Fluorescent Reporter and its Use for the Detection of Target Molecules

Abstract

FLUORESCENT REPORTER AND ITS USE FOR TARGET MOLECULE DETECTION The invention relates to a fluorescent reporter (1) comprising: a receptor (11) bound to a binding protein (12); two fluorochromes Fa and Fb; wherein the fluorochrome Fa is bound to the receptor (11) and the fluorochrome Fb is bound to the binding protein (12); and the fluorochromes Fa and Fb form a FRET donor / acceptor pair. The invention also relates to a device and a method for detecting a target molecule and / or measuring the concentration of a target molecule. Abstract figure: Fig. 1

Description

Title of the invention: FLUORESCENT REPORTER AND ITS USE FOR THE DETECTION OF TARGET MOLECULES FIELD OF INVENTION

[0001] The present invention relates to a fluorescent reporter for the detection and / or measurement of the concentration of a target molecule in a sample. STATE OF THE ART

[0002] The detection of target molecules in a sample has become essential for the search for contaminants in agri-food products, wastewater, or for medical research such as the diagnosis of numerous pathological conditions, including cancers, infectious diseases, autoimmune diseases, and allergies. The detection of target molecules using FRET technology, based on non-radiative energy transfer between two fluorochromes, conventionally requires a FRET donor / acceptor pair, each individual element of which carries a recognition molecule such as an antibody.This two-part fluorescent reporter has several drawbacks: both parts must recognize the target molecule to generate the FRET effect and thus detect the target molecule, detection is slow, and its sensitivity is fundamentally limited by the concentration of the target molecule and the affinity of the antibodies. Developing a fluorescent reporter that operates via intramolecular FRET would overcome some of these limitations.

[0003] In parallel, the in vivo application of fluorescent reporters remains very limited. In this context, the methods used are generally based on the injection of non-specific tracers or antibodies coupled to a fluorochrome. Detection is then performed by measuring the intensity of the signal present on the tissue surface. These approaches, although sometimes used, have many drawbacks. In particular, the signal obtained is strongly affected by the stability of the reporter in vivo, its biodistribution, and its specificity. Moreover, the safety of this injectable product must be systematically demonstrated. The development of a reporter system composed of a single molecule reacting to the presence of a target molecule by a change in optical properties would allow the detection of markers of interest by simple contact, without the constraints associated with injection.

[0004] When used in vivo, for example during surgery or exploration of a part of the human body by endoscopy, it is essential for the surgeon to be able to identify the tumor cells present in a certain and rapid manner. tissues. Thus, fluorescence-assisted surgery is today a rapidly growing field, but is limited by the inherent flaws of marker injection.

[0005] There is therefore a real need for fluorescent protractors that can be used during surgery or endoscopy, at the end of an optical fiber for example, without risk of biological contamination, and allowing confirmation of the diagnosis.

[0006] In in vitro applications, for example, during the rapid analysis of solutions for medical diagnostic purposes or for the detection of target molecules in an environmental sample, it is necessary to avoid biological contamination of the sample being studied by the fluorescent reporter, as is the case with fluorescent reporters developed to date. The present invention proposes a solution to this problem by providing a fluorescent reporter whose various elements are strongly bonded to one another, particularly the receiving portion, so that it does not separate from the other elements constituting the fluorescent reporter to the benefit of the target molecule to be detected, thus preventing biological contamination of the sample. Furthermore, the fluorescent reporter of the invention has the advantage of requiring no additional handling steps such as washing, secondary labeling, or other such steps.Simply placing it in contact with a sample to be analyzed is sufficient, which considerably reduces the number of sample manipulations, as well as the time required to obtain results. SUMMARY

[0007] The invention relates to a fluorescent protractor comprising: - a receptor linked to a binding protein; - two fluorochromes Fa and Fb;

[0008] wherein the fluorochrome Fa is bound to the receptor and the fluorochrome Fb is bound to the binding protein; and

[0009] the fluorochromes Fa and Fb form a FRET donor / acceptor pair.

[0010] In one embodiment, the receptor is selected from antibodies, antibody fragments, aptamers, proteins, peptides, or derivatives thereof. In one embodiment, the binding protein is selected from protein G, protein L, protein A, protein Z, protein M, immunoglobulin, a complete or partial immunoglobulin, or a derivative thereof. In one embodiment, the fluorochromes Fa and / or Fb are selected from fluorescent molecules or fluorescent proteins.

[0011] The invention also relates to a device for detecting a target molecule and / or measuring the concentration of a target molecule, comprising: - a substrate to the surface of which a grafting molecule is covalently attached; - at least one fluorescent protractor including: • a receptor linked to a binding protein; • two fluorochromes Fa and Fb;

[0012] wherein the fluorochrome Fa is bound to the receptor and the fluorochrome Fb is bound to the binding protein; and

[0013] the fluorochromes Fa and Fb form a FRET donor / acceptor pair;

[0014] in which the linking protein is linked to the grafting molecule by covalent bonding.

[0015] In one embodiment, the target molecule is a molecule for which the receptor exhibits high affinity and specificity, preferably an antigen. In one 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 of silica or polymer, a microscope slide, a glass coverslip, the outer layer of an optical fiber, or a substrate configured to be attached to the head of an optical fiber. In one embodiment, the binding protein comprises a terminal group selected from a thiol, amine, azide, alkyne, epoxide, carboxylic acid, aldehyde, aziridine, alkene, or a derivative thereof.In one embodiment, the grafting 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 a derivative thereof.

[0016] The invention also relates to a method for detecting a target molecule and / or measuring the concentration of a target molecule comprising the following steps: - Bring a sample into contact with at least one fluorescent reporter, said fluorescent reporter comprising: • a receptor linked to a binding protein; • two fluorochromes Fa and Fb;

[0017] wherein the fluorochrome Fa is bound to the receptor and the fluorochrome Fb is bound to the binding protein; and

[0018] the fluorochromes Fa and Fb form a FRET donor / acceptor pair; and

[0019] the receptor has an affinity for said target molecule; - Excite the fluorescent reporter at a given wavelength so that the donor fluorochrome is excited; - Measure the ratio between the fluorescence intensity emitted by the donor fluorochrome and the fluorescence intensity emitted by the acceptor fluorochrome; and - Determine the presence or absence of said target molecule in the sample and / or calculate the concentration of said target molecule in the sample. DEFINITIONS

[0020] In the present invention, the terms below are defined as follows: - “Antibodies” (also known as immunoglobulins, in (abbreviated Ig) refers to gamma globulin proteins found in the blood and other bodily fluids of vertebrates. These proteins are used by the immune system to identify and neutralize foreign substances, such as bacteria and viruses. Antibodies consist of two pairs of polypeptide chains, called heavy chains and light chains, arranged in a Y shape. The two ends of the Y are the regions that bind to and inactivate antigens. The term "antibody" (Ab) as used here includes monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term "immunoglobulin" (Ig) is used interchangeably with "antibody." - "Antigen" refers to a molecule that triggers a response Immune response. This immune response can involve either the production of antibodies, the activation of specific immunologically competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. - "Aptamer" refers to a synthetic oligonucleotide, most often a RNA that is capable of binding a specific ligand. - "Active configuration" (noted as "ON") refers to the configuration of the fluorescent reporter in the presence of energy transfer (FRET effect) between the fluorochromes Fa and Fb. - "Inactive configuration" (noted as "OFF") refers to the configuration of the fluorescent reporter in the absence of energy transfer (FRET effect) between the fluorochromes Fa and Fb. - "Fluorochrome" (or fluorophore) refers to a chemical substance capable of emitting fluorescence light after excitation. An "antibody fragment" comprises a portion of an intact antibody, including the variable region 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 US patent 5,641,870; Zapata et al., Protein Eng. 8 (10): 1057–1062

