Testing of capillary action using photoluminescent inorganic nanoparticles

Description

[0001] The present invention relates to the field of bioanalysis and diagnostics in vitro. Its main focus is a process in vitro detection and / or quantification of a substance of biological or chemical interest, for example proteins, antibodies, toxins and other compounds, in a liquid sample, by a capillary diffusion test using, as probes, photoluminescent inorganic nanoparticles with controlled optical and physicochemical properties.

[0002] Capillary diffusion assays, such as lateral-flow assays (LFAs), also known as strip tests, are commonly used for clinical, pharmaceutical, food, and chemical analyses. They can be used to detect the presence of many types of analytes, including antibodies, antigens, proteins, biomarkers, chemical molecules, nucleic acids, and more ([1]). When the recognition molecules used in the lateral-flow assay are antibodies, it is more commonly referred to as an immunochromatographic assay (Lateral Flow Immunoassay (LFIA)).

[0003] Capillary diffusion tests are particularly appreciated for their ease of use, speed (detection in a period of less than or equal to 15 minutes) and low cost.

[0004] In general, capillary diffusion test devices implement a capillary diffusion method in the form of a porous solid support (e.g., a nitrocellulose membrane), within which the sample to be tested, deposited at one end of the solid support, and the reagents, integrated into the commercial device, migrate by capillary diffusion.

[0005] Typically, the porous solid support of capillary diffusion test devices includes a labeling area ("Conjugate Pad" in Anglo-Saxon terminology) carrying, in liquid, lyophilized or dehydrated form, a binding reagent specific to the substance to be analyzed (or "analyte"), conjugated to a probe (or revealing species), and a detection area ("Detection Pad" in Anglo-Saxon terminology) on which is immobilized a capture reagent specific to the analyte.

[0006] The analyte-specific binding reagent is immobilized in lyophilized form but becomes mobile within the solid support when wet. Thus, when the solid support comes into contact with a liquid sample, the sample migrates by capillary diffusion through the support, carrying with it the analyte-specific binding reagent conjugated to the probe. The sample and the analyte-specific binding reagent migrate by capillary diffusion through the solid support to the detection zone containing an immobilized, analyte-specific capture reagent.

[0007] In the most common capillary diffusion assays, of the "sandwich" type, the binding reagent conjugated to the probe binds to the analyte in the sample upon contact. The analyte is then immobilized on the solid support by the capture reagent. The presence or absence of the analyte in the sample is thus measured. viathe detection of the probe immobilized at the detection zone via the analyte.

[0008] These devices also include a control zone, located downstream of the detection zone relative to the capillary diffusion direction, in which a second capture reagent specific to the labeled targeting reagent is immobilized. After migrating to the detection zone, the excess probe-coupled binding reagent, having not reacted with the analyte, migrates to the control zone and binds to the second capture reagent. The user thus has a positive control to verify the migration of the sample and reagents within the device, and therefore to confirm the proper functioning of the test.

[0009] The determination of the analyte in the sample is therefore carried out by detecting the presence, or absence, of the probe at the level of the detection zone and, possibly, the control zone.

[0010] The most commonly used probes in capillary scattering tests are gold nanoparticles ([1], [2]). These absorb light at characteristic wavelengths that correspond to their surface plasmon frequency. The surface plasmon frequency of the nanoparticles depends on their size and state of aggregation. When the gold nanoparticles are immobilized in the detection and / or control zone, the absorption linked to the surface plasmon frequency of the nanoparticles, which are in close proximity to each other, gives these zones a characteristic color, typically blue / violet, visible to the naked eye.These tests offer the advantage of obtaining rapid sample analysis results, typically in minutes, compared to the time required for conventional immunodetection techniques, such as an enzyme-linked immunosorbent assay (ELISA), which typically takes several hours. Furthermore, they do not require costly and bulky preparatory or analytical equipment.

[0011] However, these tests have the major drawback of low detection sensitivity and a lower limit of quantification. They therefore generally provide only qualitative or semi-quantitative information. In particular, these strip tests have a much lower detection sensitivity than that achievable with conventional immunoassays, such as ELISA. For example, gold nanoparticle-based strip tests typically detect concentrations on the order of a few ng / mL, while an ELISA test detects concentrations of a few pg / mL, typically two to three orders of magnitude more sensitive. For example, Cortez Diagnostics offers a strip test for the detection of troponin I (a biomarker of myocardial infarction) with a sensitivity of 1 ng / mL ([3]), while Abcam offers an ELISA test (ab200016) with a sensitivity of 7 pg / mL.

[0012] In order to improve the sensitivity of strip tests, in other words to lower the detection limit of these tests, various materials have been proposed as probes, as an alternative to gold nanoparticles, in capillary diffusion tests, and in particular modified gold nanoparticles, magnetic particles, semiconductor nanocrystals or "quantum dots", up-conversion phosphors, organic fluorophores, etc. ([1], [4]).

[0013] Thus, several gold nanoparticle-based nanomaterials have been explored as probes for capillary diffusion test devices, such as magnetic microspheres comprising a core of a nanometric Fe₂O₃ particle coated with gold nanoparticles ([5]), silica nanotubes carrying gold nanoparticles ([6]), or multi-branched gold nanoflowers (GNFs) ([7]). Der-Jiang can also be mentioned. et al([8]) which propose depositing a silver layer onto gold nanoparticles. However, these approaches require highly complex synthesis methods for these probes. Moreover, since silver oxidizes more readily than gold, probes combining silver with gold exhibit less stability for application in capillary diffusion assays in aqueous media.

[0014] The use of photoluminescent probes (also referred to simply as "luminescent probes"), such as organic fluorophores and quantum dots, has increased the sensitivity of capillary scattering assays compared to gold nanoparticle-based assays. In fact, the detection of light emission (luminescence) is generally more sensitive than the detection of absorption (as with gold nanoparticles), the latter occurring against the high background of transmitted light (e.g., for organic fluorophores [9],

[10] , and

[11] ; for QDs:

[11] -

[17] ,

[50] ).

[0015] Unfortunately, these photoluminescent probes have several disadvantages that prevent their full potential from being exploited as probes in strip tests. Among these drawbacks are, for example, the phenomenon of photobleaching in the case of organic fluorophores, which, following irreversible structural modifications induced by illumination, results in the disappearance of fluorescence; and the phenomenon of emission flicker in semiconductor nanocrystals, or "quantum dots," where the probes periodically cease emitting and are therefore unsuitable for producing a constant and reproducible signal. Other disadvantages stem, for example, from the broad emission spectrum of the luminescent probes.Indeed, an excessively broad emission spectrum makes it difficult to filter out any background signal that may be present, which affects signal quality and, in particular, the signal-to-noise ratio. In addition to the optical factors that contribute to the probe's effectiveness in a biological assay, the probe's practicality and ease of use must also be considered. For example, some particles, such as semiconductor nanocrystals, lose their luminescence characteristics after freezing, which is a drawback for storing bioconjugated semiconductor nanocrystals. The ease of coupling the probes to the molecular compound used to target the desired molecules is also an aspect to consider when choosing the appropriate probe. Thus, a number of particles, including semiconductor nanocrystals, are synthesized in organic solvents.Consequently, their use in biological applications requires additional surface preparation steps to achieve water dispersion of these particles, a process that can be complex and unstable over time (

[18] ). Indeed, the surface functionalizations used for semiconductor nanocrystals do not involve covalent bonds with the nanocrystal surface. The functionalization molecules can therefore detach and cause the analyte-specific binding reagent (antibody or other) to dissociate from the nanocrystal used as a probe. Thus, the conjugation of these nanocrystals to the analyte-specific binding reagents can take from a few weeks to a few months, depending on the type of functionalization.However, commercial use of a capillary diffusion test requires stability on the order of two years after deposition of the probe-binding reagent conjugates on the test strip.

[0016] Another drawback of these luminescent probes is that the excitation required for luminescence detection can cause the emission of stray light, thereby increasing the background signal and consequently decreasing the signal-to-noise ratio. Various approaches have been proposed to eliminate, or at least reduce, this stray emission signal, such as the use of luminescent nanoparticles containing chelates or lanthanide ion complexes, combined with delayed luminescence detection; upconversion nanoparticles; or persistent luminescence nanoparticles.

[0017] Thus, luminescent particles, loaded with chelates or lanthanide complexes, have already been proposed as luminescent probes in capillary diffusion tests.

[0018] For example, Zhang et al. (

[19] ) propose to use, as probes for the detection of the bacterium Pantoea stewartii subsp. stewartii (Pss) In a migration strip test, silica nanoparticles loaded with lanthanide chelates (Eu) were used. It is reported that these probes achieve a detection limit 100 times lower than that achievable with conventional tests using gold particles. Similarly, Xia et al. (

[20] ) use europium chelate-loaded silica particles in a lateral flow assay for the detection of hepatitis B surface antigen (HBsAg).

[0019] We can also mention the Liang publications et al. (

[21] ) and Juntunen et al.(

[22] ), which propose the use of polystyrene microparticles loaded with europium chelate, in a lateral flow test, for the detection of alpha-fetoprotein (AFP) in a serum sample, or for the detection of prostate-specific antigen (PSA) and biotinylated bovine serum albumin (biotin-BSA). Documents WO 2013 / 013214 and WO 2014 / 146215 also propose the use of polystyrene nanospheres loaded with terbium and / or europium chelates as probes in a lateral flow test strip.

[0020] It was also proposed, in document WO 2014 / 146215, to exploit the long lifetime emission (on the order of 100 µs) of these nanoparticles containing chelates or lanthanide ion complexes, to implement a delayed detection making it possible to overcome parasitic emissions whose lifetime is generally on the order of 1-10 ns.

[0021] However, luminescent particles loaded with lanthanide chelates or complexes, used as luminescent probes, typically contain only a single lanthanide ion per chelate or complex. Each chelate or complex occupies a significant portion of the nano- or microparticle, which considerably limits the number of emitting ions for a given particle size. For example, a 45 nm nanoparticle contains only about 1000 chelates and emitting ions

[47] . Furthermore, the synthesis of this type of particle containing lanthanide ion complexes or chelates involves at least two steps: the synthesis of the complex or chelate, followed by the synthesis of the particle containing the chelates. Thus, the synthesis of this type of particle is complex and, consequently, relatively expensive. Finally, the stability of this type of particle has also been questioned (

[23] ).

[0022] In recent years, another type of rare-earth-based luminescent nanoparticle has been proposed as a luminescent probe in bioimaging applications, particularly in capillary scattering assays: these are up-conversion phosphor nanoparticles, which emit visible light when excited by infrared or near-infrared sources (e.g.,

[24] -

[29] ). In the implementation of these up-conversion nanoparticles, two photons are absorbed by the nanoparticle before the luminescence emission, which corresponds to the detected signal, is observed. As an example, see the publication by Niedbala et al.(

[30] ) which offers lateral flow test strips employing UPT (Up-converting Phosphor Technology) probes, enabling higher detection sensitivity than enzyme immunoabsorption tests. Such luminescent up-conversion probes are, for example, used in the lateral flow test device proposed in US patent 2014 / 0170674.

[0023] These up-conversion phosphors have the advantage, compared to the aforementioned luminescent probes, of exhibiting resistance to photobleaching and a low level of spurious fluorescence causing background noise. Indeed, since excitation occurs at a wavelength shorter than the detection wavelength, emission from ancillary substances in the sample or from the porous solid support is practically nonexistent.

[0024] Unfortunately, these up-conversion phosphors have the major drawback of exhibiting low quantum efficiencies, and their efficiency decreases considerably at low optical power densities of the excitation source, as luminescence is proportional to the square of the excitation power density. Furthermore, a fairly large area encompassing the test band, and possibly the control band, must be excited, which reduces the excitation power density for a given power level. Consequently, reading tests using such luminescent probes requires complex equipment, combining laser diodes for excitation with other components such as lenses, filters, photomultiplier tubes, preamplifiers, and so on.

[0025] Finally, inorganic nanoparticles emitting persistent luminescence have also been proposed to overcome the spurious luminescence induced by excitation. These inorganic nanoparticles are formed from a crystalline matrix containing lanthanide ions as dopants. The distinctive feature of persistent luminescence nanoparticles lies in the fact that the dopants introduce trap states into the electronic structure of the crystal, where excited charges become trapped. Thus, luminescence emission by these nanoparticles can only occur after the release of charges from these trap states, a release that takes place through thermal activation (

[31] ). Depending on the energy of these trap states in the electronic structure, i.e., the depth of the trap, the thermal activation, and thus the lifetime of the emission, can last for hours or even days.It is thus possible to excite the nanoparticles, then insert the capillary diffusion test device into a suitable reader after the excitation has stopped, and read the test by detecting luminescence in the absence of excitation, and therefore in the absence of spurious emissions. For example, Paterson. et al. (

[32] ) obtained significantly improved detection sensitivity compared to that obtained with gold nanoparticles (detection limit approximately 10 times lower than that obtained with gold nanoparticles). This also eliminates the need for an emission filter for reading.

[0026] However, this system has the drawback of requiring a long signal acquisition time. In fact, since the emission by these nanoparticles occurs over several minutes, and in particular over several hours, or even days, depending on the case, it is necessary to wait the equivalent of this lifetime to collect a non-negligible fraction of the emitted photons. Thus, per unit of time, for example per second, the number of emitted photons will be low, which consequently requires extending the luminescence acquisition time to achieve a high level of sensitivity. Such an acquisition time is contrary to the objective of capillary scattering tests, which is to provide a rapid diagnosis. To counteract this problem, it is possible to increase the excitation power; however, this implies complex equipment, which makes it impossible to meet the requirement for a compact and inexpensive test system.

[0027] Finally, in another variant of the capillary diffusion assay, europium- and bismuth-codified YVO₄ nanoparticles were used (

[33] ). The presence of bismuth shifts the absorption of the YVO₄ matrix, which exhibits an absorption peak at 280 nm due to the O₂⁻<-V⁵⁺< charge transfer transition within vanadate VO₄³⁻< ions, towards the visible spectrum. This shift is due to the appearance of the Bi³⁺<-V⁵⁺< charge transfer transition, thus allowing the use of more common excitation sources around 350 nm. The excitation is then transferred to Eu³⁺< ions. However, this approach has the drawback of requiring complex synthesis of these nanoparticles. In particular, it is difficult to make a homogeneous solid solution of YVO 4 and BiVO 4 whose crystalline structures are different (

[34] ).This requires the use of either high-temperature hydrothermal syntheses requiring more complex equipment (autoclave) (

[33] ), or maturation processes (

[34] ).

[0028] Application JP-A-2012-032263 describes a fluoroimmunological assay kit using a fluorescent fine particle doped with 0.01% to 6% molar rare earth.

[0029] Thus, none of the probes proposed so far for capillary scattering tests are entirely satisfactory. In particular, there remains a need for a probe, compatible with implementation in a capillary scattering test device, that combines the advantages of a highly luminous probe, simple and inexpensive to synthesize, which allows for much more sensitive detection than capillary scattering systems based on gold nanoparticles, with a reading system that can be used with the naked eye or with a compact and inexpensive reader. The present invention aims precisely to provide new luminescent probes that can be used in a capillary scattering test and that meet this need.

[0030] More specifically, it proposes the use, as probes in a capillary diffusion test analysis method, of luminescent nanoparticles doped with rare earth ions, with controlled optical and physico-chemical properties.

