onto the bottom of the previously functionalized well plate as described above. A 2 mM glutaraldehyde solution is added for 15 minutes. Excess glutaraldehyde is washed off with PBS. To covalently attach the antibody to Protein G, an anti-EGFR antibody solution (labeled with Alexa 488) is incubated at 10 μg / ml. Excess antibody is washed off with PBS.

[0146] After excitation at 488 nm, two fluorescent probes S nc and S c The fluorescence intensity emitted by the antibody was measured.

[0147] Assessment of antibody-protein G binding stability Then, each fluorescent probe S nc and S c The wells were incubated in 100 μL of 0.1 M aqueous glycine solution (pH 2.5), which reduces the affinity of Protein G for the antibody, for 30 minutes at 37° C. After incubation, the glycine solution was deposited into adjacent wells.

[0148] The fluorescence intensity of the remaining assembled antibody and that of the eluted antibody were measured, which allows us to determine the amount of antibody released from the bound protein, i.e., to assess the strength of the antibody-protein bond and therefore the possibility of detachment of the fluorescent probe.

[0149] The addition of a glutaraldehyde conjugation step increases the amount of antibody bound to Protein G, inducing a covalent bond between the two. Figure 7 shows the relationship between the amount of antibody successfully bound to Protein G (white column), the amount of antibody remaining attached to Protein G after incubation with glycine (gray column), and the amount of antibody eluted after incubation with glycine (black column) with and without covalent conjugation. The following is evident: - the amount of antibody successfully bound to Protein G increases upon covalent bond formation, thus increasing the yield of usable fluorescent probe; - the amount of antibody that remains bound to Protein G is higher in the case of covalent binding, proving the strength of this binding, which means that in the case of non-covalent binding the fluorescent probe is likely to dissociate, making this type of probe unusable in vivo; After elution, the antibody is in solution. The fluorescence intensity detected in the eluate appears to be greater than the initial amount for two reasons: once it is no longer bound to Protein G, the fluorescence intensity decreases due to the FRET effect, and the laser excitation occurs on the column of solution, not on the monolayer of grafted protein.

[0150] Thus, the fluorescent probe is stabilized by the presence of a covalent bond between the antibody and the protein. The same phenomenon can be observed for smaller polypeptides.

[0151] Probe S c In this case, the amount of "residual" antibody is higher than the amount of "initial" antibody, which is due to the error range of the measuring device and does not change the above conclusion.

[0152] Target molecule detection The FRET index (acceptor fluorescence intensity / donor fluorescence intensity) was calculated for each fluorescent probe S nc and S c was measured using a fluorometer after excitation at 488 nm.

[0153] EGFR antigen diluted in PBS was added to fluorescent probe solution S nc and S c was added to each well containing 100 μg of PBS at a final concentration of 50 nM.

[0154] The FRET index (acceptor fluorescence intensity / donor fluorescence intensity) was measured for each fluorescent probe S after adding the antigen and after 1 hour of incubation at ambient temperature. nc and S c was measured after excitation at 488 nm. The magnitude of the change in response to this addition ("fold change") was determined using the formula FRET index with EGFR / FRET index without EGFR.

[0155] Figure 8 shows the fluorescent probe S nc (non-covalent bond between antibody and protein) and fluorescent probe S c The magnitude of this change is shown in Figure 1 for the case of a covalent antibody-protein bond. It is clear that the presence of a covalent antibody-protein bond increases the magnitude of the FRET index change in response to the addition of antigen, thereby increasing the sensitivity of the fluorescent probe.

[0156] Reference number 1-Fluorescent probe 11-receptor 12-Polypeptide 2-Target molecule 3-graft molecule 4-Optical Fiber 41-Port 42-Outer emission surface 43-Main body 44-ferrule 45-core 46-Cladding 5-Equipment F a - Fluorescent dye bound to receptor (inactive configuration) F' a - Fluorescent dye bound to receptor (active configuration) F b -Fluorescent dye bound to polypeptide (inactive configuration) F' b -Fluorescent dye bound to polypeptide (active construct) XX' - longitudinal axis of optical fiber

Claims

1. A device (5) for detecting a target molecule (2) and / or measuring the concentration of the target molecule (2), comprising: a substrate to the surface of which graft molecules (3) are covalently attached; at least one fluorescent probe (1), at least one receptor (11) covalently bound to a polypeptide (12); ・Two types of fluorescent dye F a and F ｂ At least one fluorescent probe (1) comprising Including, The fluorescent dye F a binds to the receptor (11), and the fluorescent dye F ｂ binds to the polypeptide (12); and The fluorescent dye F a and F ｂ form a FRET donor / acceptor pair; A device (5) in which the polypeptide (12) is covalently attached to the graft molecule (3).