[1995] ); single-stranded antibody molecules; and multispecific antibodies formed from antibody fragments. The term "antibody fragment" (or functional fragment) refers to a compound that shares qualitative biological activity with a full-length antibody. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize easily.The Fab fragment consists of an entire L chain with the variable region domain of the H chain (VH) and the first constant domain of a heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, meaning it has only one antigen-binding site. Pepsin treatment of an antibody yields a single large fragment (Fab')2, which is roughly equivalent to two Fab fragments linked by a disulfide bridge. This fragment has divalent antigen-binding activity and is still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy end of the CH1 domain, including one or more cysteines from the antibody's hinge region. Fab'-SH is the designation used here for Fab' fragments in which the cysteine ​​residue(s) of the constant domain(s) carry a free thiol group.The F(ab')2 antibody fragments were initially produced as pairs of Fab' fragments that have cysteine ​​hinges between them. Other chemical couplings of antibody fragments are also known. A ligand is a specific target molecule capable of reversibly binding to a receptor. The ligand interacts non-covalently and specifically with the receptor. This binding occurs through forces between molecules, such as ionic bonds, hydrogen bonds, and van der Waals forces. An antibody / antigen pair is an example of a receptor / ligand pair. In this description, the terms ligand, antigen, and target molecule are used interchangeably. "Optically transparent" refers to a material that absorbs less than 50%, preferably less than 20%, more preferably less than 10% of light at wavelengths between 350 nm and 1100 nm. - "Protein" refers to a functional entity made up of one or several peptides. - "Receptor" refers to a biological molecule capable of recognizing and / or to bind reversibly to a specific target molecule (or ligand). The receptor interacts non-covalently and specifically with this target molecule. Binding occurs through forces between molecules, such as ionic bonds, hydrogen bonds, and van der Waals forces. An antibody / antigen pair is an example of a receptor / ligand pair. DETAILED DESCRIPTION

[0021] The present invention relates to a fluorescent reporter comprising: - a receptor linked to a binding protein; - two fluorochromes Fa and Fb.

[0022] The fluorochrome Fa is bound to the receptor and the fluorochrome Fb is bound to the binding protein. The fluorochromes Fa and Fb form a FRET donor / acceptor pair.

[0023] Thus the fluorescent reporter includes a part responsible for the specific binding of a target molecule to be detected (receptor), two fluorochromes capable of converting the recognition of the target molecule into a measurable fluorescent signal (Fa and Fb) and a support system for one of the two fluorochromes which can also serve as an attachment allowing controlled binding on a substrate (binding protein).

[0024] When a target molecule is recognized by the fluorescent reporter, a conformational change occurs in the receptor. This conformational change affects the relative positions of the VH (heavy variable chain) and VL (light variable chain) variable fragments, as well as the constant fragments, thus altering the distance between the two fluorochromes. This leads to variations in the emission levels of the donor and acceptor fluorochromes through non-radiative energy transfer, i.e., the FRET (Förster Resonance Energy Transfer) effect. This non-radiative energy transfer allows for a modification of the optical signature of the fluorescent reporter when the distance between the two fluorochromes changes. This results in a variation in the fluorescence intensity emitted by each of the two fluorochromes, thereby converting the conformational change of the receptor induced by the recognition of the target molecule into a measurable fluorescent signal.

[0025] To form a FRET donor / acceptor pair, the two fluorochromes Faet Fb must have compatible spectral characteristics, in particular an overlap of the emission spectrum of the so-called "donor" fluorochrome with the excitation spectrum of the so-called "acceptor" fluorochrome. When the donor fluorochrome is excited, its fluorescence will then excite the acceptor fluorochrome. The efficiency of this energy transfer depends essentially on the distance between the two fluorochromes, their extinction coefficient and their quantum yield, as well as the extent of the overlap between their emission and excitation spectra.

[0026] The specific locations of the fluorochromes on the receptor and the binding protein are optimized to promote changes in their optical signature during fluorescence upon recognition and / or binding of the target molecule. Preferably, the fluorochrome Fb is grafted onto a free amine of the binding protein.

[0027] The binding protein has a certain affinity for the receptor, for example the receptor is an antibody and the binding protein is a G protein.

[0028] According to one embodiment, the receptor is linked to the binding protein by Van der Waals interactions or by covalent bonding. Advantageously, a strong bond (Van der Waals or covalent) between the receptor and the binding protein, preferably stronger than the receptor-target molecule bond, ensures that once the target molecule is recognized, the receptor will not detach from the binding protein. This prevents the fluorescent reporter from separating into two parts.Preventing separation between the receptor and the binding protein is particularly important when the fluorescent reporter is used for in vivo detection of target molecules, especially when grafted to the distal end of an optical fiber for intracorporeal exploration, as this limits the risk of leaving a portion of the fluorescent reporter (the part with the receptor) in the patient's body upon fiber removal. Furthermore, without being bound by any theory, the applicant believes that covalent binding enhances the efficiency of the FRET effect.

[0029] According to one embodiment, the receptor is chosen from antibodies, antibody fragments, aptamers, proteins, peptides, or a derivative thereof.

[0030] According to one embodiment, the receptor is capable of recognizing and / or reversibly binding to a ligand, i.e., a target molecule. The ligand corresponds to any molecule for which the receptor exhibits high affinity and specificity, and is capable of reversibly binding to a given receptor.

[0031] In a specific configuration of this embodiment, the target molecule (or ligand) is an antigen.

[0032] In a specific configuration of this embodiment, the antibody or antibody fragment is chosen from Fab, Fab', (Fab')2, scFv, or scFv-Fc.

[0033] According to one embodiment, the binding protein is preferably an immunoglobulin binding protein.

[0034] According to one embodiment, the binding protein is chosen from protein A, protein G, protein L, protein M, protein Z, immunoglobulin, a complete or partial immunoglobulin, or a derivative thereof.

[0035] According to one embodiment, the fluorochrome Fa is the donor and the fluorochrome Fb is the acceptor of the donor / acceptor pair FRET.

[0036] According to another embodiment, the fluorochrome Fa is the acceptor and the fluorochrome Fb is the donor of the FRET donor / acceptor pair.

[0037] According to one embodiment, the fluorochromes Fa and / or Fb have a fluorescence emission peak between 350 nm and 399 nm (in the UV range), between 400 nm and 499 nm (in the blue range of the visible spectrum), between 500 nm and 599 nm (in the green range of the visible spectrum) or between 600 nm and 719 nm (in the red range of the visible spectrum), between 720 nm and 850 nm (in the near-infrared range).

[0038] According to one embodiment, the fluorescence emission peaks of the fluorochromes Fa and Fb exhibit an overlap zone. Advantageously, this overlap allows for non-radiative energy transfer between the two fluorochromes.

[0039] According to one embodiment, the fluorochromes Fa and / or Fb are chosen from fluorescent molecules or fluorescent proteins.

[0040] In a specific configuration of this embodiment, a fluorescent molecule is chosen 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.