[0031] More specifically, the invention relates, according to one of its aspects, to a process in vitro detection and / or quantification of a substance of biological or chemical interest in a liquid sample, by a capillary diffusion test of the migration strip type, using, as probes, photoluminescent inorganic nanoparticles, of the following formula (II): A 1-x Ln x VO 4(1-y) (PO 4 ) y (II) in which: . A is chosen from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof; . Ln is chosen from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho) and mixtures thereof; 0.1 ≤ x ≤ 0.9, in particular 0.2 ≤ x ≤ 0.6 and more particularly x equals 0.4; and . 0 ≤ y < 1, in particular y equals 0; said method implementing the detection of luminescence, with an emission lifetime shorter than 100 ms, of nanoparticles, after absorption by one photon, by excitation of the matrix at a wavelength less than or equal to 320 nm.

[0032] In particular, luminescence detection is advantageously carried out by exciting the AVO 4(1-y) (PO 4 ) y matrix, for example YVO 4 , at a wavelength less than or equal to 300 nm, in particular between 250 and 300 nm.

[0033] According to the method of the invention, the detected signal thus corresponds to the emission of luminescence by the photoluminescent nanoparticles after absorption of a single photon; in other words, emission at a wavelength longer than that of the excitation. The emission of luminescence after absorption of a single photon is particularly distinct from the case of detecting luminescence emission by particles for absorption of two photons, as is the case with the "up-conversion" particles mentioned previously.

[0034] These nanoparticles are functionalized with recognition molecules (antibodies, nucleic acids, peptides, aptamers, etc.) which are capable of recognizing the substance to be analyzed as described below.

[0035] For the purposes of the invention, the "analysis" of the substance in a sample covers the aspect of detection or qualitative characterization of the presence or absence of said substance, and also the aspect of dosage or quantitative characterization of said substance.

[0036] The liquid sample may include a biological sample, in particular any biological or bodily fluid. It may be a sample taken from a human body, for example chosen from blood, serum, plasma, saliva, sputum, nasal swab, urine, diluted fecal matter, vaginal swab or even cerebrospinal fluid.

[0037] It may also be a solution containing biological molecules, chemical molecules or even pathogenic viruses or bacteria, such as environmental samples or samples from agri-food products.

[0038] The method of the invention can in particular be implemented for the detection and / or quantification of molecules, proteins, nucleic acids, toxins, viruses, bacteria or parasites, in a sample, in particular in a biological sample.

[0039] For example, it can be used to detect the presence of biomarkers, antibodies, DNA and / or RNA, immunoglobulins (IgG, IgM, etc.), antigens, the antigens also being biomolecules composing a virus, a bacterium or a parasite, in a biological sample.

[0040] It may also be, for example, a molecule of interest for scientific police investigations, for example an illicit chemical substance such as a drug, or a substance of interest for defense (bioterrorism agents).

[0041] It may still be a substance of interest for food safety (pathogenic bacteria such as Salmonella of the Listeria or Escherichia coli, or viruses such as norovirus or allergens), or for the environment, for example a pollutant (pesticides).

[0042] The substance of biological or chemical interest that we are trying to analyze via The capillary diffusion test according to the invention is referred to, more simply thereafter, by the expressions "substance to be analyzed" or "analyte".

[0043] The use of luminescent inorganic nanoparticles according to the invention as probes in a capillary diffusion test device, in a test strip, proves to be particularly advantageous in several respects.

[0044] Firstly, rare earth ion-doped nanoparticles, implemented according to the invention, of formula (A 1-x Ln x ) a (M p O q ) (I) described more precisely in the rest of the text, for example YVO 4 :Eu or GdVO 4 :Eu, YAG :Ce type nanoparticles, in particular the nanoparticles of formula A 1-x Ln x VO 4(1-y) (PO 4 ) y (II), exhibit particularly advantageous properties, notably with regard to their excellent photostability, which allows the acquisition of a constant and prolonged signal, and an absence of emission flicker phenomenon.

[0045] Furthermore, these nanoparticles do not lose their luminescence after freezing.

[0046] For example, rare-earth-doped yttrium vanadate-based nanoparticles have been described in detail by Riwotzki et al. (

[45] ) and Huignard et al.(

[46] ). As for document EP 1 282 824, it describes the implementation of inorganic luminescent nanoparticles, modified on the surface, as probes to detect a biological or other organic substance.

[0047] However, to the inventors' knowledge, it has never been proposed to take advantage of photoluminescent inorganic nanoparticles as defined above, doped with rare earth ions, distinct from persistent luminescent particles, and emitting after absorption of a single photon, for their use as luminescent probes in a capillary scattering test.

[0048] It was by no means foreseeable that these lanthanide ion-based nanoparticles could be used for the detection and quantification of chemical or biological substances, particularly in a capillary diffusion test, and, moreover, that they would lead to improved performance in terms of test sensitivity.

[0049] Indeed, apart from the absence of blinking, the luminescence properties of rare-earth-based nanoparticles are considered inferior to those of quantum dots. In these rare-earth-doped nanoparticles, particularly those with a metal oxide matrix, luminescence can be induced either by direct excitation of the matrix or, more rarely, by direct visible excitation of the luminescent rare-earth ions. The extinction coefficient for direct absorption of rare-earth ions is generally very low, but the extinction coefficient for excitation of the crystalline matrix is ​​much higher (

[35] ).

[0050] However, the absorption band of the crystalline matrix is ​​generally located in the UV, which presents a major drawback: biomolecules, as well as the various components of the capillary diffusion device (e.g., the capillary diffusion membrane), also absorb strongly in the UV. For example, in the case of a vanadate ion-based matrix (VO₄³⁻), the absorption peak at 280 nm coincides with the absorption of proteins, and in particular the amino acid tryptophan. Consequently, the excitation of luminescence in nanoparticles based on a rare-earth ion-doped crystalline matrix, especially in the UV, is likely to produce significant side emission signals. Such unwanted emission is incompatible with the purpose of a diagnostic test. in vitro such as a capillary diffusion strip test, which is specifically designed to identify a low-intensity signal from a complex mixture of molecules.

[0051] Against all expectations, the inventors discovered that it is possible to use such rare-earth-based photoluminescent nanoparticles according to the invention as probes in a diagnostic technique in vitro of the capillary diffusion test type, even under UV excitation conditions and in particular for excitation below 350 nm, advantageously below 320 nm, more advantageously between 250 and 320 nm and in particular between 250 and 300 nm.

[0052] Without being bound by theory, the inventors observed that detection of the luminescence of UV-excited nanoparticles in a capillary scattering assay is possible, despite the presence of a strong spurious emission signal, due to three optical properties specific to the nanoparticles used: i) a large number of luminescent lanthanide ions contained in the nanoparticles of the invention without requiring very large nanoparticles, ii) a narrow emission spectrum of rare-earth ions, which effectively eliminates spurious emissions that are generally very broad spectrally, and iii) a large Stokes shift (the shift between the absorption peak and the emission peak), typically on the order of 350 nm for Eu-doped YVO₄ or GdVO₄ nanoparticles (absorption peak at 280 nm for the vanadate matrix and peak at 280 nm for the emission peak). emission of Eu at 617 nm),This allows for effective rejection of excitation wavelengths and spurious emissions due to the migration medium or the sample containing the substance to be analyzed, which generally exhibit a low Stokes shift. In particular, this large Stokes shift allows the use of a simple high-pass filter for detection, which is less expensive than an interference filter.

[0053] Therefore, it is possible to achieve effective elimination of parasitic emissions, and the acquisition of a signal with a signal-to-noise ratio sufficient to achieve the desired detection sensitivity.

[0054] Furthermore, it turned out, contrary to all expectations, that excitation below 320 nm, and in particular around 280 nm where the absorption peak of the vanadate matrix is ​​located (see Figure 11(A)) or at 300 nm, induces spurious emissions, related to the nitrocellulose membrane typically used for lateral flow tests, which are much weaker than excitation at 380 nm (see Figure 11(B) ). Given that molecules chelating or complexing lanthanide ions typically absorb between 320 nm (

[47] ) and 400 nm (

[48] ), the use of a matrix absorbing between 250 and 300 nm, particularly between 260 and 300 nm, presents an additional advantage.

[0055] In particular, apart from the O2-<-V5+< charge transfer absorption inside vanadate ions VO43-< in a YVO4 or GdVO4 crystal, which is centered at 280 nm, or in a LaVO4 crystal, centered around 300 nm (

[40] ), in the case of Eu-containing nanoparticles, another absorption, linked to an O2-<-Eu3+< charge transfer, is possible and leads to an absorption centered around 260 nm. This has, for example, been observed in La2Hf2O7:Eu, A2Hf2O7 (A = Y, Gd, Lu) and La2Zr2O7:Eu nanoparticles (

[41] ).

[0056] Moreover, as illustrated in the examples that follow, the method of the invention makes it possible to achieve a performance of the capillary diffusion test, in terms of detection sensitivity, improved by at least an order of magnitude or even much more.

[0057] In particular, it allows not only qualitative analyses (presence or absence of the analyte in the sample), but also semi-quantitative and quantitative analyses.

[0058] Thus, this document still concerns the use, as probes in a capillary diffusion test device, of nanoparticles as defined above, to increase the detection sensitivity of said capillary diffusion test device.

[0059] Photoluminescent nanoparticles can be used as probes in any known type of capillary diffusion test, for example, lateral flow tests, whether it be a so-called "sandwich" test as schematically represented in Figure 2 , or even a so-called "competitive" test as represented in Figure 3In particular, they are suitable for implementation in capillary diffusion test devices previously proposed with gold nanoparticles as luminescent probes, without requiring modification of the characteristics of the support of the capillary diffusion test device.

[0060] In particular, as illustrated in the examples that follow, the photoluminescent nanoparticles according to the invention can, for example, have an average size similar to that of gold nanoparticles, on the order of 30 to 50 nm and, consequently, compatible with capillary diffusion means, typically a nitrocellulose membrane, adapted to the migration of particles of this size.

[0061] Alternatively, the photoluminescent nanoparticles implemented according to the invention can be larger, thereby optimizing the luminescence signal. Indeed, the number of lanthanide ions increases with the volume of the nanoparticle, and thus the emitted luminescence signal increases with the cube of the radius of a spherical particle. In this case, the migration support for the lateral flow test can contain pores adapted to the size of the chosen nanoparticles. Membranes with variable pore sizes are, for example, commercially available (e.g., the variable pore size membranes marketed under the references HF075, HF090, HF120, HF135, and HF180 by MerckMillipore).

[0062] Larger nanoparticles can, for example, be obtained by size sorting by centrifugation of the particles as exemplified, retaining only the largest particles in the size distribution, or by grinding the bulk material. Any other technique known to those skilled in the art can also be used.

[0063] Furthermore, as illustrated in the examples, while maintaining a particle size similar to that of gold particles, the nanoparticles implemented according to the invention have a large number of ions responsible for luminescence, in particular significantly higher than in the case of particles based on chelates or lanthanide complexes, and therefore make it possible to produce a high intensity emission signal and, consequently, to access improved sensitivity.

[0064] Advantageously, the detection method according to the invention makes it possible to measure qualitatively up to 10 times, in particular up to 100 times, or even up to 1000 times, below the detection limit of the same test using gold nanoparticles as probes.

[0065] Finally, the implementation of the nanoparticles implemented according to the invention as probes in a capillary diffusion test even leads to improved performance, in terms of sensitivity, compared to the results obtained with particles loaded with lanthanide chelates.

[0066] The invention relates, according to another of its aspects, to a capillary diffusion test device of the migration strip type according to claim 14.

[0067] Thus, the capillary diffusion test device according to the invention comprises photoluminescent inorganic nanoparticles of formula (II) whose luminescence, with an emission lifetime of less than 100 ms, is detected after absorption by a photon, by excitation of the matrix at a wavelength less than or equal to 320 nm, in particular less than or equal to 300 nm and more particularly between 250 and 300 nm.

[0068] More specifically, like probes, for example gold nanoparticles, classically implemented in known lateral flow test devices, the photoluminescent nanoparticles according to the invention are present in an area of ​​the test device, called the "labeling area" (more commonly called "Conjugate Pad" in Anglo-Saxon terminology), in a form coupled with at least one binding reagent specific to the substance to be analyzed, such as an antibody.

[0069] In the following text, the invention is described more particularly with reference to a conventional capillary diffusion test device of the migration strip type (known in English as a "Lateral Flow Strip"), as schematically represented in Figure 1 Of course, the method of the invention can implement any other variant of capillary diffusion test device, provided that it is adapted to the implementation, as probes, of the photoluminescent nanoparticles implemented according to the invention.

[0070] Typically, a capillary diffusion test device according to the invention may thus comprise a capillary diffusion means in a reference direction, in particular a porous solid support, such as a nitrocellulose membrane, comprising: - a liquid sample deposition zone; - a zone, located downstream of the sample deposition zone, called the labeling zone or coupling zone, loaded with the photoluminescent inorganic nanoparticles according to the invention (probes), coupled to at least one "binding reagent" (recognition molecule), for example an antibody, specific to the substance to be analyzed; - a reaction zone, also called the "detection zone," located downstream of the labeling zone, in which at least one "capture reagent" (recognition molecule), such as an antibody, specific to the substance to be analyzed, is immobilized; - a reagent migration control zone, located downstream of the detection zone; and Optionally, an absorbent pad, placed downstream of the control zone.

[0071] Examples of capillary diffusion test devices will be detailed further in the text.

[0072] The liquid sample can be analyzed directly using the capillary diffusion test device according to the invention. Analysis of a liquid sample according to the method of the invention typically comprises: (i) application of the liquid sample to be analyzed, and possibly a diluent, to the deposition zone of the capillary diffusion test device; (ii) incubation of the device until detection in the reaction zone of luminescence generated by the photoluminescent nanoparticles and / or until detection of luminescence in the migration control zone; and (iii) reading and interpretation of the results.

[0073] According to another aspect, the present invention relates to the use of a capillary diffusion test device according to the invention to detect and / or quantify a substance of biological or chemical interest in a liquid sample, in particular a biological sample.

[0074] The capillary diffusion test device can be coupled with a reader to provide the test result.

[0075] As detailed later in the text, reading the results includes detecting the luminescence generated by the immobilized nanoparticles at the detection zone, and where applicable at the control zone, of the capillary diffusion test device.

[0076] It is operated more specifically by: - excitation of immobilized photoluminescent nanoparticles; and - detection of luminescence emission.

[0077] A particularly advantageous feature is that it is possible to read the capillary diffusion test device with the naked eye, using only a suitable filter.

[0078] Alternatively, luminescence reading can be performed using simple detection equipment, for example using an emission filter and a detector such as a camera.

[0079] The emission filter can be an interference filter or a simple high-pass filter. Indeed, thanks to the large Stokes shift associated with the emission of these particles, any parasitic emission, which generally has a small Stokes shift, will be at a shorter wavelength than the emission of these nanoparticles.

[0080] Finally, the capillary diffusion test device according to the invention is adapted to multiplexed detection, in other words to the simultaneous detection by the same capillary diffusion test, of several substances in the same sample.

[0081] The invention also relates, according to another aspect, to the use of a detection method as defined above, or of a capillary diffusion test device as defined above, for diagnostic purposes in vitro. Advantageously, the possibility, by the capillary diffusion test according to the invention, of detecting low levels of certain substances in biological samples allows, for example, the use of the process of the invention for earlier detection of diseases, or for a diagnosis of the evolution of a disease or of the effect of a therapeutic treatment.

[0082] The diseases that can be diagnosed by a capillary diffusion test according to the invention are not limited and include all diseases revealed by the presence of a specific marker of the disease, of the type molecule of biological interest (protein, nucleic acid, antibody, ...), for which there is one or more specific binding partner(s) (ligand, antibody, antigens, complementary nucleic acids, aptamers, ...).