2. The device (5) according to claim 1, wherein the receptor (11) is selected from an antibody, an antibody fragment, an aptamer, a peptide, or a derivative thereof.

3. 2. The device (5) of claim 1, wherein the polypeptide (12) is a binding protein selected from protein G, protein L, protein A, protein Z, protein M, an immunoglobulin, a complete or partial immunoglobulin, or a derivative thereof.

4. The device (5) of claim 1, wherein the polypeptide (12) comprises between 2 and 100 amino acids.

5. The fluorescent dye F a and / or F ｂ The device (5) according to any one of claims 1 to 4, wherein is selected from fluorescent molecules or fluorescent proteins.

6. 5. The device (5) according to any one of claims 1 to 4, wherein the substrate is selected from a cell culture plate, a well plate, a film, a strip, an agarose gel, a cellulose gel, nanoparticles or microparticles, a microscope slide, a glass slide, a substrate configured to be attached around an optical fiber or to the head of an optical fiber.

7. 7. The device (5) according to claim 6, wherein the substrate is a polymer film.

8. 8. The device (5) according to claim 7, wherein the polymer film is selected from polyethylene terephthalate, fluorinated polyethylene-co-propylene, polymethyl methacrylate, polytetrafluoroethylene, polymethylpentene, polyvinyl chloride, styrene methyl methacrylate, polyethylene naphthalate, derivatives thereof or mixtures thereof.

9. 5. The device (5) according to any one of claims 1 to 4, wherein the graft molecule (3) comprises at least two reactive groups selected from maleimide, N-hydroxysuccinimide (NHS) ester, sulfo-N-hydroxysuccinimide ester, sulfo-NHS, azide, alkyne, epoxide, carboxylic acid, aldehyde, aziridine, alkene, or derivatives thereof.

10. 5. The device (5) of claim 1, further comprising an optical fiber (4) and a probe head, the probe head having a body and a light-emitting surface, at least a portion of which is transparent and forms a port (41), and the substrate being the port (41).

11. A fluorescent probe (1), - at least one receptor (11) covalently bound to the polypeptide (12); - Two types of fluorescent dyes F a and F ｂ Including, The fluorescent dye F a binds to the receptor (11), and the fluorescent dye F ｂ binds to the polypeptide (12); and The fluorescent dye F a and F ｂ form a FRET donor / acceptor pair (1).

12. Fluorescent probe (1) according to claim 11, wherein the receptor (11) is selected from an antibody, an antibody fragment, an aptamer, a peptide, or a derivative thereof.

13. Fluorescent probe according to claim 11 or 12, wherein the polypeptide (12) is a binding protein selected from protein G, protein L, protein A, protein Z, protein M, immunoglobulins, complete or partial immunoglobulins, or derivatives thereof.

14. The fluorescent probe according to claim 11 or 12, wherein the polypeptide (12) comprises 2 to 100 amino acids.

15. A method for detecting and / or measuring the concentration of a target molecule (2), comprising: The following steps: - contacting the sample with at least one fluorescent probe (1), said fluorescent probe (1) comprising: at least one receptor (11) covalently bound to a polypeptide (12); ・Two types of fluorescent dye F a and F ｂ Including, The fluorescent dye F a binds to the receptor (11), and the fluorescent dye F ｂ binds to the polypeptide (12); and The fluorescent dye F a and F ｂ form a FRET donor / acceptor pair; and contacting the sample with at least one fluorescent probe (1), wherein the receptor (11) has affinity for the target molecule (12); - exciting said fluorescent probe (1) at a predetermined wavelength so as to excite the donor fluorescent dye; - measuring the ratio between the intensity of the fluorescence emitted by the donor fluorescent dye and the intensity of the fluorescence emitted by the acceptor fluorescent dye; and - determining the presence or absence of said target molecule (2) in said sample and / or calculating the concentration of said target molecule (2) in said sample; A method comprising:

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