[0041] Dans une configuration spécifique de ce mode de réalisation, une protéine fluorescente est choisie parmi la protéine fluorescente verte (GFP, « Green Fluorescent Protein »), 22G, aceGFP, amFP486 (« GFP-like fluorescent chromoprotein amFP486 », « Anemonia manjano FP486 »), amm2CP, avGFP, AvicFPl, cFP484 (« GFP-like fluorescent chromoprotein 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 cqFP6lI », « Entacmaea quadricolor FP611 »), HcRed (« GFP-like non-fluorescent chromoprotein », « Heteractis crispa Red »), KikG, KO, LanYFPn, Montipora sp20 (« Cytochrome c oxidase subunit 1 »), mRed7 (« Rod shape-determining protein MreD », « mine drainage metagenome 7 »), NpR3784g, RpBphPl (« Rhodopseudomonas palustris BphPl »),RpBphP2 (« Rhodopseudomonas palustris BphP2 »), RpBphPô (« Rhodopseudomonas palustris BphP6 »), TeAPCalpha, zFP538 (« GFP-like fluorescent chromoprotein FP538 », « Zoanthus FP538 »), AausFPl, vsfGFP-0, LanYFP, bfloGFPal, RRvT, dLanYFP, dVFP, ccalYFPl, , efasGFP, pcDronpa (Green), aeurGFP, Skylan-S (On), mVenus-Q69M, tdTomato, PlamGFP, eechGFPl, mNeonGreen, Kaede (Green), mClover3, Cio ver, VFP, pcDronpa2 (Green), moxNeonGreen, tdimer2(12), Dronpa (On), YPet, Skylan-NS (On), ffDronpa (On), eechGFP2, gfasGFP, Gamillus (On), sarcGFP, vsfGFP-9, mEos3.1 (Green), pmeaGFPl, mVFP, pmimGFPl, pmimGFP2, mEos4a (Green), pcDronpa2 (Red), pdaelGFP, pmeaGFP2, mScarlet, mCitrine, ccalGFP3, phiYFP, SYFP2, Citrine2, mVFPl, Gamillus0.4, SHardonnay, aacuGFP2, mVenus, mEos4b (Green), mKOΰ, fabdGFP, mGeos-C (On), rsKame (On), TurboRFP, afraGFP, stylGFP, phiYFPv, FoldingReporterGFP, Citrine, dendFP (Green), PSmOrange (Orange), anobGFP, mRuby3, RFP611, Topaz, SEYFP, mScarlet-I, mWasabi, iq-mVenus, meffRFP, d2EosFP (Green), eqFP578, EYFP-Q69K, mTFPl, ccalRFPl, eechGFP3, cgreGFP, SuperfolderGFP, meffGFP, mEos3.2 (Green), pporGFP, muGFP, Venus, mGeos-E (On), Gamillus0.2, mEosFP-M159A (Green), pporRFP, pcDronpa (Red), dlEosFP (Green), moxGFP, oxGFP, EosFP (Green), amilFP513, KO, mGeos-S (On), tdKatushka2, mOrange, mEYFP, anmlGFPl, meffCFP, AzamiGreen, obeGFP, TagRFP, dimer2, dTomato, mEos2 (Green), usGFP, M355NA, cgfTagRFP, mGeos-F (On), EYFP, Gamillus0.3, Kohinoor (On), amilFP593, ccalOFPl, obeYFP, mGeos-M (On), moxVenus, oxVenus, WasCFP, mEos4a (Red), mUkG, NowGFP, mEosFP (Green), mRuby2, ppluGFP2, mAvicFPl, dTFP0.2, AvicFPl, scubRFP, mK02, UnaG, mEos4b (Red), GFPmut2, mRuby, Emerald, mEmerald, ppluGFPl, meleRFP, mGeos-L (On), OFP, mmilCFP, KikGRl (Green), TurboGFP, mApple, CyRFPl (CyRFPl), E2-Red / Green, ccalGFPl, mPapaya, moxCerulean3, GFP(S65T), eqFPôll, 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, CyOFPl, mEos2 (Red), SGFP2(206A), LanFPl, 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, AcGFPl, NijiFP (Orange), SPOON (on), Cerulean, amilFP490, dTFPO.l, PATagRFP1297 (On), mT-Sapphire, T-Sapphire, NijiFP (Green), mAmetrine, mNectarine, mStrawberry, SGFP1, cgfmKate2, BrUSLEE, rsGreenl (Bright), mlrisFP (Green), G2, mTurquoise, moxDendra2 (Green), iq-mCerulean3, PATagRFP (On), mKate2, dlEosFP (Red), Turquoise-GL, MiCy, mBlueberry2, FusionRed-M, dendFP (Red), Dendra2-T69A (Green), anobCFPl, Î+GFP, mCerulean2.N, LSSmOrange, mCerulean2.D3, cpT-Sapphirel74-173, Aquamarine, oxCerulean, mEosFP (Red), mEosFP-F173S (Green), KikGRl (Red), mNeptune2.5, EBFP1.5, Dendra2-T69A (Orange), EosFP (Red), . aacuGFPl, bsDronpa (On), Dendra2 (Green), mKateS158A, IrisFP (Green), ZsGreen, mCerulean.B24, obeCFP, Katushka, Padron0.9 (On), mNeptune2, AausGFP, TagCFP, mCerulean.B, LanFP2, mKateS158C, mEos3.1 (Red), Dronpa-2 (On), D10, shBFP-N158S / L173I, Kaede (Red), sg25, d2EosFP (Red), mCerulean2.N(T65S), avGFP, E2-Orange, BDFPE6, DsRed-Max, mCerulean.B2, mlrisFP (Red), CGFP, Dendra2 (Red), Dronpa-3 (On), Sapphire, EBFPE2, rsEGFP2 (On), FusionRed, EBFP2, CyPet, eforCP, mEos3.2 (Red), mKikGR (Red), mBeRFP, mCyRFPl, mKillerOrange, RFP630, mCherry2, moxBFP, oxBFP, KillerOrange, moxDendra2 (Red), mClavGR2 (Red), cFP484, rsEGFP (On), KCY-G4219, GamillusO.l, mMaple (Red), SCFP3A, IrisFP (Orange), RFP637, mCardinal, mRFPl-Q66T, mKalamal, mCerulean, mKateM41GS158C, SBFP2, KCY-R1, mPapaya0.7, mCherry, iq-EBFP2, eqFP650, Gl, sgl2, mMiCy, mEos2-A69T (Green), SCFP3B, DsRed-Express2, mKate, mScarlet-H, Dendra (Green), mClavGR2 (Green), td-RFP639, Azurite, Dendra (Red), miRFP670-2, PA-GFP (On), ÎRFP713 / V256C, miRFP680, mECFP, SuperNovaRed, mNeptune, DsRed.T4, BFP.A5, mCRISPRed, ECFP, ZsYellowl, Neptune, mRaspberry, rsFolder (Green), PAmCherry2 (On), DsRed-Express, moxMaple3 (Red), ÎRFP670, mRFPl, mMaple3 (Red), RFP639, iq-mApple, emiRFP670, miRFP670, shBFP, SiriusGFP, AvicFP4, W7, H9, SCFP2, mRFPl-Q66S, mTangerine, IrisFP-M159A (Green), KillerRed, mMaple (Green), dsFP483, GZnP3, PS-CFP2 (Green), sgi 1, mRFPl-Q66C, miRFP670nano, mEosFP-F173S (Red), miRFP682, LSSmCherryl, rsFolder2 (Green), 1RFP682, R3-2+PCB, amFP486, PSmOrange (Far-red), mGarnet2, miRFP, Jred, mMaroonl, PS-CFP2 (Cyan), mGarnet, zFP538, PAmCherryl (On), miRFP670vl, W1C, dTG, rsFusionRedl (On), P4-1, emiRFP703, miRFP703, mKelly2, mStable, dKeima, mEos2-A69T (Orange), iRFP702, deGFP2, PSmOrange2 (Far-red), miRFP702, lanRFP-î”S831, laRFP, W2, mKellyl, SCFP1, miRFP713, ÎFP2.0, pHuji, mIFP, iFPl.4, SuperNovaGreen, HcRed-Tandem, EBFP, iRFP713, SOPP3, SOPP2, miniSOG, HcRed7, miRFP720, RDSmCherryO.l, P4-3E, mTFP0.3, mMaple3 (Green), mCarmine, ÎRFP720, SOPP, MaroonO.l, moxMaple3 (Green), PS-CFP (Cyan), BFP, mBlueberryl, eechRFP, PS-CFP (Green), deGFP3, PAmCherry3 (On), RDSmCherryl, deGFPl, PAmKate (On), LSS-mKate2, Wi-Phy, rsFusionRed2 (On), miRFP709, eqFP670, mBanana, KFP1 (On), sg50, mPlum, rsTagRFP (ON), deGFP4, iq-mKate2, sg42, Pp2FbFPL30M, TagRFP675, mRojoB, AQ143, Sinus, mKeima, TagRFP657, ECGFP, SNIFP, tKeima, Pp2FbFP, rsFusionRed3 (On), AsRed2, LSS-mKatel, AvicFP2 (pre-conversion), ECFPH148D, dKeima570, mHoneydew, cpCitrine, rsCheny (On), mNeptune681, mGrapel, mGinger2, mGrape3, mNeptune684, mGingerl, RDSmCherry0.2, mGrape2, mRojoA, SBFP1, mRouge, P4, RDSmCheny0.5, GZnP3 (GZnP3(apostate)), rsChenyRev (On), Sandercyanin, HcRed, mRtms5, . anm2CP, mTFPl-Y67W, Ultramarine, 10B, 11, 22G, (3-F)Tyr-EGFP, 5B, 6C, Ala, A44-KR, aacuCP, AausFP2, AausFP3, AausFP4 (On), acanFP, aceGFP-G222E-Y220L, aceGFP-h, Achilles, AdRed, AdRed-C148S, ahyaCP, alajGFPl, alajGFP2, alajGFP3, amCyanl, amFP495, amFP506, amFP515, amilCP, amilCP580, amilCP586, amilCP604, amilFP484, amilFP497, amilFP504, amilFP512, amilFP597, anmlGFP2, apulCP584, apulFP483, AQ14, asCP562, asFP499, asulCP, atenFP, avGFP454, avGFP480, avGFP509, avGFP510, avGFP514, avGFP523, AvicFP3 (pre-conversion), bfloGFPcl, BFP5, BFPsol, Bluel02, BRI, cEGFP, CFP, CFP4, cgigCP, cgigGFP, CheGFPl, CheGFP2, CheGFP3, CheGFP4, Clomeleon, Cloverl.5, cpasCP, cp-mKate, Cyll.5, dClavGRl.6, dClover2, dClover2A206K, dfGFP, dhorGFP, dhorRFP, dimerl, dis2RFP, dis3GFP, dPapayaO.l, DrCBD, d-RFP618, Dronpa-C62S, DspRl, DsRed.Ml, DsRed-Timer, DstCl, EaGFP, echFP, echiFP, eGFP203C, eGFP205C, EnhancedCyan-EmittingGFP, EYFP-F46L, fcFP, fcomFP, Flamindo2, FP586, Fpaagar, Fpag_frag, Fpcondchrom, FPmann, FPmcavgr7.7, FPrfl2.3, Gamillus0.5, GCaMP2, GCaMPôf (in presence of Ca2+), gdjiCP, gfasCP, GFP-151pyTyrCu, GFPhal, GFP-Tyrl51pyz, GFPxmlô, GFPxmlôl, GFPxml62, GFPxml63, GFPxml8, GFPxml81uv, GFPxml8uv, GFPxml9, GFPxml91uv, GFPxml9uv, gtenCP, HcRedl-Blue, hcriCP, hcriGFP, hfriFP, hmGFP, HriCFP, HriGFP, iLov, jRGECOla (in absence of Ca2+), jRGECOla (in presence of Ca2+), Katushka-9-5, KCY-G4219-38L, KCY-R1-158A, KCY-R1-38H, KCY-R1-38L, KikG, KOFP-7 (KOFP-7), laesGFP, laGFP, LEA, mcl, mc2, mc3, mc4, mc5, mc6, McaGl, McaGlea, McaG2, mcavFP, mcavGFP, mcavRFP, mcCFP, mcFP497, mcFP503, mcFP506, mCherryl.5, mClavGRl, mClavGRl.l, mClavGR1.8, mCloverl.5, mcRFP, meffCP, mEos2-NA, meruFP, MfaGl, miniS0G2, 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, mPapayaO.6, mPlum-E16P, mRed7, mRed7Ql, mRed7QlSl, mRed7QlSlBM, mRFPl.l, mRFP1.2, mRFP1.3, mRFP1.4, mRFP1.5, mTFP*, mTFP0.4, mTFP0.5, mTFPO.6, mTFP0.8, mTFP0.9, mTFPl-Y67H, mTurquoise-146G, mTurquoise-146S, mTurquoise2-G, mTurquoise-DR, mTurquoise-GL, mTurquoise-GV, mTurquoise-RA, NpR3784g, OFPxm, Pli, P9, Padron(star) (On), PdaCl, PDM1-4, pHluorin2 (acidic), pHluorin2 (alkaline), pHluorin, psupFP, ptilGFP, Q80R, RCaMP, R-FlincA, rfloGFP, rfloGFP2, rfloRFP, RFP618, roGFPl, roGFPl-Rl, roGFPl-R8, roGFP2, RpBphPl, RpBphP2, RpBphPô, rrenGFP, rrGFP, rsCherryRevl.4 (On), RSGFP1, RSGFP2, RSGFP3, RSGFP4, RSGFP6, RSGFP7, Rtms5, SAASoti (Red), SAASoti (Green), scleFPl, scleFP2, scubGFPl, scubGFP2, secBFP2, sfCherry, sfCherry2, sfCherry3C, SH3, ShG24, spisCP, stylCP, SuperfoldermTurquoise2, SuperfoldermTurquoise2ox, . sympFP, TeAPCÎi, tPapayaO.Ol, 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.