[0083] Examples include infectious diseases (bacterial, parasitic, or viral, such as AIDS), inflammatory and autoimmune diseases, cardiological, neurological, or oncological diseases (for example, solid cancers such as breast or prostate cancer).

[0084] The most significant gains in sensitivity (by a factor of 100 or 1000) allow performance to approach that of an ELISA test or one of its variants. Thus, the method of the invention is particularly well-suited to cases where sensitive ELISA-type detection is required but not readily available.

[0085] This makes it possible to have a rapid diagnosis at the point of intervention (POC for "Point Of Care" in English).

[0086] The method of the invention can thus be useful for the diagnosis of infectious diseases or other common diseases, for example in developing countries, in rural and / or remote areas for the diagnosis of infectious diseases or other common diseases.

[0087] It can also prove particularly useful in emergency situations (SAMU, SMUR), enabling rapid diagnosis, especially when the patient's life may be at risk (heart failure, venous thrombosis, inflammatory syndrome, systemic bacterial infection (sepsis), acute pancreatitis). In such situations, it can be used to perform a rapid test with a sensitivity comparable to that of an ELISA test before arrival at the hospital, saving time in diagnosis and patient management, and thus improving the patient's prognosis.

[0088] Furthermore, a capillary diffusion test, using particles according to the invention as probes, can be particularly useful for patients who require regular diagnostic testing to adjust their medication dosage (for example, in the case of immunomodulators or immunosuppressants). Indeed, performing a test strip, rather than a blood test or other more invasive examination, offers the advantage of improving patient comfort, reducing diagnostic costs, enabling more frequent detection and quantification, and thus allowing for better adjustment of medication dosages.

[0089] Of course, the method of the invention is not limited to the aforementioned applications. It can thus be implemented for the detection of nucleic acids (GMOs in seeds for example), or for the detection of a pollutant or pathogen in the environment, for example in water, or in food intended for human or animal consumption.

[0090] The applications of the method of the invention can thus extend from immunological fields to molecular genetics or the detection of DNA and RNA. It can be used to label one or more RNA strands from a biological sample with a partially complementary fragment bound to a nanoparticle, and then detect them by hybridization with complementary fragments from another region grafted onto the substrate of a strip, following an approach similar to Affymetrix-type DNA microarrays. One advantage of the invention lies in the absence of the amplification step usually required for these approaches.

[0091] The method of the invention can still be implemented for detection in vitro illicit chemical substances, for example drugs or any other substance of interest to the police or defense.

[0092] It can still be used for the detection and / or quantification of a substance of interest, including a pathogen, in an agri-food product or in the environment.

[0093] Other features, variations and advantages of the capillary diffusion test method and device according to the invention will become clearer from the following description, examples and figures, which are given by way of illustration and not limitation of the invention.

[0094] In the following text, the expressions "between ... and ...", "ranging from ... to ..." and "varying from ... to ..." are equivalent and are meant to mean that the boundaries are included, unless otherwise stated.

[0095] Unless otherwise indicated, the expression "including one" should be understood as "comprising at least one". PHOTOLUMINOUS INORGANIC NANOPARTICLES

[0096] As previously stated, the process of the invention uses, as probes in a capillary diffusion test device of the migration strip type, photoluminescent inorganic nanoparticles exhibiting specific optical and physicochemical properties.

[0097] The photoluminescent nanoparticles implemented according to the invention are formed from a crystalline matrix doped with rare-earth ions. The "crystalline matrix" is characteristic of a crystalline solid, in which certain atoms are replaced by other atoms, called "substituted ions." The substituted ions make it possible to modify a chemical or physical property of the crystalline matrix, in particular to impart an optical emission quality to the nanoparticle.

[0098] The rare earth ions in the nanoparticles are not in the form of rare earth ion complexes or chelates; these are formed from rare earth ions in combination with appropriate organic ligands, as described, for example, in the Yuan publication et al. (

[36] ).

[0099] Nanoparticles can be doped with rare earth ions of the same or different types.

[0100] Advantageously, the imperfect crystallinity of nanoparticles, as described in more detail below, makes it possible to overcome the "quenching" effect.

[0101] According to another characteristic of the photoluminescent nanoparticles implemented according to the invention, they are capable of emitting luminescence after absorption of a single photon, which corresponds to the detected signal.

[0102] Moreover, the emission of luminescence by nanoparticles, unlike so-called persistent luminescence nanoparticles (

[32] ), does not involve “trap” states.

[0103] Thus, the nanoparticles implemented according to the invention exhibit a luminescence emission lifetime shorter than 100 ms, in other words, strictly less than 100 ms (

[35] ,

[39] ,

[49] ). The emission lifetime is understood as the lifetime of the excited state of the emitting nanoparticle, and more specifically of the emitting rare-earth ions, and is determined in practice by the duration of luminescence photon emission after the excitation is stopped, i.e., the characteristic time of the exponential decay of luminescence after the excitation is stopped.

[0104] The emission lifetime of an emitting nanoparticle is distinct from the emission lifetime of luminescence before photodegradation or photobleaching of the nanoparticles.

[0105] The nanoparticles implemented according to the invention have an emission lifetime of less than 100 ms, or even less than 10 ms, or even less than 1 ms.

[0106] Advantageously, the nanoparticles implemented according to the invention have an emission lifetime greater than or equal to 5 µs, in particular greater than or equal to 10 µs, especially greater than or equal to 20 µs, or even greater than or equal to 50 µs.

[0107] It is possible to take advantage of the emission lifetime of the particles of the invention (a few hundred µs in the case of Y 1-x Eu x VO 4 particles, compared to the lifetimes of conventional fluorophores on the order of nanoseconds) to perform time-resolved detection, in particular delayed emission detection, with sufficient temporal resolution (on the order of 10 µs or even on the order of 100 µs), using simple and inexpensive equipment. For example, it is possible to modulate the current of an LED (Light Emitting Diode) that can be used for excitation and to record a series of images of the strip instead of a single image, and then analyze the signal over time so as to eliminate any residual spurious emission with a short lifetime (on the order of 10 ns or less). , the photoluminescent nanoparticles implemented according to the invention are of the following formula (II): A -x Ln x VO 4(1-y) (PO 4 ) y (II) in which: . A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu) and mixtures thereof, in particular A represents Y; . Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodynium (Pr), holmium (Ho) and mixtures thereof, preferably Ln represents Eu; . 0.1 ≤ x ≤ 0.9, in particular 0.2 ≤ x ≤ 0.6 and more particularly x equals 0.4; and . 0 ≤ y < 1, in particular y equals 0 said process implementing the detection of luminescence, with a lifetime shorter than 100 ms, by nanoparticles, after absorption by one photon.

[0108] According to a particular embodiment, the nanoparticles implemented according to the invention correspond to the aforementioned formula (II) in which y is 0. In other words, the nanoparticles can be of formula A 1-x Ln x VO 4 (III), in which A, Ln and x are as defined previously.

[0109] A, in formula (II) or (III) above, may be more particularly chosen from yttrium (Y), gadolinium (Gd), lanthanum (La), and mixtures thereof. In particular, A represents Y or Gd. According to a particular embodiment, A in formula (II) or (III) above represents yttrium (Y).

[0110] Ln, in formula (II) or (III) above, may be more particularly chosen from europium (Eu), dysprosium (Dy), samarium (Sm), ytterbium (Yb), erbium (Er), neodymium (Nd), and mixtures thereof. In particular, Ln is chosen from Eu, Dy, Sm, and mixtures thereof. According to another particular embodiment, Ln in formula (II) or (III) above represents Eu.

[0111] Thus, according to one embodiment, the nanoparticles implemented as luminescent probes according to the invention are of formula Y 1-x Eu x VO 4 (IV) in which 0.1 ≤ x ≤ 0.9, in particular 0.2≤x≤0.6 and more particularly x is 0.4.

[0112] According to one embodiment, it is possible to exploit the direct absorption of rare-earth ions in cases where the corresponding electronic transition is allowed. In these cases, direct absorption is stronger than when the corresponding electronic transition is forbidden, although it generally remains weaker than the absorption of the oxide matrix. Two examples of rare-earth ions that fall into this category are Eu²⁺ and Ce³⁺. These ions can, for example, be found as constituents of the following inorganic nanoparticles: LaPO₄ or YAG in the case of Ce³⁺, and Sr₂O₄ in the case of Eu²⁺.

[0113] The photoluminescent nanoparticles implemented according to the invention can have an average size greater than or equal to 5 nm and strictly less than 1 µm, in particular between 10 nm and 500 nm, preferably between 20 nm and 200 nm and in particular between 20 nm and 100 nm.

[0114] The photoluminescent nanoparticles implemented according to the invention thus have a sufficient volume to contain a large number of rare earth ions, and therefore emit a sufficient luminescent signal to allow the detection of low concentrations of analyte.

[0115] Preferably, the nanoparticles implemented according to the invention comprise at least 10 3< rare earth ions, in particular between 1000 and 6,000,000 rare earth ions, in particular between 5,000 and 500,000 and more particularly between 20,000 and 100,000 rare earth ions.

[0116] As an example, a spherical nanoparticle Y 0.6 Eu 0.4 VO 4 with a diameter of 30 nm contains 70,000 Eu 3+ ions (calculation of the number of ions according to the Casanova reference). et al.

[37] ) .

[0117] The average size can be measured by transmission electron microscopy. Transmission electron microscopy images allow the shape of nanoparticles (spherical, ellipsoidal) to be determined, and their average dimensions to be deduced. In the case of generally spherical particles, the average size refers to the average diameter of the particles. In the case of ellipsoidal particles, the average size refers to the average size of a sphere with the same volume as the ellipsoid. It is generally assumed that the third axis of the ellipsoid, not visible in transmission images which are 2D projections, has a length equal to the axis of smallest size.

[0118] According to a particular embodiment, the nanoparticles are of an overall elongated ellipsoidal shape (“prolate” in English).

[0119] In particular, they can exhibit a major axis length, denoted a,between 20 and 60 nm; and a minor axis length, denoted b, between 10 and 30 nm. In particular, nanoparticles can have an average major axis length, a, of 40 nm and an average minor axis length, b, of 20 nm.

[0120] Advantageously, the nanoparticles implemented according to the invention exhibit low polydispersity. Preferably, the polydispersity index, which can be deduced from dynamic light scattering measurements, should be strictly less than 0.2. When this is not the case after the synthesis or functionalization of the particles, lower polydispersity can be obtained by size sorting by centrifugation or by any other technique known to those skilled in the art.

[0121] According to a particular embodiment, the product between the doping rate in rare earth ions, for example in europium (Eu), and the quantum yield of the emission by the nanoparticle is maximized.

[0122] This maximization of the product between the Ln ion doping level (x) and the quantum yield can be achieved using high Ln ion doping, for example between 0.2 and 0.6, and particularly 0.4, without decreasing the quantum yield. This is achieved, in particular, by limiting the transfer processes between doping ions that lead to concentration extinction. Specifically, to maintain a high quantum yield, the nanoparticle exhibits imperfect crystallinity. Indeed, excellent crystallinity promotes transfer processes between doping ions, especially when they are in close proximity, as is the case with high doping levels, and consequently favors ion de-excitation processes via non-radiative processes related to the surface and the presence of the solvent.In particular, a synthesis process at room temperature, or at least at a temperature not exceeding 600°C, is favorable for the imperfect crystallinity required for these nanoparticles.

[0123] The crystallinity of nanoparticles is considered "imperfect" when the coherence length, determined by the X-ray diffractogram in at least one given crystallographic direction, is less than 80% of the particle size in that direction as measured from transmission electron microscopy images. Different types of imperfect crystallinity can be considered: polycrystalline structure, defects, porosity, etc.

[0124] Advantageously, the nanoparticles implemented according to the invention are capable of emitting more than 10⁸ photons each before emission ceases, in particular more than 10⁹, or even more than 10¹⁰ photons. In many cases, particularly with Eu-doped YVO₄ or GdVO₄ particles, no cessation (extinction) of emission is observed under continuous illumination. In other words, advantageously, the nanoparticles according to the invention do not exhibit irreversible photodegradation or photobleaching.

[0125] Advantageously, the nanoparticles implemented according to the invention exhibit good colloidal stability in solution.

[0126] The stability of nanoparticles in solution is particularly critical to meet the requirements for reproducibility of detection results from the use of these particles as probes in a capillary diffusion test device.

[0127] In particular, good colloidal stability of the nanoparticles ensures, during the migration of the liquid sample in the porous support of the capillary diffusion test, the migration of the luminescent nanoparticles, where applicable, linked to the substance to be analyzed, to the detection zone and, possibly, to the control zone of the device.

[0128] The "zeta potential" is one of the key indicators of a suspension's stability. It can be directly measured, for example, using equipment such as the Zetasizer Nano ZS from Malvern. This instrument uses optical devices to measure the velocity of particles as a function of the applied electric field.

[0129] In particular, the nanoparticles implemented according to the invention advantageously exhibit, at the end of their synthesis, a zeta potential, denoted ζ, less than or equal to -28 mV, in aqueous medium at pH ≥ 5. In particular, the nanoparticles exhibit a zeta potential ζ, in aqueous medium at pH ≥ 6.5, in particular at pH ≥ 7, and especially at pH ≥ 8, less than or equal to - 30 mV.

[0130] The "zeta potential," denoted ζ, can be defined as the potential difference between the core of the solution and the shear plane of the particle. It is representative of the stability of a suspension. The shear plane (or hydrodynamic radius) corresponds to an imaginary sphere around the particle within which the solvent moves with the particle as the particles move through the solution. The zeta potential can be determined by methods known to those skilled in the art, for example, by moving the particle and its solubilization layer in an electric field.

[0131] This negative zeta potential of the nanoparticles, less than or equal to -28 mV at pH ≥ 5, increases the electrostatic repulsion between nanoparticles in aqueous solution, thereby suppressing flocculation. Indeed, it is empirically known to those skilled in the art that a high absolute zeta potential, particularly above 28 mV, generally eliminates flocculation effects in media with low ionic strength.

[0132] It is understood that the zeta potential measurements are carried out after purification of the aqueous suspension of particles, and therefore for an aqueous suspension with an ionic conductivity strictly less than 100 µS.cm⁻¹. The ionic conductivity of the suspension, allowing an assessment of the ion concentration in said suspension, can be measured at room temperature (25°C) by any known conductivity meter.

[0133] According to a particular embodiment, the luminescent nanoparticles implemented according to the invention may have one or more surface molecules, allowing them to be kept in suspension, thanks to a high zeta potential.

[0134] According to a particular embodiment, the nanoparticles used according to the invention may have tetraalkylammonium cations on their surface. Such nanoparticles, and their synthesis process, are described, for example, in application no. FR1754416.

[0135] As an example, the photoluminescent nanoparticles implemented according to the invention may have the formula Y 0.6 Eu 0.4 VO 4 on the surface of which tetramethylammonium cations are optionally immobilized.

[0136] The nanoparticles implemented according to the invention, of the aforementioned formula (II), are predominantly crystalline and polycrystalline in nature, in particular with an average crystallite size, deduced by X-ray diffraction, of between 3 and 40 nm. Preparation of nanoparticles

[0137] The rare earth ion-doped crystalline matrix photoluminescent nanoparticles implemented according to the invention can be prepared by any conventional method known to those skilled in the art.

[0138] In particular, they can be prepared by colloidal synthesis. Aqueous colloidal synthesis methods are well known to those skilled in the art (Bouzigues et al., ACS Nano 5, 8488-8505 (2011)

[49] ). These aqueous syntheses have the advantage of eliminating any subsequent solvent transfer step.