[0042] According to one embodiment, the link between the fluorochrome Fa and the receptor is a covalent bond. In a specific configuration of this embodiment, this bond is of the NHS-NH2, maleimide-SH or their derivatives type, the NHS or maleimide group being located on the fluorochrome Fa and the amine or thiol being on the receptor.

[0043] According to an embodiment in which the fluorochrome Fa is a fluorescent protein, the receptor and Fa are linked as a fusion protein. In this embodiment, the receptor and Fa are encoded by the same gene.

[0044] According to one embodiment, the link between the fluorochrome Fb and the binding protein is a covalent bond. In a specific configuration of this embodiment, this bond is of the NHS-NH2, maleimide-SH or their derivatives type, the NHS or maleimide group being located on the fluorochrome Fb and the amine or thiol being on the binding protein.

[0045] According to one embodiment, in the inactive configuration of the fluorescent reporter (OFF configuration), the distance between the fluorochromes Fa and Fb does not allow the FRET effect, i.e., the distance between the fluorochromes Fa and Fb is greater than or less than the distance allowing the FRET effect; whereas in the active configuration (ON configuration), the distance between the fluorochromes Fa and Fb allows the FRET effect. In other words, the distance between the fluorochromes Fa and Fb in the inactive configuration (i.e., fluorescent reporter at rest, OFF configuration) is greater than or less than the distance between the fluorophores Fa and Fb in the active configuration (ON configuration). The fluorescent reporter is at rest when the receptor is not bound to a target molecule; in the case where the receptor is an antibody, the fluorescent reporter is at rest when the antibody recognition site is free.The fluorescent reporter is in the active configuration when the receptor is bound to a target molecule; in the case where the receptor is an antibody, the fluorescent reporter is in the active configuration when the antibody recognition site is not free, i.e., an antigen is recognized and bound to the antibody. There is also a configuration where the ON configuration occurs when the antibody does not detect its target molecule and which switches to the OFF configuration when the receptor is bound to the target molecule (FRET without a target molecule and FRET stopped with one).

[0046] The present invention also relates to a device for the detection of a target molecule and / or the measurement of the concentration of a target molecule.

[0047] The device comprises: - a substrate to the surface of which a grafting molecule is covalently attached; - at least one fluorescent protractor including: • a receptor linked to a binding protein; • two fluorochromes Fa and Fb;

[0048] wherein the fluorochrome Fa is bound to the receptor and the fluorochrome Fb is bound to the binding protein; and

[0049] the fluorochromes Fa and Fb form a FRET donor / acceptor pair.

[0050] The linking protein is linked to the grafting molecule by covalent bonding.

[0051] A strong bond (Van der Waals or covalent type) between the receptor and the binding protein, stronger than the receptor-target molecule bond, and a covalent bond between the binding protein and the grafting molecule ensure that, once the target molecule is recognized, the receptor will not detach from the binding protein. This prevents the fluorescent reporter from separating into two parts.

[0052] During in vitro detection of target molecule, avoiding separation between the receptor and the binding protein is particularly important in order to avoid inducing contamination of the sample.

[0053] During in vivo target molecule detection, avoiding separation between the receptor and the binding protein is particularly important when the fluorescent reporter is used for in vivo detection of target molecules, especially when grafted to the distal end of an optical fiber for intracorporeal exploration, as this limits the risk of leaving part of the fluorescent reporter (the part with the receptor) in the patient's body at the time of fiber removal.Such biological contamination can have various adverse effects, similar to those observed with direct antibody injection, such as discomfort like headaches, nausea, or fatigue; reactions like fever or chills; allergic-like symptoms, including skin reactions (itching, rash, hives), respiratory reactions (bronchospasm, cough, dyspnea), cardiovascular reactions (hypotension), and tumor lysis syndromes (in cases of large tumors). These adverse effects are due to both the nature of the ligand and that of the receptor.

[0054] The role of the grafting molecule is to ensure the correct orientation of the receptor so that its recognition sites are accessible to the target molecule.

[0055] The embodiments relating to the fluorescent reporter or the various elements of said reporter (receptor, binding protein, fluorochromes) apply to the device of the invention.

[0056] According to one embodiment, the target molecule (or ligand) is a molecule for which the receptor has a high affinity and specificity. Preferably, the target molecule (or ligand) is an antigen.

[0057] According to one 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 made of silica or polymer, a microscope slide, or a glass coverslip. A device according to this embodiment is intended for the detection and / or measurement of the concentration of target molecules in vitro, i.e., in solution or on a substrate.

[0058] According to one embodiment, the device further comprises an optical fiber and a scanning head. The head is attached either removably or rigidly to the optical fiber (via a ferrule). An optical fiber, in a known manner, comprises a sheath enclosing one or more fiber cores, and its distal end, intended for scanning, is in the form of a rigid ferrule firmly attached to the end of the fiber sheath and whose external face, transverse to the fiber axis, is transparent. A device according to this embodiment is intended for the detection and / or measurement of the concentration of a target molecule in vivo, i.e., in the patient's body, for example, by endoscopy or during surgery.

[0059] The "exploration head" is the part of the fiber that acts as a probe or, more generally, performs 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 fiber end is inserted. It may be removable or fixed to the optical fiber. The ferrule of the optical fiber serves only as a mechanical fastening means for the exploration head.

[0060] In a specific configuration of this embodiment, the substrate is selected from the periphery of an optical fiber or a substrate configured to be fixed to the head of an optical fiber, preferably an optically transparent substrate configured to be fixed to the distal end of an optical fiber (i.e., the exploration end), more precisely, to the distal end of the exploration head. Preferably, the fluorescent reporter is grafted onto a film, preferably an organic film such as a polymer film, at the distal end of the exploration head.

[0061] In the case of a removable exploration head, the ferrule and the exploration head are joined by mechanical assembly (crimping, fitting, screwing, clipping, quarter-turn locking), or respectively comprise means for mutually cooperative fixation (male-female cooperative means). The attachment of the head to the ferrule is designed so that the head and the ferrule cannot separate during use, particularly when the fiber has been introduced into the patient's body.

[0062] In a specific configuration of this embodiment, the exploration head has a body and an external face, referred to as the emission face, at least a portion of which is transparent, forming a window, and intended to be positioned opposite the core(s) of the optical fiber for the passage of light. Preferably, a polymer film is used as the transparent portion so that it is functionalized with the fluorescent protractor. Preferably, the body is made of a polymeric material, glass, ceramic, stainless steel, composite material, or a combination of these materials, and the external emission face is made of a polymer, glass, ceramic, silica, composite material, or hybrid material.

[0063] According to an embodiment in which the optical fiber comprises a core partially covered by a metallic cladding, a portion of the core not being covered by the cladding, the fluorescent reporter is grafted onto the surface of the uncovered portion of the core. This embodiment makes it possible to obtain a surface wave on the longitudinal part of the optical fiber.

[0064] According to one embodiment, the linking protein comprises a terminal group selected from thiol, amine, azide, alkyne, epoxide, carboxylic acid, aldehyde, aziridine, alkene, or a derivative thereof.

[0065] According to one embodiment, the grafting 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 a derivative thereof. The grafting molecule enables the covalent bond between the substrate and the fluorescent reporter. Preferably, the two reactive groups are located at each end of the molecule.

[0066] According to one embodiment, the substrate is covered with a layer of organic or inorganic material chosen from zirconia, titanium dioxide, epoxy, organosilane such as, for example, amine organosilane, thiol organosilane, azide organosilane, alkyne organosilane, carbonyl organosilane, or organosilane having a carbon-carbon double bond.

[0067] The present invention also relates to a method for detecting a target molecule and / or measuring the concentration of a target molecule.

[0068] The method comprises the following steps: - Bring a sample into contact with at least one fluorescent reporter, said fluorescent reporter comprising: • a receptor linked to a binding protein; • two fluorochromes Fa and Fb;

[0069] wherein the fluorochrome Fa is bound to the receptor and the fluorochrome Fb is bound to the binding protein; and

[0070] the fluorochromes Fa and Fb form a FRET donor / acceptor pair; and

[0071] the receptor has an affinity for said target molecule; - Excite the fluorescent reporter at a given wavelength so that the donor fluorochrome is excited; - Measure the ratio between the intensity of the fluorescence emitted by the donor fluorochrome and the intensity of the fluorescence emitted by the acceptor fluorochrome; - Determine the presence or absence of said target molecule in the sample and / or calculate the concentration of said target molecule in the sample.

[0072] When a target molecule is recognized by the receptor, a conformational change occurs in the receptor, inducing a variation in the distance between the fluorochromes Fa and Fb, leading to a non-radiative transfer from the donor fluorochrome to the acceptor fluorochrome (FRET effect). The variations in the intensity of the fluorescence peaks of the donor and acceptor fluorochromes are thus a direct consequence of the concentration of the target molecule in the sample. An increase or decrease in the fluorescence intensity emitted by the donor fluorochrome is therefore expected if the target molecule is present in the sample; this results in an increase or decrease in the ratio between the fluorescence intensity emitted by the donor fluorochrome and the fluorescence intensity emitted by the acceptor fluorochrome (hereafter referred to as the FRET index).If the intensity of the fluorescence peaks remains unchanged (FRET index zero), this indicates the absence of the target molecule in the sample. Therefore, determining the presence or absence of said target molecule in the sample and / or calculating its concentration in the sample depends on interpreting the change in the FRET index. The reverse is also possible.

[0073] Detection of the target molecule is advantageously rapid.

[0074] The embodiments relating to the fluorescent reporter, the various elements of said reporter (receptor, binding protein, fluorochromes), the device or the various elements of said device apply to the implementation of the method according to the invention. In particular, the method according to the invention is implemented by the device of the invention.

[0075] According to one embodiment, the fluorescent report is brought into contact with the sample by any means of contact. Preferably, the contact takes place in solution or by direct contact.

[0076] In a specific configuration of this embodiment, the contact by direct touch lasts a few seconds. In another specific configuration of this embodiment, for example in solution, the contact in solution lasts less for one hour, preferably less than 30 minutes, and more preferably less than 5 minutes. The longer the contact time, the clearer the optical signal.

[0077] According to one 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 spectrometry.

[0078] According to one embodiment, the method also includes a preliminary calibration step in which the device is brought into contact with a clean sample. Advantageously, this step allows for the determination of a reference FRET index measurement for the chosen Fa / Fb pair. Subsequently, comparison between this reference measurement and the FRET index measured in contact with the sample suspected of containing the target molecule will allow a conclusion to be drawn regarding the presence or absence of the target molecule and its concentration in the tested sample.

[0079] The presence, absence, or detection of a quantity of target molecule above or below a certain threshold will allow the sample to be characterized, and to be considered or not as healthy.

[0080] According to one embodiment, the sample can be any sample having the possibility of containing a target molecule as an object of detection or measurement and can be a liquid sample or a solid sample.

[0081] According to one embodiment, the sample is chosen from a solution, a cell culture (for example eukaryotic or prokaryotic), whole blood, plasma, blood serum, sweat, or any biological liquid or fluid, an organic tissue, or an organ.

[0082] In a specific configuration of this embodiment, a liquid sample can be used directly as a detection or measurement object or can be diluted with, for example, a buffer solution or a saline solution, and then used as a detection or measurement object. Examples of liquid samples include, but are not limited to, cell cultures (e.g., eukaryotic or prokaryotic), culture supernatants, cell extracts, bacterial extracts, bodily fluids such as, for example, serum, plasma, saliva, sweat, cerebrospinal fluid or urine, industrial wastewater, or an agri-food liquid such as, for example, milk.

[0083] In a specific configuration of this embodiment, a solid sample is selected from an organic tissue, an organ. The solid sample may be dissolved, suspended, or immersed in a liquid, such as a buffer solution or saline solution, in a state capable of coming into contact with the free fluorescent reporter and is then used as a sample. Preferably, the solid sample undergoes no treatment before being brought into contact with the fluorescent reporter.

[0084] According to one embodiment, the detection threshold of the target molecule is dependent on the affinity of the receptor with the target molecule, the detection threshold is on the order of a few pmol.L 1 (picomolar), preferably on the order of a few fmol.L 1 (femto molar).

[0085] According to one embodiment, the method can be implemented: - In vitro, for example in suspension, in solution, or on a substrate (e.g., multipluite plate, microscope slide, or strip); or - In vivo, for example by endoscopy, or during surgery.

[0086] According to one embodiment, the device also includes an excitation means configured to excite the sample and / or the fluorescent reporter, and / or an optical data collection means configured to collect data from the fluorescence of the fluorochromes Fa and Fb.

[0087] In a specific configuration of this embodiment, the excitation means is a light source capable of irradiation with a given wavelength such as, for example, a mercury lamp, a xenon lamp, an LED, a UV lamp or a laser light source.

[0088] In a specific configuration of this embodiment, the optical data collection means is a microscope, a fluorometer, a cytometer, or a spectrophotometer.

[0089] The present invention also relates to the use of the fluorescent reporter according to the invention and / or the device according to the invention for the detection of a target molecule and / or the measurement of the concentration of a target molecule in a sample.

[0090] According to one embodiment, a fluorescent reporter according to the invention and / or the device according to the invention are used for the detection of a target molecule and / or the measurement of the concentration of a target molecule in an in vitro sample.

[0091] According to one embodiment, the fluorescent reporter according to the invention and / or the device according to the invention are used for the detection of a target molecule and / or the measurement of the concentration of a target molecule in an in vivo sample.

[0092] In a specific in vivo embodiment, the fluorescent reporter according to the invention and / or the device according to the invention are used for the detection of tumor cells, the search for infection, or the detection of markers that may aid in the diagnosis or monitoring of the progression of a disease. In a specific in vivo embodiment, the device is an optical fiber comprising a head to which the fluorescent reporter is attached, or a catheter at the distal end of which the fluorescent reporter is attached. Preferably, the fluorescent reporter according to the invention and / or the device according to the invention are used in endoscopy. Endoscopy allows examination of the interior of a cavity using a backscattered light detection technique with a fiber optical. An endoscope comprises a flexible sheath housing 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 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, light detectors also arranged in alignment with the optical fibers and at the proximal end being intended to receive the light emitted by the fluorescent protractor and transmitted back via the fibers.

[0093] According to one embodiment, a fluorescent reporter according to the invention and / or the device according to the invention are used for the detection of a target molecule and / or the measurement of the concentration of a target molecule in industrial water, wastewater, or an agri-food liquid such as milk, for example. In a particular configuration of this embodiment, a fluorescent reporter according to the invention and / or the device according to the invention are used for the detection of drug or pesticide residues, or the detection of pathogens. DESCRIPTION OF THE FIGURES

[0094] [Fig. 1] is a diagram representing the transition of the fluorescent reporter from an inactive (off) configuration to an active (on) configuration during the recognition of a target molecule.

[0095] [Fig.2] represents a detection device according to a particular embodiment.

[0096] [Fig.3A] is an illustration of the optical spectra obtained by fluorometer analysis of the response of the fluorescent reporter to different concentrations of antigen.

[0097] [Fig.3B] is an illustration of the dose-response curve obtained with the fluorescent protractor, by measuring the FRET indices from the curves shown in [Fig.3A].

[0098] [Fig.4] is an illustration of the FRET response obtained upon the addition of an antigen to the fluorescent reporter (here TrkB) and a non-relevant molecule, BSA, where the conformational change does not take place.

[0099] [Fig.5A] is an illustration of the optical spectra measured after exposure of the fluorescent reporter by a 488nm laser in the presence of cell lines expressing or not the target antigen.