[0139] As an example, nanoparticles of formula A 1-x Ln x VO 4(1-y) (PO 4 ) y (II) can be formed by co-precipitation reaction, in aqueous medium, from precursors of said elements A and Ln, and from precursors of orthovanadate ions (VO 4 3-< ) and possibly of phosphate ions (PO 4 3-< ).

[0140] The precursors of elements A and Ln can typically be in the form of salts of these elements, for example nitrates, chlorides, perchlorates, or acetates, particularly nitrates. The precursors of elements A and Ln, and their quantity, are of course chosen appropriately with regard to the nature of the desired nanoparticle.

[0141] For example, the synthesis of nanoparticles of formula Y 1-x Eu x VO 4 (IV) can implement, as precursor compounds of yttrium and europium, yttrium nitrate (Y(NO 3 ) 3 ) and europium nitrate (Eu(NO 3 ) 4 ).

[0142] Such a colloidal synthesis process for photoluminescent nanoparticles implemented according to the invention is, for example, described in application no. FR1754416. Advantageously, as described in application no. FR1754416, the co-precipitation reaction can be carried out in the presence of an effective amount of tetraalkylammonium cations.

[0143] The synthesis of luminescent nanoparticles implemented according to the invention, in particular of larger sizes, greater than a few tens of nanometers, can be carried out by any other approach known to a person skilled in the art, for example by grinding. SURFACE FUNCTIONALIZATION OF LUMINOUS NANOPARTICLES Coupling of nanoparticles with a binding reagent

[0144] Like the probes classically used in lateral flow test devices, the luminescent nanoparticles used according to the invention are coupled to at least one binding reagent specific to the substance to be analyzed.

[0145] The function, and consequently the nature, of the binding reagent coupled to the luminescent probes varies according to the nature of the capillary diffusion test, in particular the lateral flow test implemented, as detailed later in the text, especially depending on whether it is a so-called "sandwich" test or a "competitive" test.

[0146] Thus, a "binding reagent" is defined as any chemical, biochemical, or biological compound capable of specifically binding to the biological or chemical substance of interest being sought in a "sandwich" test, or to the capture reagent in the detection zone in competition with the biological or chemical substance being sought in a "competitive" test. As detailed later in the text, the binding reagent is also capable of specifically binding to the second capture reagent immobilized in the control zone of the lateral flow test device.

[0147] By "to bind" or "bonding" we mean any strong bond, for example covalent, or, preferably, a set of weak bonds, for example of the antigen / antibody type.

[0148] The nature of the binding reagent coupled to the luminescent nanoparticles used as probes according to the invention is of course chosen with regard to the substance to be analyzed in the sample.

[0149] Advantageously, the photoluminescent nanoparticles implemented according to the invention are perfectly suited to a wide variety of biological targets, the specificities being dependent on the nature of the binding reagent(s) grafted onto the surface of the nanoparticle.

[0150] The binding reagent can be specifically chosen from among a polyclonal or monoclonal antibody, an antibody fragment, a nanobody, an antigen, an oligonucleid, a peptide, a hormone, a ligand, a cytokine, a peptidomimetic, a protein, a carbohydrate, a chemically modified protein, a chemically modified nucleic acid, a chemically modified carbohydrate that targets a known cell surface protein, an aptamer, a protein and DNA / RNA assembly, or a chloroalkane used by HaloTag-type tags. A SNAP-Tag or CLIP-Tag approach can also be used.

[0151] According to a particular embodiment, it is an antibody or fragment of an antibody, a peptide, a chemically modified nucleic acid or an aptamer, in particular an antibody.

[0152] Suitable antibody fragments include at least one variable domain of an immunoglobulin, such as single variable domains Fv, scFv, Fab, (Fab') 2< and other proteolytic fragments or "nanobody" (single-domain antibodies such as VHH fragments obtained from camelid antibodies or V NAR obtained from cartilaginous fish antibodies).

[0153] The term "antibody" according to the invention includes chimeric antibodies, human or humanized antibodies, recombinant and modified antibodies, conjugated antibodies, and their fragments.

[0154] The binding reagent can also be derived from a molecule known to bind a cell surface receptor. For example, the targeting fragment can be derived from low-density lipoproteins, transferrin, EGF, insulin, PDGF, fibrinolytic enzymes, anti-HER2, anti-HER3, anti-HER4, annexins, interleukins, interferons, erythropoietins, or colony-stimulating factors. Coupling of the particle with the binding reagent

[0155] It is the responsibility of a person skilled in the art to implement appropriate coupling / grafting methods to adequately prepare the particles coupled to one or more binding reagents. The quantity of binding reagent(s) used for surface functionalization of the luminescent nanoparticles is adjusted according to the quantity of particles.

[0156] Typically, it is desirable for each nanoparticle to be coupled to several binding reagents, preferably at least five binding reagents, and more preferably at least ten binding reagents.

[0157] The binding reagent can be grafted directly, or via a spacer (also referred to as "linker" or "spacer"), to the nanoparticle.

[0158] The methods of coupling (also called grafting) particles to biomolecules are well known to those skilled in the art. These methods generally involve coupling by covalent bonding, surface complexation, electrostatic interactions, encapsulation, or adsorption.

[0159] In some cases, including the case of coupling by covalent bond, the particles can be pre-functionalized by chemical groups capable of then reacting with another chemical group carried by the bonding reagent to form a covalent bond.

[0160] Examples of chemical groups that may be present on the surface of nanoparticles include carboxyl, amino, thiol, aldehyde and epoxy groups.

[0161] Amino groups can be provided by molecules such as amino organosilanes, like aminotriethoxysilane (APTES). The advantage of APTES lies in the fact that it forms via Covalent bonds form a capsule around the nanoparticle. The amines provided by APTES are thus very stable over time. The amino groups can be transformed into carboxyl groups by reaction with succinic anhydride.

[0162] Carboxyl groups can be provided by molecules such as citric acid or polyacrylic acid (PAA).

[0163] Carboxyl groups can be activated by any technique known to those skilled in the art, in particular by reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), to then react with amine functions on the surface of a polypeptide and form a covalent amide bond, when the linking reagent is a protein or antibody.

[0164] The functionalization of nanoparticles by APTES can be advantageously achieved following the coating of the nanoparticles by a layer of silica.

[0165] In other cases, the particles may be pre-coupled to molecules capable of enabling subsequent coupling with a binding reagent.

[0166] For example, the particles can be coupled to streptavidin suitable for coupling with a biotinylated targeting agent.

[0167] As an example, application no. FR1754416 illustrates the coupling of nanoparticles with biotinylated antibodies, by coupling streptavidin-coupled nanoparticles with biotinylated antibodies. This can also be achieved directly by coupling antibodies to nanoparticles functionalized with APTES as described above (transformation of amino groups into carboxyl groups, activation of the carboxyl groups, and direct reaction with the amino groups on the surface of the antibodies).

[0168] The coupling of the binding reagent to the surface of the nanoparticles can also be done by any other method known to those skilled in the art.

[0169] It can also be advantageously achieved by coating the nanoparticles with a silica layer, followed by a coating reaction using APTES, whose amine groups react with a bifunctional spacer agent containing two NHS groups. Subsequently, the nanoparticles coupled to the bifunctional spacers can react with the amine groups on the surface of a protein (antibody, streptavidin, etc.). This type of coupling process is notably described in the Casanova publications. et al. (

[38] ) and Giaume et al. (

[39] ). Migration agent

[0170] Depending on the charge and nature of their surface, their shape and size, the luminescent nanoparticles implemented according to the invention can also be coated by an agent, referred to in the following text as a "migration agent", which facilitates their migration within the capillary diffusion test device, for example within the nitrocellulose membrane.

[0171] A person skilled in the art is able to appropriately functionalize the nanoparticles with one or more migration agents. In particular, it is understood that this migration agent must not disrupt the coupling of the nanoparticles with the binding reagent as described previously, and especially the ability of the latter, during the capillary diffusion test, to bind specifically to the analyte or to the capture reagent competing with the analyte.

[0172] Migration agents can be chosen from among stealth or passivation agents.

[0173] Such agents may be, for example, polyethylene glycol (PEG) or poly(ethylene oxide) (PEO) chains, particularly silanized; PEO-poly(propylene oxide)-PEO; poly(ethylene oxide) chains grafted with poly(L-lysine) chains ("poly(L-lysine)-grafted-Poly(Ethylene Glycol)" (PLL-g-PEG)); dextran chains grafted with poly(L-lysine); poly(p-xylylene) (parylene); poloxamers (triblock copolymers whose central part is a propylene oxide block and the ends are polyethylene oxide blocks, for example those marketed by BASF under the name Pluronic®); poloxamines; polysorbates and polysaccharides (e.g. chitosan, dextran, hyaluronic acid and heparin), poly(D,L-lactide-co-glycolide) (PLGA), polylactic acids (PLA), polyglutamic acids (PGA), poly(caprolactone) (PCL), N-(2-hydroxypropyl)-methacrylate copolymers (HPMA) and polyamino acids;anionic surfactants; cationic surfactants; non-ionic surfactants and zwitterionic detergents.;

[0174] Preferably, the migration agents are chosen from silanized PEG chains, poloxamers, and polylactic acids (PLA). These agents can be deposited on the surface of the nanoparticles by any approach known to those skilled in the art. For example, they can be adsorbed or covalently attached.

[0175] The coupling of nanoparticles with one or more migrating agents can be performed simultaneously with that of the binding reagent(s), for example, by choosing a migrating agent bearing an amino group when the coupling of the binding reagent to the nanoparticles occurs via a reaction with the amino groups of the binding reagent. In this case, the quantity of migrating agents must be adjusted relative to the quantity of binding reagents so that the nanoparticle has a sufficient number of both migrating agents and binding reagents on its surface. CAPILLARY DIFFUSION TESTING DEVICE

[0176] As previously stated, the photoluminescent nanoparticles as defined above are implemented according to the invention as probes in a capillary diffusion test, as defined in the claims, for example in a "lateral flow" test.

[0177] The term "lateral flow" refers to a liquid flow in which all dissolved or dispersed compounds are transported by capillarity, preferably at equivalent velocities and a regular flow rate, laterally through a diffusion medium.

[0178] The method of the invention can be implemented with any conventional capillary diffusion test device, for example, one known for gold nanoparticle probes. The capillary diffusion test device can, in particular, adopt any configuration; it can thus have a linear, radial, T-shaped, L-shaped, cross-shaped, etc. configuration.

[0179] The rest of the text refers more specifically to figures 1 to 3 and 5 attached, which relate to a lateral flow test device of the migration strip type.

[0180] Also, the device implemented according to the invention can be adapted to a "sandwich" type test or, alternatively, to a "competition" test, as detailed later in the text.

[0181] Typically, a capillary diffusion test device, in particular a lateral flow test device according to the invention, as shown in Figure 1 , includes a capillary diffusion means in a reference direction (X), in particular a porous solid support (10), comprising: - a zone (1) for depositing the liquid sample, and optionally a diluent; - a zone (2), located downstream of the deposit zone, called the "labeling zone", loaded with the photoluminescent inorganic nanoparticles according to the invention (probes) coupled with at least one binding reagent specific to the substance to be analyzed; - a reaction zone (3), also called the "detection zone", located downstream of the labeling zone (2), in which at least one capture reagent specific to the substance to be analyzed is immobilized; and - a control zone (4), located downstream of the detection zone (3), in which at least one second capture reagent specific to the binding reagent specific to the analyte is immobilized.

[0182] In a "sandwich" assay, the analyte-specific capture reagent from the detection zone and the probe-coupled binding reagent are chosen to bind respectively and specifically with the analyte, e.g., at two different epitope sites of the analyte.

[0183] In a "competitive" assay, the probe-coupled binding reagent is identical or analogous to the analyte, to bind with the detection zone capture reagent, in competition with the analyte.

[0184] The migration control zone (4) indicates to the user that at least part of the sample has passed through the porous solid support of the test device.

[0185] The lateral flow test setup generally also includes an absorbent pad (5) (or "Absorbent Pad" in Anglo-Saxon terminology), positioned downstream of the reaction zone and the control zone, with one end in fluidic contact with the porous support. The absorbent pad maintains capillary migration and collects excess liquid sample.

[0186] The terms "upstream" and "downstream" refer to the direction (X) of capillary diffusion in the test, this migration taking place from the deposition zone (1) (at the upstream functional end) to the detection zone (3), and ending at the absorbent pad (5) (at the downstream functional end) when the latter is present.

[0187] Each of the different zones of the porous solid support of the lateral flow test device is in fluidic communication with the adjacent zone(s).

[0188] By "fluidic contact" between two elements, we mean, as is customary for capillary diffusion test devices, that the two elements are in physical contact, allowing the migration of a liquid from the first element into the second. Preferably, this contact is achieved by one element overlapping the other, as schematically represented in Figure 1 .

[0189] The term "capillary diffusion medium" refers more specifically to a porous solid support (10) that allows the migration of a liquid by simple capillary diffusion. The porosity of this support enables the capillary diffusion (or lateral migration) of the sample and / or reagents in their liquid or wet state. The porous support can be chosen from those already used in known lateral flow test devices. Examples include nitrocellulose, polyester, glass fibers, cellulose fibers, polysulfone ether (PES), cellulose ester, PVDF, etc.

[0190] The capillary diffusion medium may consist of one or more distinct parts, the different parts of the support being made of different materials. When the capillary diffusion medium consists of different parts or different materials, these elements are arranged so as to allow the continuity of capillary flow within the capillary diffusion medium.

[0191] Typically, the capillary diffusion medium consists of a porous solid support elongated along the (X) direction of capillary diffusion.

[0192] Advantageously, it is a porous support (10) in the form of a strip or band. In particular, it can be an immunochromatographic strip made up of several superimposed or overlapping membranes.

[0193] According to a particular embodiment, the porous support is a nitrocellulose membrane. Examples of nitrocellulose membranes include Millipore™< HF240, Millipore™< HF180, Millipore™< HF135, Millipore™< HF120, Millipore™< HF090, Millipore™< HF075, Sartorius™< CN140, Sartorius™< CN150, FF120 HP membranes (GE), FF80HP membranes (GE), AE membranes (GE), and Immunopore membranes (GE).

[0194] The size of the porous solid support in a lateral flow test device can vary. For example, it can be a strip 30 to 200 mm long, preferably 60 to 100 mm, and 2 to 10 mm wide, preferably 4 to 5 mm.

[0195] The lateral flow test device according to the invention can for example consist of a chromatographic strip fixed on a rigid support (6).

[0196] The rigid support (6) can be made of various materials such as cardboard, laminated cardboard, or more preferably plastics. Preferably, the rigid support is made of polystyrene.

[0197] Advantageously, a specific material corresponds to each zone of the capillary diffusion medium.

[0198] The sample deposition zone (1) (also known as the "Sample Pad" in Anglo-Saxon terminology) can advantageously be made of a porous absorbent material. Indeed, the deposition zone of the capillary diffusion medium is intended to receive a liquid sample, for example, to be brought into contact with a urine stream or a blood sample. This material is chosen from among suitable absorbents known to those skilled in the art and already used in conventional lateral flow tests.

[0199] The inorganic photoluminescent nanoparticles (7) as described above are implemented at the level of the labeling zone (2) (also known as the "Conjugate Pad" in Anglo-Saxon terminology) of the capillary diffusion medium, as shown in figures 2 And 3 .

[0200] As previously stated, these nanoparticles (7) are coupled with at least one binding reagent specific to the substance to be analyzed.

[0201] In a "sandwich" assay, the binding reagent is likely to bind specifically to the analyte during the lateral flow test. This could be, for example, an antibody specific to the analyte.

[0202] In a conventional competitive assay, the binding reagent is designed to bind specifically to the capture reagent in the detection zone (3), competing with the analyte. The binding reagent can be, for example, the analyte itself or a suitable analog. A suitable analog is defined as one that binds specifically to the analyte's specific capture reagent.