[0100] [Fig.5B] is an illustration of the FRET indices obtained from the spectra shown in [Fig.5A]. ILLUSTRATIVE METHODS OF IMPLEMENTING THE INVENTION

[0101] The recognition of a target molecule 2 by the fluorescent reporter 1 is illustrated in [Fig. 1], the fluorescent reporter 1 comprises: - a receptor 11 bound to a binding protein 12; and - two fluorochromes Fa and Fb;

[0102] Fluorochrome Fa is bound to receptor 11 and fluorochrome Fb is bound to binding protein 12. Fluorochromes Fa and Fb form a FRET donor / acceptor pair. Receptor 11 is bound to binding protein 12 with a stronger bond than that which can bind receptor 11 to a recognition molecule 2.

[0103] Before the target molecule 2 is recognized by the fluorescent reporter 1, the latter is in an inactive ("OFF") configuration, i.e., there is no FRET effect between the two fluorochromes Fa and Fb. In this configuration, the donor fluorochrome emits light by fluorescence because it is excited, while the acceptor fluorochrome does not. Upon recognition of the target molecule 2 by the fluorescent reporter 1, the latter then takes on an active ("ON") configuration. A conformational change occurs in the receptor 11, modifying the distance separating the two fluorochromes Fa and Fb, thus inducing a non-radiative energy transfer (FRET effect) between the two fluorochromes. This energy transfer occurs from the donor fluorochrome to the acceptor fluorochrome: the fluorescence intensity of the donor fluorochrome decreases, and that of the acceptor fluorochrome increases; in this configuration the fluorochromes are noted F'a and F'b.The variation in their emission spectrum due to the FRET effect can be measured to detect and / or measure the concentration of target molecule 2.

[0104] This embodiment is particularly advantageous because it allows rapid detection of the target molecule 2 while avoiding degradation of the fluorescent reporter 1 because the receptor 11-binding protein 12 binding prevails over the receptor 11-target molecule 2 binding.

[0105] In an embodiment illustrated in [Fig.2], the target molecule detection device comprises: - an optical fiber 4 comprising: • a sheath 46; • at least one core 45 with longitudinal axis XX'; • a ferula 44 • an exploration head comprising a body 43 and an external face 42, called the emission face, at least part of which is transparent forming a window 41, and intended to be opposite the core(s) 45 of the optical fiber 4 for the passage of light; and - a fluorescent protractor 1 comprising: • a receptor bound to a binding protein; and • two fluorochromes Fa and Fb; the fluorochrome Fa is bound to receptor 11 and the fluorochrome Fb is bound to binding protein 12, The fluorochromes Fa and Fb form a FRET donor / acceptor pair.

[0106] 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, constituting the scanning end of the optical fiber 4 from which the light necessary for illuminating the fluorescent protractor will be emitted. This same optical fiber is also used for collecting the light response of the protractor and conducts the return light beam to a housing at the proximal end of the fiber.

[0107] The exploration end of the optical fiber 4 is provided in a known manner with a ferrule 44 forming a rigid tip pierced in its center and in which the sheath 46 of the optical fiber 4 is fixed.

[0108] The window 41 on the external face 42 of the exploration head is functionalized by the fluorescent reporter 1 via a grafting molecule 3. The fluorescent reporter 1 can thus recognize a target molecule present in the cavity explored by the optical fiber 4, in particular a target molecule that the exploration head would encounter during its use, such as lodged in a cavity of the human body, i.e. on human tissue, to detect cancer cells by observation and / or measurement of the variations in the emission spectra of the fluorochromes Fa and Fb.

[0109] Preferably illustrated in [Fig.2], the exploration head comprises a hollow cylindrical body 43 with the same longitudinal median axis XX' in the assembled position of the head on the optical fiber body 4, and an external distal end face 42 which is transverse to the axis of the cylindrical body and from which the light exiting the core 45 of the optical fiber 4 is intended to be emitted.

[0110] This embodiment is particularly advantageous because it allows for rapid detection of the target molecule in a cavity of the human body (for example, by endoscopy or during surgery) while avoiding degradation of the fluorescent reporter 1 during recognition of the target molecule. This notably prevents leaving portions of said fluorescent reporter 1 in the cavity to be examined, which would lead to contamination of the human body being examined. EXAMPLES

[0111] The present invention will be better understood by reading the following examples which illustrate the invention in a non-limiting way.

[0112] Example 1a In vitro detection of a target molecule - Case of the detection of an antigen in solution

[0113] Preparation of the fluorescent protractor

[0114] A solution of the antibody is incubated with the binding protein. The latter may be in solution or immobilized on a substrate. The incubation must be long enough to ensure optimal binding between the binding protein and the antibody. Chemical fixation may or may not be performed during this step to covalently bind the support protein and the antibody.

[0115] Target molecule detection

[0116] The solution containing the antigen is mixed with a solution containing the reporter. In parallel, solutions of known antigen concentrations are diluted in the same reporter solution, thus preparing a standard curve. After incubation, the spectral properties are measured by a fluorometer, or any other instrument capable of such measurements. The FRET indices are calculated for the standard curve and the sample, thereby allowing the concentration of the latter to be calculated. The results obtained for the standard curve are presented in Figures 1a and 1b.

[0117] The results shown in Figures 4, 5A, and 5B were obtained by depositing cells expressing or not expressing the antibody target onto a microscope slide to which the fluorescent protractor had been previously attached. Laser excitation and signal collection were performed using an optical fiber and a spectrophotometer. The results allow the amount of target molecule present on the cell surface to be determined (low to zero in HEK cells, moderate in A549 cells, high in A431 cells).

[0118] Example 1b In vitro detection of target molecule

[0119] Example 1a has been reproduced but the fluorescent protractor has been modified according to the Table I.

[0120] [Tables 1] Fa receptor Fb binding protein Ligand Ligand detection Anti-Trk B antibody Alexa 488 G protein Alexa 546 TrkB recognizes combination yes Anti-Trk B antibody Alexa 488 G protein Alexa 594 TrkB recognizes combination yes Anti-EGFR antibody (clone AY3) Alexa 488 G protein Alexa 546 EGFR recognizes combination yes Anti-EGFR antibody (clone MA-12693) Alexa 488 G protein Alexa 546 EGFR recognizes combination yes Anti-EGFR antibody (clone MA-12693) CF Dye 503R G protein CF Dye 55 5 A4 cell line cells 31 yes Anti-EGFR antibody (clone MA-12693) CF Dye 503R G protein CF Dye 59 4 Cells of A4 cell line 31 yes Anti-EGFR antibody (clone MA-12693) CF Dye 503R G protein CF Dye 55 5 A5 cell line 49 yes Anti-EGFR antibody (clone MA-12693) CF Dye 503R G protein CF Dye 59 4 A5 cell line 49 yes

[0121] Table I: Fluorescent protractor compositions

[0122] Example 2 In vitro detection of target molecule

[0123] Substrate grafting

[0124] An organosilica (thiolated silica) / zirconia sol is deposited on a substrate, typically the well of a plate, and is heat-treated at 80°C for 12 hours. A first bonding molecule containing a maleimide group and an amine group is dissolved in DMSO, and this solution is added to the well for 1 hour with stirring. After rinsing and washing, a second solution containing another bonding molecule, containing an NHS group and a maleimide group in DMSO, is added to the well for 1 hour with stirring. After rinsing and washes, the substrate is prepared to bind the first fluorescent binding protein (classically a G protein ending with a cysteine).

[0125] Preparation of the fluorescent protractor

[0126] A solution of the antibody is incubated with the binding protein immobilized on a substrate. The incubation must be long enough to ensure optimal binding between the binding protein and the antibody. Chemical fixation may or may not be performed during this step to covalently bind the support protein and the antibody. Then, the excess antibody is washed off.

[0127] Target molecule detection

[0128] A solution containing the target antigen is added to the well and will be detected in the same manner as in the solution test of Example 1.

[0129] Example 3a In vivo detection of target molecule

[0130] Grafting onto a capsule or an optical fiber

[0131] Once the fluorescent reporter is ready, according to the method defined previously, it is grafted either directly onto the distal end of the optical fiber or onto a transparent window positioned in a capsule for assembly with an optical fiber. The window is typically made of a PET (polyethylene terephthalate) or FEP (fluoropolyethylene-copropylene) polymer film. The grafting is carried out in the same way as in Example 2. That is, first via a silica / zirconia thin-film deposition step on the window or on the end of the target, and then via a surface functionalization step with linker molecules containing maleimide / NHS groups.

[0132] The method of grafting the fluorescent reporter onto the end of the fiber or onto the window is comparable to the method of grafting onto the substrate of Example 2.

[0133] Target molecule detection

[0134] The window or optical fiber is brought into contact with a fabric or other surface interface on which the target molecule is likely to be located, and simultaneously, laser beam illumination is delivered through the optical fiber to the fluorescent reporter. The reflected beam is collected by the same optical fiber and sent to a spectrophotometer for analysis.

[0135] The FRET index is calculated to determine whether the interaction between the fiber (or the window) and the fabric is positive (presence of the target molecule) or negative (absence of the target molecule). This detection is performed within a few seconds.

[0136] Example 3b Target molecule detection

[0137] Example 3a has been reproduced but the fluorescent protractor has been modified according to the Table II.

[0138] [Tables2] Fa receptor, Fb binding protein, Ligand, Ligand detection, Antibody, TrkB, Alexa 488, G protein, Alexa 546, HEK or HCT cells 116 or A 431, or human patient tumor, Yes, Antibody, TrkB, Alexa 488, G protein, Alexa 594, HEK or HCT cells 116 or A 431, or human patient tumor, Yes, Antibody, TrkB, Alexa 488, A protein, Alexa 546, HEK or HCT cells 116 or A 431, Yes, Antibody, TrkB, Alexa 488, A protein, Alexa 594, HEK or HCT cells 116 or A 431, Yes, Antibody, TrkB, Alexa 488, G protein, Alexa 546, HEK or HCT cells 116 or A 431, or human patient tumor, Yes, Antibody TrkB Alexa 488 G Protein Alexa 594 HEK or HCT 116 or A 431 cells, or human patient tumor Yes Anti-EGFR antibody (cio ne AY3) Alexa 488 G Protein Alexa 546 HEK or HCT 116 or A 431 cells, or human patient tumor Yes Anti-EGFR antibody (cio Alexa 488 G Protein Alexa 546 HEK or HCT 116 cells Yes MA-126 93) or A 431, or human patient tumor Anti-EGFR antibody (MA-126 93) CF Dye 50 3R G protein CF Dye 555 HEK or HCT 116 or A 431 cells, or human patient tumor Yes Anti-EGFR antibody (MA-126 93) CF Dye 50 3R G protein CF Dye 594 HEK or HCT 116 or A 431 cells, or human patient tumor Yes Anti-EGFR antibody (MA-126 93) CF Dye 50 3R G protein CF Dye 555 HEK or HCT 116 or A 431 cells, or human patient tumor Yes Anti-EGFR antibody (MA-126 93) CF Dye 50 3R Protein G CF Dye 594 HEK or HCT 116 or A 431 cells, or human patient tumor Yes

[0139] Table II: Fluorescent protractor compositions DIGITAL REFERENCES

[0140] 1 - Fluorescent reporter

[0141] 11 - Receiver

[0142] 12 - Binding protein

[0143] 2 - Target molecule

[0144] 3 - Grafting molecule

[0145] 4 - Optical fiber

[0146] 41 - Hublot

[0147] 42 - External emission face

[0148] 43 - Body

[0149] 44-Ferule

[0150] 45 - Heart

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] 46 - Sheath 5 - Device Fa - Fluorochrome bound to the receptor (inactive configuration) F'a - Fluorochrome bound to the receptor (active configuration) Fb - Fluorochrome bound to the binding protein (inactive configuration) F'b - Fluorochrome bound to the binding protein (active configuration) XX' - Longitudinal axis of the optical fiber

Claims

Demands

1. Fluorescent reporter (1) comprising: - a receptor (11) linked to a binding protein (12) by a covalent bond; - two fluorochromes Fa and Fb; wherein the fluorochrome Fa is linked to the receptor (11) and the fluorochrome Fb is linked to the binding protein (12); and the fluorochromes Fa and Fb form a FRET donor / acceptor pair.

2. Fluorescent reporter (1) according to claim 1, wherein the receptor (11) is selected from antibodies, antibody fragment, aptamer, proteins, peptides, or a derivative thereof.

3. Fluorescent reporter (1) according to claim 1 or claim 2, wherein the binding protein (12) is selected from protein G, protein L, protein A, protein Z, protein M, immunoglobulin, a complete or partial immunoglobulin, or a derivative thereof.

4. Fluorescent reporter (1) according to any one of claims 1 to 3, wherein the fluorochromes Fa and / or Fb are selected from fluorescent molecules or fluorescent proteins.

5. A device (5) for detecting a target molecule (2) and / or measuring the concentration of a target molecule (2) comprising: - a substrate to the surface of which a grafting molecule (3) is covalently attached; - at least one fluorescent reporter (1) comprising: • a receptor (11) linked to a binding protein (12) by a covalent bond; • two fluorochromes Fa and Fb; wherein the fluorochrome Fa is linked to the receptor (11) and the fluorochrome Fb is linked to the binding protein (12); and the fluorochromes Fa and Fb form a donor / acceptor pair FRET; wherein the binding protein (12) is linked to the grafting molecule (3) by a covalent bond,

6.

7.

8. in which the linking protein (12) comprises a terminal group selected from thiol, amine, azide, alkyne, epoxide, carboxylic acid, aldehyde, aziridine, alkene, or a derivative thereof, and the grafting 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 a derivative thereof. Device according to claim 5, wherein the target molecule (2) is an antigen. A device according to claim 5 or claim 6, 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, preferably spherical, preferably of silica or polymer, a microscope slide, a glass coverslip, the outer layer of an optical fiber, or a substrate configured to be attached to the head of an optical fiber. A method for detecting a target molecule (2) and / or measuring the concentration of a target molecule (2) comprising the following steps: - Bring a sample into contact with at least one fluorescent reporter (1), said fluorescent reporter (1) comprising: • a receptor (11) linked to a binding protein (12) by a covalent bond; • two fluorochromes Fa and Fb; in which the fluorochrome Fa is bound to the receptor (11) and the fluorochrome Fb is bound to the binding protein (12); and the fluorochromes Fa and Fb form a FRET donor / acceptor pair; and the receptor (11) has an affinity for said target molecule (12); Excite the fluorescent reporter (1) at a given wavelength so that the donor fluorochrome is excited; Measure the ratio between the intensity of fluorescence emitted by the donor fluorochrome and the intensity of fluorescence emitted by the acceptor fluorochrome; and Determine the presence or absence of said target molecule (2) in the sample and / or calculate the concentration of said target molecule (2) in the sample.

Images