[0203] The binding reagent, coupled to the luminescent probes, is also capable of binding specifically with the second capture reagent (9) immobilized in the control zone.

[0204] Preferably, as previously mentioned, the luminescent nanoparticles also have on their surface at least one agent designed to facilitate their migration within the lateral flow test device, such as a stealth or passivation agent, for example polyethylene glycol. These agents will thus facilitate the migration of the nanoparticles, where applicable, bound via the analyte-binding reagent, within the porous support, for example within the nitrocellulose membrane, to the detection zone (3).

[0205] Inorganic photoluminescent nanoparticles coupled to at least one analyte-specific binding reagent are immobilized in the dry state in the capillary diffusion medium, but are free to migrate by capillary diffusion in the wet state.

[0206] Thus, during the test, the sample which migrates by capillary diffusion through the capillary diffusion medium carries the nanoparticles coupled with the specific binding reagent of the substance to be analyzed.

[0207] A first capture reagent, specific to the substance to be analyzed, is immobilized at the detection zone (3) (also known as the "Detection Pad" in Anglo-Saxon terminology) of the capillary diffusion means of the lateral flow test device according to the invention. It is appropriately chosen for its ability to bind specifically with the analyte.

[0208] As part of a "sandwich" test ( Figure 2 ), the capture reagent for the detection zone can be of the same nature as the binding reagents as described previously for coupling to photoluminescent nanoparticles. It can be, for example, an antibody (8) having a high affinity for the substance to be analyzed.

[0209] In a "competitive" test, the capture reagent is also able to bind to the binding reagent coupled to the luminescent probes (e.g. identical or analogous to the analyte).

[0210] The analyte (11) and the capture reagent (8) typically form a ligand / receptor, antigen / antibody, DNA / RNA, DNA / DNA or DNA / protein pair.

[0211] Thus, if the analyte is an antigen or a hapten, the capture reagent is, for example, an antibody specific to the analyte; or, if the analyte is an antibody, the capture reagent is the antigen recognized by the antibody or an antibody that specifically recognizes the analyte. If the analyte is a nucleic acid, the capture reagent is, for example, a complementary DNA probe.

[0212] The capture reagents are deposited and immobilized at the detection zone, so that they are not mobile in the wet state. This immobilization can be achieved using techniques known to those skilled in the art, for example by electrostatic interactions in the case of nitrocellulose or charge-modified nylon membranes, or by hydrophobic interactions in the case of poly(vinylidene fluoride) (PVDF) or polyethersulfone (PES) membranes.

[0213] In one particular embodiment, the detection zone (3) may comprise one or more spatially separated regions on the capillary diffusion medium, for example in the form of bands (one or more test line(s) "T"), functionalized with one or more capture reagents. The use of several "test lines" is particularly advantageous when using the test for multiplexed detection, i.e., for the simultaneous detection of several substances in the same sample.

[0214] The capillary diffusion test device typically includes a control zone (4), located downstream of the detection zone (3), used to confirm the validity of the test, in which at least one second capture agent (9), specific to the binding reagent coupled to the probes, is immobilized.

[0215] This second capture agent is appropriately chosen for its ability to bind specifically to the probe-conjugated binding reagent. For example, it could be a secondary antibody or an antibody-specific antigen used as the analyte-specific binding reagent in a "sandwich" assay.

[0216] As with the first capture reagent in the detection zone (3), this second capture reagent is immobilized in the control zone (4) in such a way that it is not mobile in the wet state.

[0217] The capillary diffusion means can optionally be fixed to a solid support (6) such as a plate or cassette, usually made of plastic.

[0218] A capillary diffusion test device according to the invention may, in particular, include a housing (12) in which the test strip is placed, said housing preferably being closed, except at certain openings provided, as schematically represented in Figure 8 In particular, an opening (14) is provided above the sample deposition area. Another opening, constituting the reading window (13), can, for example, be provided at the level of the detection zone (3) and the possible control zone (4). Alternatively, two windows can be provided, for observing the detection zone and the control zone respectively.

[0219] Alternatively, to make these areas visible, the case can be transparent or have one or more transparent parts.

[0220] Of course, various device configurations, known for conventional capillary diffusion test devices, can be implemented. For example, the housing containing the test strip can have at least one recessed area on its upper surface, the base of which rests on the surface of the strip, forming a well or volume for the liquid sample.

[0221] The capillary diffusion test procedure according to the method of the invention includes, in particular: (i) application of the liquid sample to be analyzed, and possibly a diluent, to the deposition zone (1) of the capillary diffusion test device; (ii) incubation of the device until detection in the reaction zone (3) of the luminescence generated by the photoluminescent nanoparticles and / or until detection of the luminescence in the migration control zone (4); and (iii) reading and interpretation of the results.

[0222] The liquid sample to be analyzed can be deposited directly onto the deposition area (1) of the capillary diffusion medium of the device.

[0223] A "liquid sample" is defined as any sample in which the substance to be analyzed is in solution or suspension. This liquid sample may include any biological or bodily fluid. The liquid sample may also have been obtained from a biological or bodily fluid. It may also be a liquid extract of a solid sample.

[0224] Typically, the liquid sample is urine, whole blood, plasma, serum, or diluted fecal matter.

[0225] In one particular embodiment, a diluent is used with the sample to be analyzed, especially when the liquid sample is plasma, serum, whole blood, nasal or vaginal smear, or sputum, for example. The diluent is deposited in the device's application zone.

[0226] It can be mixed with the sample to be analyzed prior to sample application. Alternatively, the diluent can be applied before or after the sample. This diluent migrates through the porous support, carrying with it the sample and the probes coupled to the binding reagent. Typically, this diluent consists of a buffered saline solution. It may also include a detergent or any other component necessary for the reaction.

[0227] The capillary diffusion test device is then incubated for a sufficient time for the capillary diffusion migration of the liquid sample from the deposition area to the control area.

[0228] More specifically, the procedure of a "sandwich" type lateral flow test, schematically represented in figure 2 , is the following.

[0229] When the porous support is brought into contact with the liquid sample containing the analyte (11), the latter migrates by capillary diffusion through this support to the labeling zone where the analyte-specific binding reagent coupled to the probes (7) is located. The analyte (11) thus binds to the luminescent probes (7) via the binding reagent.

[0230] If the substance to be analyzed is present, it will then be immobilized in the detection zone (3) of the capillary diffusion test device by the first capture reagent (8) attached to this zone. This will therefore result in the immobilization of the luminescent probes in the detection zone (3).

[0231] The presence or absence of the substance to be analyzed in the sample is thus measured by detecting luminescent probes at the detection zone (3). More specifically, the luminescence detected at the detection zone increases with, and is particularly proportional to, the concentration of the analyte in the sample.

[0232] Excess probes, that is, nanoparticles coupled with a binding reagent that did not react with the analyte, migrate to the control zone. In this control zone, the binding reagent binds to the second capture agent (9), causing the excess probes to become immobilized in the control zone (4). The user thus has a positive control to verify the migration of the sample and reagents within the device, and therefore to confirm the proper functioning of the test.

[0233] According to another variant of the method of the invention, the test implemented is of the "competition test" type. In a competition test, as schematically illustrated in Figure 3-a If the analyte is absent from the sample, the luminescent probes will be immobilized in the detection zone by the binding of their binding reagent to the capture reagent of the detection zone. Conversely, if the analyte is present, it will bind, in competition with the binding reagent of the luminescent probes, to the capture reagent of the detection zone.

[0234] Thus, in the context of a competitive test, the luminescence detected at the detection zone decreases with, in particular is inversely proportional to, the concentration of the analyte in the sample.

[0235] According to yet another variant of a "competition-based" type test, as represented in Figure 3-b, the analyte is already immobilized at the capture sites of the detection zone, while the binding reagent coupled to the luminescent nanoparticles of the labeling zone is able to bind specifically with the analyte, like a "sandwich" test.

[0236] If the sample contains the analyte, it binds to the luminescent nanoparticles, similar to a sandwich test. via the binding reagent, and therefore cannot bind to the detection zone. Conversely, probes coupled to the binding reagent that has not reacted with the analyte can bind to the detection zone via The analyte is immobilized at the level of the capture reagents in the detection zone. Here again, the luminescence signal detected at the detection zone will decrease with, in particular, the concentration of the analyte in the sample.

[0237] The reading of the results, according to one or the other of the aforementioned variants of capillary diffusion test, is therefore done by detection of the luminescence generated by the immobilized nanoparticles, at the end of the test, at the level of the capillary diffusion test device, in particular immobilized, at the end of the migration of the sample at the level of the detection zone (3) and, possibly, at the level of the control zone (4).

[0238] Of course, the invention is by no means limited to the implementation of a capillary diffusion test device, as schematically shown in the attached figures.

[0239] Other variations of capillary diffusion test devices for implementing the method according to the invention may be considered, provided they are suitable for using the photoluminescent nanoparticles implemented according to the invention as detection probes. For example, these may be capillary diffusion test devices of the "Dipstick Lateral Flow" or "Vertical Lateral Flow" type, etc.

[0240] According to one embodiment, it is possible to detect several substances in the sample with a single test (so-called "multiplexed" detection).

[0241] For example, in a "sandwich" assay, it is possible to immobilize, within the reaction zone (3), several capture reagents specific to each of the substances to be analyzed, in distinct regions (e.g., several test lines "T"). In this case, the nanoparticles used as detection probes can be coupled to two or more types of capture reagents specific to each of the substances to be analyzed. In the presence of the different analytes, the probes will bind to each of the distinct regions containing the capture reagents specific to each analyte. The presence and value of the luminescence signal in each of the reaction zones will correspond to the presence and concentration of the corresponding analyte. This is a case of spatial multiplexing.

[0242] Alternatively, it is also possible to implement, in the labeling zone (2), different probes doped with different lanthanide ions emitting at different wavelengths, each probe being coupled with a binding reagent specific to each of the substances to be analyzed, and, in a single reaction zone (3), several capture reagents specific to each of the substances to be analyzed. This is a multiplexing method using several emission colors. In this case, UV excitation of the crystalline matrix is ​​particularly advantageous because it allows different lanthanide ions, which emit at different wavelengths, to be excited with the same excitation wavelength.

[0243] The two approaches, spatial multiplexing and multi-color emission multiplexing, can be combined to perform, for example, the detection of four analytes with two probes emitting two different colors, each coupled to two types of reagents specific to two of the four substances to be analyzed, and two reaction zones, each containing binding reagents specific to two of the four substances to be analyzed. Thus, the signal at the two different colors on the first reaction zone will indicate the presence and concentration of the first two analytes; the signal at the two different colors on the second reaction zone will indicate the presence and concentration of the other two analytes. LIGHT DETECTION

[0244] Similar to conventional capillary diffusion tests, the evaluation of the test (detection and / or quantification) implemented according to the method of the invention is carried out by observation of the detection zone and, possibly, the control zone.

[0245] More specifically, the test results are read by detecting the luminescence generated by the probes immobilized at the level of the detection zone and / or the control zone, preferably at the level of the detection zone and the control zone. Detection device

[0246] The observation of the detection and control zones of the capillary diffusion test device according to the invention more particularly implements a step (i) of excitation of the photoluminescent nanoparticles and a step (ii) of detection of the emission of luminescence.

[0247] According to another aspect, the invention relates to a diagnostic set in vitroincluding at least: - a capillary diffusion test device according to the invention as defined above; and - a device for detecting the luminescence generated by the probes immobilized at the level of the detection zone and, optionally, the control zone of the device.

[0248] A simple detection setup, including an illumination device with an excitation source, makes it possible to observe the presence of luminescent probes.

[0249] The excitation must be compatible with the absorbance characteristics of the nanoparticles. Excitation can be carried out in the UV, visible, or near-infrared range.

[0250] It can be operated using a non-coherent excitation source such as a lamp, a light-emitting diode, or a laser.

[0251] The excitation source can directly excite the rare earth ions and / or the nanoparticle matrix in which the rare earth ions are embedded. Preferably, the rare earth ions are excited by exciting the matrix (e.g., AVO₄, or another metal oxide matrix) of the photoluminescent nanoparticles immobilized in the detection zone and, optionally, the control zone, followed by energy transfer to the rare earth ions within said nanoparticles. In the vast majority of cases, matrix excitation is performed in the UV range.

[0252] In particular, for nanoparticles of formula A 1-x Ln x VO 4(1-y) (PO 4 ) y (II) as described above, especially where y is 0, luminescence detection can be performed by exciting the matrix at a wavelength strictly less than 350 nm, particularly less than or equal to 320 nm, and more particularly less than or equal to 300 nm. In the context of the invention, the matrix is ​​excited at a wavelength less than or equal to 320 nm.

[0253] In the case of the AVO₄ matrix, and in particular the YVO₄ matrix of nanoparticles with the formula Y₁₋ₓEuₓVO₄, (IV), excitation can be performed at wavelengths between 230 and 320 nm, especially between 250 and 310 nm, and more specifically between 265 and 295 nm. Excitation of the nanoparticle matrix is ​​particularly advantageous since the matrix absorption coefficient (or the extinction coefficient for nanoparticles in solution) is much higher than that corresponding to the direct excitation of luminescent ions. Moreover, contrary to expectations, the background noise generated by UV excitation does not prevent the detection of the signal from the nanoparticle emission, even when the analyte is present in low concentrations, as illustrated in the following examples.

[0254] In the case of oxide matrices containing Eu, excitation can be carried out at a wavelength between 210 and 310 nm, in particular between 230 and 290 nm and more particularly between 245 and 275 nm.

[0255] In this case, excitation can be achieved using a UV lamp, a UV light-emitting diode (LED), or a UV laser. The excitation power and intensity required for probe detection can be easily obtained with a UV lamp or a UV LED. Preferably, excitation is achieved using an LED, as this results in minimal energy loss and therefore minimal heat dissipation.

[0256] Also advantageously, excitation is achieved homogeneously across the surface of the strip, particularly in the detection and control zones. This homogeneity can be achieved with a UV lamp but also with several lower-power LEDs arranged around the detection and control zones. For example, as shown in Figure 7 Four groups of four LEDs can be used. The diagram of the Figure 7 indicates one of the possible schemes for arranging several LEDs around the detection and control zones of the strip.

[0257] The excitation power density can be between 0.5 and 20 mW / cm², in particular between 1 and 10 mW / cm².

[0258] In the context of a commercial application of a capillary diffusion test according to the invention, assuming that the heat generated during excitation can be efficiently dissipated, a factor of merit can be defined, taking into account the ratio between the detection sensitivity obtained with a given excitation power and the cost of the excitation source required to achieve that excitation power. Advantageously, the diagnostic system according to the invention allows for the optimization of this factor of merit.

[0259] According to a particularly advantageous embodiment, the reading of the results, in particular in the context of a qualitative characterization of the analyte, can be carried out by direct observation with the naked eye of the capillary diffusion test device, in particular of the detection zone and, possibly, of the control zone, in particular by implementing an emission filter.

[0260] The emission filter allows selection of the characteristic emission band of the luminescent ions, thus excluding non-specific signals. For example, in the case of Y 1-x Eu x VO 4 nanoparticles, the emitted light intensity can be detected at the luminescence wavelength of Eu 3+ in the YVO 4 matrix, namely 617 nm. The emission filter can be an interference filter or a high-pass filter.

[0261] Alternatively, the result can be read using a simple detection apparatus. This may include an emission filter and a detector.

[0262] The detector is a photon detector.

[0263] This can be a single detector, in particular of the photomultiplier, photodiode, avalanche photodiode type, or a detector of the array type of photosensitive devices consisting of a 2D surface of detection pixels such as a CCD or EM-CCD camera or a CMOS camera.

[0264] Preferably, this is a 2D detection device, such as a camera. This allows for obtaining a 2D image of the strip used for the lateral flow test.

[0265] For example, it could be the CCD or CMOS sensor of a smartphone.

[0266] Advantageously, the capillary diffusion test according to the invention offers a short signal acquisition time. In particular, luminescence measurement can be performed in a few seconds, especially in less than one second, and in particular in less than 100 ms. Preferably, the signal acquisition time should be compatible with the image acquisition time of a smartphone camera. Analysis of the results of the lateral flow test

[0267] The luminescence results can then be interpreted.

[0268] The analysis of the results of the lateral flow test may consist of the simple determination of the presence of the probes (qualitative measurement) at the level of the detection and / or control area, for example by simple visual observation with the naked eye or by visual reading of the 2D image of the strip, obtained for example with a CCD camera, for example from the photograph recorded by a smartphone.

[0269] It allows us to conclude whether or not the substance targeted by the test is present in the analyzed sample.

[0270] For example, in the context of a "sandwich" type lateral flow test, the qualitative interpretation of the results may be as follows: . if two bands are present: the test is positive; . if only the control band is present: the test is negative; . if only the test band is present: the test is invalid; . if no band is present: the test is invalid.

[0271] Advantageously, the detection method according to the invention makes it possible to measure qualitatively up to 10 times, in particular up to 100 times, or even up to 1000 times, below the detection limit of the same capillary diffusion test using gold nanoparticles as probes.

[0272] The analysis of the results of the capillary diffusion test may also include a quantitative characterization of the substance to be analyzed, in other words a determination of the concentration of said substance within the sample, by interpretation of the luminescence results.

[0273] The detection system implemented according to the invention may then further include any means of analyzing the emission of luminescence, for example a converter allowing the recording and exploitation of the luminescence signal.

[0274] The interpretation of the luminescence measurement can be carried out by reference to a pre-established standard or calibration.

[0275] As seen previously, in a sandwich test, the luminescence signal of the detection area increases, in particular is proportional, to the concentration of the analyte, whereas it may be inversely proportional in a competition test.

[0276] Quantification by reference to a calibration can, for example, be achieved using several control strips, called calibration strips, containing different concentrations of the substance to be analyzed.

[0277] The interpretation of the luminescence measurement can notably exploit the ratio between the luminescence signal of the detection zone and that of the control zone.

[0278] These means of interpreting luminescence can, for example, be combined within a smartphone application, allowing analysis of the image obtained, and the provision of a quantitative value for the result obtained.

[0279] Alternatively, the detection system implemented according to the invention may use a 2D detector, an image recording system and image analysis software.

[0280] Alternatively, the 2D detector can be integrated into the reader, and the recorded image can then be transferred to a smartphone or other system enabling image analysis.

[0281] An analysis of the results ( via Analytical software (for example), particularly for quantitative characterization of the analyte, might include determining the signal corresponding to the detection zone, the control zone, and the background signal. The luminescence value of the background signal is subtracted from the values ​​of the other two zones. Then, the ratio between the signal in the detection zone and the signal in the control zone is calculated.

[0282] More specifically, an analysis of the results ( viaan analysis software, for example, or advantageously via an application embedded on the detection device (smartphone or other), particularly for a quantitative characterization of the analyte, may, for example, include (i) the determination of the luminescence level in the detection zone, LD, the luminescence level in the control zone, LC, and the luminescence level in a marker-free zone LB (background signal) identified by the user, (ii) the calculation of the raw signals SD and SC in the form SD / C = (LD / C - LB), (iii) the implementation of an SD / C maximization algorithm allowing optimal localization of the detection and control zones, (iv) the calculation of the ratiometric signal R=SD / SC or R= SD / (SD +SC) and (v) the comparison of the value of R to a calibration table allowing the determination of the absolute concentration of analyte.Automated positioning of the detection zones (step iii) eliminates user-introduced bias and provides a reproducible quantitative measurement. Alternatively, the position of the detection and control bands can be selected fully automatically without user intervention. In this latter case, it is crucial that the positioning of the detection and control bands on the test strip and the positioning of the strip in the reader are always identical.

[0283] In a "sandwich" test, the analysis of the results preferably includes the calculation of the ratio R = SD / SD + SC. Indeed, in a "sandwich" test, the higher the analyte concentration, the stronger the SD signal and the weaker the SC signal (fewer probes remain available to migrate to the control zone).

[0284] All the excitation, luminescence detection and results analysis elements can be grouped within a housing, designated as the reader of the capillary diffusion test device, for example a strip reader.

[0285] An opening can, for example, be provided in the strip reader to insert one or more strips for the purpose of reading the test result.

[0286] An opening containing a USB connection or equivalent can also be provided so as to be able to transfer the recorded images to a data analysis device.

[0287] The method according to the invention advantageously allows the detection of a substance of interest in a sample at a concentration strictly below 5 ng / mL, in particular below 0.5 ng / mL, or even below 0.05 ng / mL. These performance levels depend, of course, on the analyte, as well as on the efficiency of the specific binding reagent used.

[0288] Advantageously, the detection method according to the invention makes it possible to measure quantitatively up to 10 times, in particular up to 100 times, or even up to 1000 times, below the limit of quantification of the same capillary diffusion test using gold nanoparticles as probes.

[0289] The examples and figures shown below are given solely for illustrative purposes and are not intended to limit the invention. FIGURES

[0290] Figure 1 : Schematic representation, in cross-sectional view, of a lateral flow test strip; Figure 2 : Schematic representation of a "sandwich" type test, before the application of a liquid sample to be analyzed including the analyte (11) at the level of the deposition zone (1) (top figure) and at the end of the test (bottom figure); Figure 3 : Schematic representation of the course of a "competition" test according to two variants, before the application of a liquid sample to be analyzed including the analyte (11) at the level of the deposition zone (1) (top figure) and at the end of the test (bottom figure); Figure 4 : Images obtained by transmission electron microscopy (TEM) of the nanoparticles obtained according to example 1.1.a. (Scale bar: 60 nm ( figure 4a) and 5 nm ( figure 4b ), respectively); Figure 5 : Nanoparticle size histogram determined from TEM images for a set of approximately 300 nanoparticles according to example 1.1.a.; Figure 6: Photographs of strips, according to the test in Example 3, following the migration of a solution containing the h-FABP antigen at 5, 0.5, and 0.05 ng / mL, illuminated by a UV lamp. The detection band is visible on the left and the control band on the right. The absorbent buffer is visible at the right edge of the images. The luminescence signals visible in the photographs were analyzed using ImageJ. The results are shown in the following: Figure 7 . Figure 7 : Results of the ratio R= SD / SD +SC measured by ImageJ for liquid samples containing 5, 0.5, 0.05 and 0 ng / mL of h-FABP tested by test strips according to example 3. The points represent the mean value of R and the error bars the associated standard deviation for 3 and 2 strips, respectively; Figure 8 : Schematic top view representation of a housing containing a lateral flow test strip; Figure 9Diagram of the strip reader using four groups of four UV LEDs (LEDs #1 to LEDs #4) for nanoparticle excitation. The strip can be inserted into the reader at the insertion rail (20). Reading is performed through an opening in the cover, in which a filter is positioned to select the nanoparticle emission (centered at 617 nm in this example) and reject the excitation wavelength (centered at 280 nm in this example). This filter can be either an interference or a high-pass filter. A camera, for example, the CCD or CMOS camera of a mobile phone, is positioned in front of this opening to record an image. Figure 10Illustration of the analysis of a test strip using a dedicated Android (Samsung) application. Left: Black and white image of the test strip with rectangles within which the cumulative luminescence levels are calculated, from top to bottom, for the detection zone, the background signal zone, and the control zone. Right: Photograph of the mobile phone screen on which the Android analysis application is running. The black and white image of the test strip is visible, along with the rectangles within which the cumulative luminescence level is calculated and the "Capture," "Measure," "Adjust," and "Save" functions. Figure 11: (A)Absorbance spectrum of a Y 0.6 Eu 0.4 VO 4 nanoparticle solution synthesized according to the example. (B) Emission spectrum of a nitrocellulose membrane bonded to a backing card, such as that used for the lateral flow tests in the example, inserted into a quartz cell, excited at 280, 300, and 380 nm (excitation slit width: 5 nm). The emission is much more intense following excitation at 380 nm across the entire spectrum, and particularly at 617 nm, the wavelength at which the signal from the Y 0.6 Eu 0.4 VO 4 nanoparticle-based probes is detected. Figure 12 : Excitation spectrum of Y 0.6 Eu 0.4 VO 4 nanoparticles (left part of the figure) with the emission wavelength fixed at 617 nm and emission spectrum (right part of the figure) with the excitation wavelength fixed at 278 nm. Figure 13 Excitation spectrum of YVO 4:Dy 5% nanoparticles ( figure 13-a ) with the emission wavelength fixed at 572 nm and emission spectrum ( figure 13-b ) with the excitation wavelength fixed at 278 nm. Figure 14 Excitation spectrum of YVO 4 nanoparticles: Sm 3% ( figure 14-a ) with the emission wavelength fixed at 600 nm and emission spectrum ( figure 14-b ) with the excitation wavelength fixed at 278 nm. Figure 15 Excitation spectra of Y 0.6 Eu 0.4 VO 4 , Lu 0.6 Eu 0.4 VO 4 , LuVO 4 :Dy 10%, La 0.6 Eu 0.4 VO 4 and GdVO 4 :Dy 20% nanoparticles, for an emission wavelength fixed at 617 nm for nanoparticles containing Eu 3+< ions and at 573 nm for nanoparticles containing Dy 3+< ions. Figure 16 : Emission spectrum of Lu 0.6 Eu 0.4 VO 4 nanoparticles for an excitation wavelength of 278 nm (excitation of the LuVO 4 matrix). The emission shows a main peak at 617 nm and two other peaks at 593 and 700 nm. Figure 17: Emission spectrum of LuVO 4:Dy 10% nanoparticles for an excitation wavelength of 278 nm (excitation of the LuVO 4 matrix). The emission has two main peaks at 483 and 573 nm. Figure 18 : Emission spectrum of La 0.6 Eu 0.4 VO 4 nanoparticles for an excitation wavelength of 278 nm (excitation of the LaVO 4 matrix). The emission shows a main peak at 617 and two other peaks at 593 and 700 nm. Figure 19 : Emission spectrum of GdVO 4:Dy 20% nanoparticles for an excitation wavelength of 278 nm (excitation of the GdVO 4 matrix). The emission has two main peaks at 483 and 573 nm. Figure 20 : Absorbance spectra of nanoparticles Y(VO 4 ) 1-y (PO 4 ) y :Eu 20% for y=0, y=0.05, y=0.2, y=0.5 and y=1. The initial concentrations before dilution are on the order of 50 mM in vanadate ions. Figure 21 :Emission spectra of nanoparticles Y(VO4)1-y(PO4)y:Eu 20% for y=0, y=0.05, y=0.2, y=0.5 and y=1 for an excitation wavelength fixed at 278 nm. The initial concentrations before dilution are on the order of 50 mM vanadate ions. Figure 22 Migration of Lu 0.6 Eu 0.4 VO 4 -SA and Lu 0.9 Dy 0.1 VO 4 -SA nanoparticles on dipstick strips containing immobilized BSA-Biotin on the control line, in the absence of antigen. The strips are observed under illumination with a 312 nm UV lamp. Emission is detected through an interference filter (Semrock FF01-620 / 14-25 and FF03-575 / 25 for the emission of Eu 3+< and Dy 3+< ions, respectively); image taken with an iPhone 6 smartphone. Two clear bands are observed on the control line. To the right of the image, the emission of the nanoparticles that migrated to the absorbent pad is visible. EXAMPLE 1. Preparation of photoluminescence probes 1.1. Synthesis of photoluminescent inorganic nanoparticles 1.1.a Synthesis of Y nanoparticles 0, 6 Eu 0, 4 VO 4

[0291] Ammonium metavanadate ions (NH₄VO₃) are used as a source of metavanadate ions, with orthovanadate (YO₄³⁻) being obtained. in situ following a reaction with a base, here tetramethylammonium hydroxide, N(CH3)4OH. Yttrium and europium nitrates were used as sources of Y3+ and Eu3+ ions.

[0292] A 10 mL aqueous solution of NH4VO3 at 0.1M and 0.2 M of N(CH3)4OH (solution 1) is freshly prepared.

[0293] A volume of 10 mL of another solution (solution 2) of Y(NO 3 ) 3 and Eu(NO 3 ) 3 at 0.1 M in ions (Y 3+< + Eu 3+< ) is added dropwise using a syringe pump into solution 1 at a flow rate of 1 mL / min.

[0294] The molar concentration ratio between Y(NO3)3 and Eu(NO3)3 is chosen according to the desired ratio between Y3+< and Eu3+< ions in the nanoparticle, typically the Y3+<:Eu3+< molar ratio is 0.6:0.4.

[0295] Upon addition of the Y(NO3)3 / Eu(NO3)3 solution, the solution becomes diffusive and appears white / milky without precipitate formation. The synthesis continues until the total amount of Y(NO3)3 / Eu(NO3)3 solution has been added.

[0296] The final 20 mL solution must now be purified to remove excess counterions. This is done by centrifugation (typically three times) at 11,000 g (Sigma 3K10, Bioblock Scientific) for 80 minutes, each followed by sonication redispersion (Bioblock Scientific, Ultrasonic Processor, maximum power 130 W operating at 50% for 40 s), until a conductivity strictly below 100 µS·cm⁻¹ is achieved. The conductivity is measured using a chemical conductivity meter.

[0297] The synthesis of Y 0.6 Eu 0.4 VO 4 nanoparticles, on the surface of which tetramethylammonium cations are immobilized, can be described as follows: NH 4 VO 3 + 2 (N(CH 3 ) 4 )OH ⇆ VO 4 3-< +2 N(CH 3 ) 4 +< + NH 4 +< VO 4 3-< + 2 N(CH 3 ) 4 +< + NH 4 +< + 0.6 Y(NO 3 ) 3 +0.4 Eu(NO 3 ) 3 →Y 0.6 Eu 0.4 VO 4 + 2 N(CH 3 ) 4 +< + NH 4 +< + 3NO 3 -<

[0298] Visual observation of the nanoparticle solution, after being left to stand for 16 hours in a bottle, shows a uniformly diffusing solution.

[0299] The final solution remains very stable in water, even after several months at the final pH of the synthesis (approximately pH 5). The solution remains stable even in the synthesis medium (before the removal of excess counter-ions), despite its high ionic strength (>0.1 M).

[0300] After elimination of counter-ions, the zeta potential of the nanoparticles, determined with a DLS-Zeta Potential device (Zetasizer Nano ZS90, Malvern), is -38.4 mV at pH 7.

[0301] Observation of nanoparticles by TEM ( figure 4 ) shows that the nanoparticles are elongated ellipsoid in shape. The dimensions of the nanoparticles are determined from TEM images for a set of approximately 300 nanoparticles ( figure 5 ). Nanoparticles have a major axis length, denoted a , between 20 and 60 nm, with an average value of about 40 nm, and a short axis length, denoted b, between 10 and 30 nm, with an average value of about 20 nm.

[0302] The excitation and emission spectrum of the Y 0.6 Eu 0.4 VO 4 nanoparticles is shown in figure 12 The excitation spectrum has a peak at 278 nm and the emission spectrum has a main peak at 617 nm and two peaks at 593 and 700 nm.

[0303] The Eu³⁺< ions in the YVO₄ matrix can be replaced by other luminescent lanthanide ions. In this case, the excitation and absorption spectrum around the absorption peak of the vanadate VO₄³⁻< ions, linked to a VO₄ charge transfer transition, remains unchanged. The emission spectrum is characteristic of the emission spectrum of each lanthanide ion. 1.1.b Synthesis of nanoparticles Y 0.95 Dy 0.05 VO 4 (YVO 4:Dy (for illustrative purposes only)

[0304] The synthesis is identical to that of example 1.1.a. except that solution 2 consists of Y(NO3)3 and Dy(NO3)3 at 0.1 M ions (Y3+< + Dy3+< ). Solution 2 is added dropwise using a syringe pump to solution 1 at a flow rate of 1 mL / min.

[0305] The molar concentration ratio between Y(NO3)3 and Dy(NO3)3 is chosen according to the desired ratio between Y3+< and Dy3+< ions in the nanoparticle; here the molar ratio Y3+<:Dy+< is 0.95:0.05.

[0306] The excitation and emission spectra of these nanoparticles are shown in the Figure 13 The emission of Dy 3+< ions has two main peaks at 483 and 573 nm. 1.1.c Synthesis of nanoparticles Y 0.97 Sm 0.03 VO 4 (YVO 4 :Sm 3%) (for illustrative purposes only)

[0307] The synthesis is identical to that of example 1.1.a except that solution 2 consists of Y(NO3)3 and Sm(NO3)3 at 0.1 M ions (Y3+< + Sm3+< ). Solution 2 is added dropwise using a syringe pump to solution 1 at a flow rate of 1 mL / min.

[0308] The molar concentration ratio between Y(NO3)3 and Sm(NO3)3 is chosen according to the desired ratio between Y3+< and Sm3+< ions in the nanoparticle; here the molar ratio Y3+<:Sm3+< is 0.97:0.03.

[0309] The excitation and emission spectra of these nanoparticles are shown in the Figure 14 .

[0310] Furthermore, the Y³⁺ ions in the YVO₄ matrix can be replaced by other ions such as Gd³⁺, Lu³⁺, and La³⁺ (see examples below). For all these GdVO₄, LuVO₄, and LaVO₄ matrices, the excitation and absorption spectrum around the absorption peak of the vanadate VO₄³⁻ ions associated with a V⁵⁺ → -O²⁻ charge transfer transition remains unchanged compared to the YVO₄ matrix. Additionally, in these GdVO₄, LuVO₄, and LaVO₄ matrices, the Eu³⁺ ions can be replaced by other luminescent lanthanide ions. The emission spectrum is characteristic of each lanthanide ion. Various representative combinations of matrices and luminescent lanthanide ions are shown below. 1.1.d Synthesis of Lu 0.6 Eu 0.4 VO 4 nanoparticles

[0311] The synthesis is identical to that of example 1.1.a except that solution 2 consists of Lu(NO3)3 and Eu(NO3)3 at 0.1 M ions (Lu3+< + Eu3+< ). Solution 2 is added dropwise using a syringe pump to solution 1 at a flow rate of 1 mL / min.

[0312] The molar concentration ratio between Lu(NO3)3 and Eu(NO3)3 is chosen according to the desired ratio between Lu3+< and Eu3+< ions in the nanoparticle; here the Lu3+<:Eu3+< molar ratio is 0.6:0.4.

[0313] The excitation spectrum of the Lu 0.6 Eu 0.4 VO 4 nanoparticles is shown in the figure 15 and the emission spectrum is presented in the figure 16 The emission spectrum of Eu 3+< ions in the LuVO 4 matrix is ​​practically unchanged compared to that in the YVO 4 matrix ( figure 12 ) and has a main peak at 617 nm and two peaks at 593 and 700 nm. 1.1.e Synthesis of LuVO 4:Dy 10% nanoparticles

[0314] The synthesis is identical to that of example 1.1.a except that solution 2 consists of Lu(NO3)3 and Dy(NO3)3 at 0.1 M ions (Lu3+< + Dy3+< ). Solution 2 is added dropwise using a syringe pump to solution 1 at a flow rate of 1 mL / min.

[0315] The molar concentration ratio between Lu(NO3)3 and Dy(NO3)3 is chosen according to the desired ratio between Lu3+< and Dy3+< ions in the nanoparticle; here the Lu3+<:Dy3+< molar ratio is 0.9:0.1.

[0316] The excitation spectrum of LuVO 4:Dy 10% nanoparticles is shown in the figure 16 and the emission spectrum is presented in the figure 17 The emission spectrum of Dy 3+< ions in the LuVO 4 matrix is ​​practically unchanged compared to that in the YVO 4 matrix ( figure 13 ) and exhibits two emission peaks at 483 and 573 nm. 1.1.f Synthesis of nanoparticles La 0.6 Eu 0.4 VO 4

[0317] The synthesis is identical to that of example 1.1.a except that solution 2 consists of La(NO3)3 and Eu(NO3)3 at 0.1 M ions (La3+ + Eu3+). Solution 2 is added dropwise using a syringe pump to solution 1 at a flow rate of 1 mL / min.

[0318] The molar concentration ratio between La(NO3)3 and Eu(NO3)3 is chosen according to the desired ratio between La3+< and Eu3+< ions in the nanoparticle; here the molar ratio La3+<:Eu3+< is 0.6:0.4.

[0319] The excitation spectrum of the La 0.6 Eu 0.4 VO 4 nanoparticles is shown in the figure 15 and the emission spectrum is presented in the figure 18 The emission spectrum of Eu 3+< ions in the LaVO 4 matrix is ​​practically unchanged compared to that in the YVO 4 matrix ( figure 12 ) and has a main peak at 617 nm and two peaks at 593 and 700 nm. 1.1.g Synthesis of GdVO 4:Dy 20% nanoparticles

[0320] The synthesis of these nanoparticles is carried out from an orthovanadate precursor as follows. A 10 mL aqueous solution of 0.1M NaVO4 (solution 1) is freshly prepared and its pH is adjusted between 12.6 and 13 with a 1 M NaOH solution.

[0321] A volume of 10 mL of another solution (solution 2) of Gd(NO3)3 and Dy(NO3)3 at 0.1 M in ions (Gd3+< + Dy3+< ) is added dropwise under stirring using a syringe pump into solution 1 at a flow rate of 1 mL / min.

[0322] The molar concentration ratio between Gd(NO3)3 and Dy(NO3)3 is chosen according to the desired ratio between Gd3+< and Dy3+< ions in the nanoparticle; here the molar ratio Gd3+<:Dy3+< is 0.8:0.2.

[0323] Upon addition of the Gd(NO3)3 / Dy(NO3)3 solution, a milky precipitate forms. The synthesis continues until the Y(NO3)3 / Eu(NO3)3 solution is fully added. The solution is stirred for 30 minutes until the pH stabilizes at 8-9.

[0324] The final 20 mL solution must be purified, as in example 1.1.a, to remove excess counter-ions. To do this, centrifugations (typically three) at 11000 g (Sigma 3K10, Bioblock Scientific) for 15 minutes, each followed by redispersion by sonication (Bioblock Scientific, Ultrasonic Processor with a maximum power of 130 W operating at 50% for 40 s), are used until a conductivity strictly less than 100 µS.cm-1 is reached.

[0325] The excitation spectrum of GdVO 4:Dy 20% nanoparticles is shown in the figure 15 and the emission spectrum is presented in the figure 19The emission spectrum of Dy 3+< ions in the GdVO 4 matrix is ​​practically unchanged compared to that in the YVO 4 matrix ( figure 13 ) and exhibits two emission peaks at 483 and 573 nm. 1.1.h Synthesis of nanoparticles Y(VO 4 ) 1-y (PO 4 ) y :In 20%

[0326] Nanoparticles containing a mixture of VO4 3-< and PO4 3-< ions in the matrix at different VO4 3-<:PO4 3-< ratios have also been synthesized.

[0327] The synthesis is identical to that of Example 1.1.a except that solution 1 consists of 0.1·y M Na₃PO₄, 0.1·(1-y) M NH₄VO₃ at a total concentration of 0.1 M (VO₃⁻ + PO₄³⁻) ions, and 0.2·(1-y) M N(CH₃)₄OH. A 10 mL aqueous solution with the above concentrations (solution 1) is freshly prepared. NPs with y=0, y=0.05, y=0.2, y=0.5, and y=1 were prepared.

[0328] PO4 3- ions do not exhibit absorption at 278 nm. Thus, nanoparticles containing 100% PO4 3- ions do not exhibit an absorption peak at 278 nm (see Figure 20 ). The emission spectra of these nanoparticles are presented in the figure 21 and are identical for all y values ​​other than 1 (no emission observed for y=1). They exhibit a main peak at 617 nm and two additional peaks at 593 and 700 nm. These emission spectra are practically identical. 1.2. Covalent coupling of nanoparticles with proteins (anti-h-FABP antibodies)

[0329] The Y 0.6 Eu 0.4 VO 4 nanoparticles, obtained as described in point 1.1.a., are coupled with antibodies according to the following protocol. 1.2.1. Coating of nanoparticles with a silica layer

[0330] Following nanoparticle synthesis, the nanoparticle solution is centrifuged at 17,000 g for 3 minutes to precipitate any nanoparticle aggregates, and the supernatant is collected. Size selection is then performed. This involves several centrifugations at 1900 g for 3 minutes. Each centrifugation is followed by redispersion of the nanoparticles using a sonicator, and then the size of the nanoparticles is determined using a DLS-Zeta Potential instrument (Zetasizer Nano ZS90, Malvern).

[0331] A 25 mL volume of Y 0.6 Eu 0.4 VO 4 particles with a 20 mM vanadate ion concentration is prepared. A 2.5 mL volume of another pure sodium silicate solution (Merck Millipore 1.05621.2500) is added dropwise using a pipette to coat the surface of the particles. This solution is left to act under stirring for at least five hours.

[0332] The solution is then purified to remove excess silicate and sodium counterions. It is centrifuged at 11,000 g (Sigma 3K10, Bioblock Scientific) for 60 minutes and then redispersed by sonication (Bioblock Scientific, Ultrasonic Processor, operating at 50% capacity at 400 W). This step is repeated until the solution's conductivity falls below 100 µS / cm. 1.2.2. Grafting of amines onto the surface of nanoparticles

[0333] In a 500 mL three-necked round-bottom flask, place 225 mL of absolute ethanol and add 265 µL of APTES (3-Aminopropyltriethoxysilane) (Mw 221.37 g / mol Sigma Aldrich), resulting in a final concentration of 1.125 mM. This amount corresponds to 5 equivalents of vanadate. A condenser is then connected to the flask. The entire assembly is placed on a heating mantle and under a fume hood. The mixture is heated under reflux at 90°C. A colloidal solution of nanoparticles (vanadate ion concentration [V] = 3 mM) in 75 mL of water at pH 9 is added dropwise using a peristaltic pump at a flow rate of 1 mL / min to one of the three-necked inlets. The system is heated with stirring for 24 h.

[0334] After 48 hours, we use a rotary evaporator (Rotavapor R-100, Buchi) to partially concentrate the nanoparticles. The solution is rotated in a suitable flask and heated in a bath at 50°C.

[0335] The recovered solution is purified by several centrifugations in an ethanol:water solvent (3:1). After purification, size sorting is carried out following the protocol described above. 1.2.3. Carboxyl grafting onto the surface of amino nanoparticles

[0336] Before starting the grafting process, a solvent transfer is carried out.

[0337] The grafting protocol is as follows.

[0338] Transfer the amino nanoparticles (NPs) from the EtOH:H₂O buffer to DMF or DMSO by performing several centrifugations (13,000 g, 90 min). The pellet is redispersed by sonication between each centrifugation (20 s at 75%). Measure and determine the NP concentration.

[0339] Recover the NPS in 5 mL of DMF, then add 10% succinic acid anhydride to a glass beaker (i.e., 0.5 g in the 5 mL). Allow to react for at least one night under an inert atmosphere, while stirring.

[0340] Wash the carboxylated NPs at least 2 times by centrifugation (13000 g for 60 min, Legend Micro 17R, Thermo Scientific) to remove DMF and excess succinic acid anhydride.

[0341] Resuspend the carboxylated particles in water or MES buffer at pH 6 by sonication (Bioblock Scientific, Ultrasonic processor). 1.2.4. Coupling of nanoparticles with anti-h-FABP antibodies

[0342] The coupling of surface-grafted nanoparticles with COOH is carried out according to the protocol below: 1. Prepare a fresh mixed solution of EDC / Sulfo-NHS (concentrations of 500 and 500 mg / mL, respectively) in MES buffer (pH 5-6). 2. To 3 mL of the previously prepared solution, add 90 nM of NPs (here, the nanoparticle concentration is calculated from the vanadate ion concentration according to the Casanova reference). et al.

[37] ) and allow to react for 25 min at room temperature while stirring. 3. Rapidly wash the NPs by at least 2 centrifugations (13000 g for 60 min, Legend Micro 17R, Thermo Scientific) with MilliQ water to remove excess reagents. 4. Recover the last pellet after sonication in sodium phosphate buffer at pH 7.3. Add the required amount of protein (anti-h-FABP antibody, Ref 4F29, 10 E1, Hytest) required depending on the desired ratio (Protein:NPs), typically 2 µM for a ratio of 20:1, and 5 mg / mL of mPEG-silane (MW: 10 kDa, Laysan Bio 256-586-9004). 5. Allow this solution to react for 2 to 4 hours at room temperature while stirring. 6. Add the blocking agent (1% glycine) so that it reacts with the free COOHs and blocks the residual reaction sites on the surface of the NPs. Allow to react for 30 minutes. 7. Wash the protein-coupled NPs several times by centrifugation using centrifuge filters (Amicon Ultra 0.5 mL, Ref UFC501096, Millipore) with PBS pH 7.2. Transfer the NPs to their storage medium: phosphate buffer + Tween 20 (0.05%) + 0.1% glycine + 10% glycerol. Take 100 µL for BCA testing. The remaining solution is aliquoted and frozen at -80°C.

[0343] Similarly, all the nanoparticles synthesized according to examples 1.1.b to 1.1.h can be coupled with antibodies, in the same way as for the Y 0.6 Eu 0.4 VO 4 nanoparticles. 1.3. Passive coupling of nanoparticles with proteins (anti-h-FABP antibodies)

[0344] Passive coupling of nanoparticles with antibodies, instead of the covalent coupling of example 1.2, can also be achieved as follows. - Centrifuge a 1 mL solution of nanoparticles (5 mM vanadate ion concentration) for 15 min at 15,000 g. - Resuspend the pellet in 800 µL of MilliQ water and then redisperse by sonication (Bioblock Scientific, Ultrasonic Processor, maximum power of 130 W operating at 50% for 40 s). - Add 100 µL of an antibody solution at 250 µg / mL in 2 mM potassium phosphate buffer, pH 7.4. - Incubate under rotation for 1 hour. - Add 100 µL of 20 mM potassium phosphate buffer, pH 7.4 / 1% BSA. - Centrifuge for 15 min at 15,000 g and discard the supernatant. - Resuspend the pellet in 1 mL of 2 mM potassium phosphate buffer, pH 7.4 / 0.1% BSA. Redisperse by sonication (Bioblock Scientific, Ultrasonic Processor, maximum power 130 W operating at 50% for 40 s). - Centrifuge for 15 min at 15,000 g and discard the supernatant. - Resuspend the pellet in 250 µL of 2 mM potassium phosphate buffer, pH 7.4 / 0.1% BSA.Redisperse by sonication (Bioblock Scientific, Ultrasonic Processor, maximum power of 130 W operating at 50% for 40 s). 2. Preparation of the test using "sandwich" type strips

[0345] To develop rapid tests to determine the presence of a protein in a qualitative or quantitative way, it is necessary to optimize the different parameters and make compromises between reaction time and test sensitivity.

[0346] The manufacture of the strip test, as shown in Figure 1 This is achieved through the combination of four essential parts: • The essentially inert glass fiber is used as a marking area (2) ("Conjugate Pad" in Anglo-Saxon terminology) (GFDX 103000, Millipore). • As a deposition area ("Sample Pad" in Anglo-Saxon terminology) (1) ( RefCFSP173000 (Millipore) uses polyesters with modified surfaces. These have the advantage of low non-specific interactions with proteins, excellent tensile strength, and good handling. Nitrocellulose (NC) membrane (HF180MC100, Millipore) is used as a capillary diffusion medium (10). It has optimal properties for fluid migration and protein immobilization. The NC membrane is bonded to a non-porous adhesive plastic backing card (6). Cellulose (CFSP173000, Millipore) is used as an absorbent pad (5) due to its high absorption capacity.

[0347] For the detection of h-FABP (“human fatty-acid binding protein”) which is a cardiac biomarker: Before the assembly of the different components, the antibodies must be deposited on the NC membrane. 1. A solution of mouse monoclonal antibodies directed against h-FABP (ref 4F29, 9F3, Hytest) is diluted in PBS (pH 7.4) to a concentration of 1 mg / mL. This solution will be used for the test strip (3). Another solution of goat polyclonal IgG antibodies ( Refab6708, Abcam) directed against mouse antibodies is diluted in PBS (pH 7.4) to a concentration of 1 mg / mL. This is used for the control strip (4). 2. The antibody solutions are dispensed onto the NC membrane using a dispenser (Claremont Bio Automated Lateral Flow Reagent Dispenser (ALFRD)). Using a syringe pump, a volume of 0.7 µL / 2 mm is dispensed for each strip along the length of the NC membrane (approximately 30 cm in length, from which several strips will be made). Allow to dry for 1 h at 37°C. 3. After antibody dispensing, incubate the NC membrane with 1% BSA diluted in PBS (pH 7.4) + 0.04% Tween 20 for 30 min at 37°C to passivate the binding sites not occupied by antibodies. 4. Deposit the Ac coupled to the NPs onto the labeling area (“conjugate pad”) using the dispenser. A volume of 3 µL / 4 mm is applied along the entire length of the fiberglass membrane.Allow to dry for 1 hour at room temperature before blocking with 1% BSA diluted in PBS (pH 7.4). Allow to dry at room temperature. Strip assembly

[0348] 1. Assemble the different structures (the cellulose which serves as an absorbent pad and deposition area, the labeling area on which Ac+NPs are deposited) onto the adhesive parts of the NC on which the Acs are already immobilized. The components are fixed to the plastic support of the NC in the following order: labeling area (“conjugate pad”), deposition area (“sample pad”), and finally absorbent pad (“absorbent pad”). For better capillary migration of the fluid, the different components are mounted so as to overlap each other as shown in the illustration ( Figure 12. Cut the assembled membrane into individual 4 mm wide pieces using a paper cutter. 3. The strips are then stored in aluminum pouches in the presence of a desiccant in an atmosphere with a humidity below 30%. 3. Strip test procedure

[0349] The strip is prepared by using Y 0.6 Eu 0.4 VO 4 nanoparticles coupled to the antibodies prepared in example 1.2.

[0350] Several concentrations of h-FABP (Ref. 8F65, Hytest) were measured, ranging from 5 ng / mL to 0.05 ng / mL. Recombinant h-FABP was diluted to the desired concentrations with buffer or serum. 1. Bring the prepared test strip, as described in point 2 above, and the samples to be analyzed to room temperature before performing the test. 2. Place 400 µL of the sample into a vertically positioned vial. 3. Immerse the test strip in the vial with the sample pad facing downwards. Tap the strip at the bottom to initiate migration. Keep the strip upright in the vial for 10 minutes. 4. Read the test strip results using a UV lamp (Vilber Lourmat, VL-8.MC 8W at 312 nm and 8W at 254 nm) (digital photography and analysis using ImageJ, see Figures 6 And 7 ) or using the reader presented in the Figures 8 And 9 . There Figure 10This illustrates the analysis of the results from a test strip using a dedicated application on an Android mobile phone. The reader uses four groups of four 278 nm LEDs and an interference filter (620 / 15, Semrock) for detection. A high-pass filter such as an RG605 filter (Schott) can also be used for detection.

[0351] The absorption spectrum of nanoparticles is presented in the Figure 11 (A) The absorption peak is located at 280 nm with a full width at half maximum (FWHM) of approximately 50 nm. The emission spectrum of the UV lamp is centered at 310 nm with a FWHM of 40 nm. The emission spectrum of the UV LEDs is centered at 278 nm with a FWHM of 10 nm. Qualitative interpretation of the results

[0352] The qualitative interpretation of the results is as follows: If two bands are present: the test is positive. If only one control band is present: the test is negative. If only one test band is present: the test is invalid. If no band is present: the test is invalid. Quantitative interpretation of the results

[0353] There Figure 10This demonstrates an example of quantitative analysis using a digital photo taken with a mobile phone and a dedicated application. Tapping "Capture" on the phone screen triggers the recording of a black and white image. Then, after tapping "Measure," the application prompts the user to point their finger at the detection zone and then the control zone on the phone screen. The application then calculates the cumulative luminescence level within a rectangle containing the detection zone (LD) and the cumulative luminescence level within a rectangle of the same size containing the control zone (LC). Tapping "Adjust" optimizes the position of the two rectangles, corresponding to the detection and control zones, to maximize the measured signal.The cumulative emission level within a rectangle of the same size located midway between the detection and control zones is used to determine the background signal, LB, and to calculate the SD / C signals = LD / C - LB. The application then calculates and displays the ratio R = SD / SC or R = SD / (SD + SC). The R value can be compared to a calibration table to also provide a concentration value in ng / mL. The result can be saved using the "Save" function for later comparison with subsequent results.

[0354] For the strip test of each sample containing the h-FABP antigen, three strips were prepared. For the sample not containing the antigen, only two strips were prepared.

[0355] The graph of the figure 7represents the results obtained for the ratio R=SD / (SD +SC), measured by ImageJ for the different liquid samples containing 5, 0.5, 0.05, and 0 ng / mL of h-FABP. The points on the figure 7 represent the mean values ​​of R, and the error bars, the associated standard deviation for the different strips tested (three strips in the case of samples containing h-FABP; two strips in the case of the sample not containing it).

[0356] Thus, the method according to the invention advantageously allows the detection of h-FABP in a sample at a concentration of 5 ng / mL or less, in particular at 0.5 ng / mL or less, and even down to a value as low as 0.05 ng / mL. In other words, h-FABP can be detected at a concentration of 330 pM or less, in particular at 33 pM or less, and even down to a concentration as low as 3.3 pM. 4. Migration of Lu 0.6 Eu 0.4 VO 4 -SA and Lu 0.9 Dy 0.1 VO 4 -SA nanoparticles on "dipstick" type "sandwich" strips

[0357] Lu 0.6 Eu 0.4 VO 4 and Lu 0.9 Dy 0.1 VO 4 nanoparticles synthesized according to Examples 1.1.d and 1.1.e, respectively, were coupled with streptavidin (SA) according to Example 1.3 (passive coupling). Dipstick strips were prepared according to Example 2 by immobilizing BSA-Biotin on the nitrocellulose membrane to recognize the NPs coupled with streptavidin. Strip tests were performed with the Lu 0.6 Eu 0.4 VO 4-SA and Lu 0.9 Dy 0.1 VO 4-SA nanoparticles according to Example 3, in the absence of antigen. The strips were visualized under UV excitation (312 nm). Two bright, intense bands were formed at the control line ( Figure 22 ). References

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Claims

1. In-vitro method for detecting and / or quantifying a biological or chemical substance of interest in a liquid sample, by a capillary action test of lateral flow strip type using, as probes, photoluminescent inorganic nanoparticles of the following formula (II):         A1-xLnxVO4(1-y)(PO4)y     (II) in which: . A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof; . Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho) and mixtures thereof; . 0.1 ≤ x ≤ 0.9, in particular 0.2 ≤ x ≤ 0.6 and more particularly x has a value of 0.4; and . 0 ≤ y < 1, in particular y has a value of 0; said method employing detection of the luminescence, with an emission lifetime shorter than 100 ms, of the nanoparticles, after one-photon absorption, by excitation of the matrix at a wavelength less than or equal to 320 nm.

2. Method according to the preceding claim, in which detection of the luminescence is effected by excitation of the matrix at a wavelength of less than or equal to 300 nm, in particular between 250 nm and 300 nm.

3. Method according to either one of the preceding claims, in which the liquid sample is a biological sample, in particular a sample taken from a human, and more particularly a sample selected from blood, serum, plasma, saliva, urine, nasal swab, vaginal smear, expectoration, diluted faecal matter and cerebrospinal fluid.

4. Method according to any one of the preceding claims, for detecting and / or quantifying molecules, proteins, nucleic acids, toxins, viruses, bacteria or parasites in a sample, in particular in a biological sample.

5. Method according to any one of the preceding claims, in which said photoluminescent nanoparticles have an average size greater than or equal to 5 nm and strictly less than 1 µm, in particular between 10 nm and 500 nm and preferably between 20 nm and 200 nm.

6. Method according to any one of the preceding claims, in which Ln is selected from Eu, Dy, Sm, Yb, Er, Nd and mixtures thereof, in particular from Eu, Dy, Sm and mixtures thereof, and more particularly is Eu; and / or A is selected from Y, Gd, La and mixtures thereof, in particular A represents Y or Gd, in particular A represents Y.

7. Method according to any one of the preceding claims, in which said nanoparticles have tetraalkylammonium cations on their surface in an amount such that said nanoparticles have a zeta potential, denoted ζ, less than or equal to - 28 mV, in an aqueous medium of pH ≥ 5, and with ionic conductivity strictly less than 100 µS.cm-1.

8. Method according to any one of the preceding claims, in which said nanoparticles are of formula A1-xLnxVO4 (III), in which A, Ln and x are as defined according to any one of Claims 1, 6 and 7, in particular of formula Y1-xEuxVO4 (IV), in which 0.1 ≤ x ≤ 0.9, in particular 0.2 ≤ x ≤ 0.6 and more particularly x has a value of 0.4.

9. Method according to any one of the preceding claims, said method using a capillary action test device of lateral flow strip type, in which said photoluminescent inorganic nanoparticles are coupled to at least one binding reagent specific to the substance to be analysed, in particular an antibody or antibody fragments, a peptide, a chemically modified nucleic acid or an aptamer, said photoluminescent inorganic nanoparticles being in particular functionalized on the surface with one or more agents intended to facilitate their migration within the capillary action test device, in particular selected from stealth agents or passivating agents, and more particularly selected from silanized PEG chains, poloxamers and polylactic acids (PLA).

10. Method according to any one of the preceding claims, using a capillary action test device, comprising: - a zone (1) for deposition of the liquid sample, and optionally of a diluent; - a zone (2), arranged downstream of the deposition zone, called "labelling zone", loaded with said photoluminescent inorganic nanoparticles coupled to at least one binding reagent specific to the substance to be analysed; - a reaction zone (3), also called "detection zone", arranged downstream of the labelling zone (2), in which at least one capturing reagent specific to the substance to be analysed is immobilized; - a control zone (4), located downstream of the detection zone, in which at least one second capturing reagent specific to the binding reagent specific to the substance to be analysed is immobilized; and optionally, an absorbent pad (5), arranged downstream of the reaction zone and of the control zone, said method comprising at least the following steps: (i) applying the liquid sample to be analysed, and optionally a diluent, at the level of the deposition zone (1) of the capillary action test device; (ii) incubating the device until the luminescence generated by the photoluminescent nanoparticles is detected in the reaction zone (3) and / or until the luminescence is detected in the migration control zone (4); and (iii) reading and interpreting the results.

11. Method according to any one of the preceding claims, in which reading of the results of the capillary action test is effected by detecting the luminescence generated by the probes immobilized, at the end of the test, at the level of the capillary action test device, in particular immobilized at the level of the detection zone (3), and optionally at the level of the control zone (4), of the device as defined in Claim 10, the reading of the results of the capillary action test being more particularly effected by direct, naked eye observation of the capillary action test device, in particular of the detection zone and, optionally, of the control zone of the capillary action test device as defined in Claim 10, in particular using an emission filter; or using detection equipment comprising an emission filter and a photon detector, in particular a CCD or CMOS camera.

12. Method according to any one of the preceding claims, in which said nanoparticles are of formula Y1-xEuxVO4, (IV), detection of the luminescence being effected by excitation of the YVO4 matrix at a wavelength between 230 and 320 nm, in particular between 250 and 310 nm and more particularly between 265 and 295 nm.

13. Method according to any one of the preceding claims, in which interpretation of the results, in particular for obtaining a quantitative characterization of said substance of interest, comprises determining the signal corresponding to the detection zone, the control zone and the background signal of the capillary action test device as defined in Claim 10, subtracting the value of luminescence of the background signal and then determining the ratio of the signal from the detection zone to the signal from the control zone.

14. Capillary action test device of lateral flow strip type, useful for detecting and / or quantifying a biological or chemical substance of interest in a liquid sample, said device comprising - a zone (1) for deposition of the liquid sample, and optionally of a diluent; - a zone (2), arranged downstream of the deposition zone, called "labelling zone", loaded with said photoluminescent inorganic nanoparticles coupled to at least one binding reagent specific to the substance to be analysed; - a reaction zone (3), also called "detection zone", arranged downstream of the labelling zone (2), in which at least one capturing reagent specific to the substance to be analysed is immobilized; - a control zone (4), located downstream of the detection zone, in which at least one second capturing reagent specific to the binding reagent specific to the substance to be analysed is immobilized, and comprising, as probes, photoluminescent inorganic nanoparticles of the following formula (II):         A1-xLnxVO4(1-y)(PO4)y     (II) in which: . A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof; . Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho) and mixtures thereof; . 0.1 ≤ x ≤ 0.9, in particular 0.2 ≤ x ≤ 0.6 and more particularly x has a value of 0.4; and . 0 ≤ y < 1, in particular y has a value of 0; said nanoparticles being able to emit luminescence, with an emission lifetime shorter than 100 ms, after one-photon absorption, by excitation of the matrix at a wavelength less than or equal to 320 nm, in particular less than or equal to 300 nm and more particularly between 250 and 300 nm.

15. Device according to the preceding claim, in which said nanoparticles are as defined according to any one of Claims 5 to 9.

16. In-vitro diagnostic kit, comprising at least: - a capillary action test device as defined according to either one of Claims 14 and 15; and - a device for detecting the luminescence generated by the probes immobilized at the level of the device, at the end of the test, in particular at the level of the detection zone and / or control zone of the capillary action test device.

17. Use of a method as defined according to any one of Claims 1 to 13 or of a capillary action test device as defined according to either one of Claims 14 and 15, for purposes of in-vitro diagnostics.

18. Use of a method as defined according to any one of Claims 1 to 13 or of a capillary action test device of lateral flow strip type as defined according to either one of Claims 14 and 15, for detecting and / or quantifying a substance of interest, in particular a pathogen, in an agri-food product or in the environment.

19. Use of a method as defined according to any one of Claims 1 to 13 or of a capillary action test device of lateral flow strip type as defined according to either one of Claims 14 and 15, for detecting and / or quantifying in-vitro an illegal chemical substance, in particular a drug, or any other substance of interest for the police or defence.