Ultra-sensitive detection method using photoluminescent particles

Abstract

Ultra-sensitive detection method using photoluminescent particles The present invention relates to an ultra-sensitive in vitro detection and / or quantification method of a substance of biological or chemical interest in a sample by detecting the luminescence emission emitted by photoluminescent inorganic nanoparticles, comprising at least the steps of arranging a vanadate or vanadate / phosphate matrix photoluminescent inorganic nanoparticle of particular formula coupled to a coupling agent with the substance of interest, bringing said photoluminescent particles into contact with the substance of interest under coupling conditions of the substance of interest with the coupling agent, and exciting the matrix of photoluminescent inorganic nanoparticles by radiation with wavelengths between 240 nm and 330 nm;the detection of luminescence emission by photoluminescent inorganic nanoparticles, and the determination of the presence and / or concentration of the substance of interest by interpretation of said measurement of luminescence emission by the particles. Figure for the abstract: Fig. 1;

Description

Title of the invention: Ultrasensitive detection method using photoluminescent particles

[0001] The present invention relates to the field of research, bioanalysis and in vitro diagnostics. More particularly, it relates to a method for the ultra-sensitive in vitro detection and / or quantification of substances of biological or chemical interest, for example biomarkers, proteins, peptides, hormones, antibodies, DNA, RNA and other compounds, in a sample, in particular a biological sample, by detecting the emission of luminescence by photoluminescent inorganic nanoparticles with controlled optical and physicochemical properties. technical field

[0002] The detection and / or quantification of the concentrations of biomarkers, antibodies or DNA and RNA in biological samples (blood, serum, saliva, urine, cerebrospinal fluid, etc.) are essential for medical diagnosis.

[0003] In the field of research, in vitro or ex vivo diagnostics, medical analysis and bioanalysis, a number of methods have been proposed to detect and / or quantify the presence of specific substances.

[0004] These methods generally rely on the use of a probe designed to detect and / or quantify a concentration in solution. These probes are coupled to a recognition compound, or targeting agent, which allows them to bind to the molecular species to be analyzed. This recognition compound can be a molecule, DNA, an aptamer, a protein, or an antibody. The probes that have bound to the molecular species to be analyzed via the recognition compound can then be detected using one or more methods based, for example, on their luminescence, absorbance, chemical reactivity, radioactivity, etc.

[0005] The most commonly used biochemical assays, particularly for protein detection, are enzyme-linked immunosorbent assays (ELISAs), which generally rely on the use of horseradish peroxidase as the enzyme to trigger a reaction with a substrate and quantify the resulting chemical reaction by measuring the absorbance of the reaction product in solution. The choice of the molecular recognition compound to which the probe is coupled is crucial for the effectiveness of these probes. More specifically, the effectiveness of these methods depends on the specific affinity of the recognition compound for the target substance. As As examples, the referenced publications [1] and [2] detail the characteristics of these mechanisms.

[0006] Luminescent probes generally lead to more sensitive detection than probes detected by their absorbance because, in the first case, the measurements of the light intensity are made on a black background, while, in the second, it is a matter of measuring a variation of the light intensity (measurements on a light background).

[0007] Other assay methods currently proposed include electrochemiluminescence assay (ECLIA), fluorescence immunoassay (FIA) and radioimmunoassay (RIA).

[0008] However, these methods have various drawbacks which limit their final detection sensitivity, in particular limitations in terms of luminescence properties of the probes used (ECLIA, FIA), safety risks, expensive equipment and the need for specialized users (no automated machines) to conduct RIA type tests.

[0009] In particular, currently available luminescent probes have several disadvantages that prevent their full potential as diagnostic probes from being exploited. Among these drawbacks are, for example, the phenomenon of photobleaching in the case of organic fluorophores, which, following irreversible chemical changes induced by illumination, results in the disappearance of fluorescence; and the phenomenon of emission flicker for semiconductor nanocrystals, or "quantum dots," whereby the probes periodically cease emitting and are therefore unsuitable for producing a constant signal. Other disadvantages arise, for example, from the broad emission spectrum of luminescent probes.Indeed, an excessively broad emission spectrum makes it difficult to filter out any background signal that may be present, and affects signal quality, particularly 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 after freezing, which is a drawback for storing bioconjugated agents. The ease of coupling the probes to the molecular compound used to target the desired molecules is also an aspect to consider when selecting the appropriate probe. Thus, a number of particles, including semiconductor nanocrystals, are synthesized in organic solvents.It follows that use for biological applications requires additional surface preparation steps to achieve dispersion of these particles in water, a process that can be complex to implement and unstable over time. [3]. Their functionalization with chemical groups, allowing coupling to molecular compounds for recognition of target molecules, is also based on weak chemical bonds, which consequently limits their stability and is detrimental to the reproducibility of detection tests.

[0010] Furthermore, the colloidal properties of the particles / probes are crucial for conducting biological tests. In fact, solutions with good colloidal stability are able to provide homogeneous media for the tests, and therefore better reproducibility of the test results.

[0011] Finally, complexity and cost are important aspects in the choice of probes for diagnostics. For example, gold nanoparticles, and their properties in terms of surface plasmon resonance, have been proposed as diagnostic probes, but have not taken off as probes for in vitro diagnostics, possibly because of the complexity of the detection method [4], or possibly because of a high cost.

[0012] On the other hand, the currently available in vitro detection methods do not give complete satisfaction, particularly in terms of detection sensitivity that can be achieved, in order to broaden the scope of application of in vitro diagnostic methods, for example by allowing earlier detection of diseases or by allowing a diagnosis on the evolution of a disease or on the effect of a therapeutic treatment.

[0013] To improve detection sensitivity, two commercial ultrasensitive immunoassay techniques have been developed. These are the methods developed by Quanterix and Singulex, notably as explained in patent applications US7914734. They rely on the use of functionalized magnetic beads as reactive surfaces for capturing target molecules. Quanterix's technology then captures individual beads functionalized with antibodies directed against the antigens. Each bead is trapped in a well, and their analysis is performed by an ELISA-type assay. Singulex, on the other hand, uses the beads to concentrate the trapped analytes and then determines their concentration by counting fluorescent signals using a confocal detection device and an excitation laser that scans the sample helically.

[0014] While these two techniques offer higher detection sensitivity than the conventional detection technologies discussed previously, they are highly complex and expensive. They require the use of equipment specifically designed for these detection techniques and are incompatible with current automated in vitro diagnostic devices.

[0015] Semiconductor nanocrystals or “quantum dots” have also been proposed in ultrasensitive detection tests ([5], [6], [7], [8] and [9]). However, the effectiveness of these tests is limited by the drawbacks associated with this type of probe. Luminescent materials, as discussed previously: complexity and high cost of their synthesis and functionalization, unsatisfactory colloidal stability, loss of luminescence properties after freezing.

[0016] Finally, methods based on the use of gold nanoparticles by exploiting phenomena such as surface plasmon detection, fluorescence quenching, silver deposition on gold nanoparticles etc. ([9] to

[13] ) allow high detection sensitivities to be achieved but are generally highly complex.

[0017] There therefore remains a need to develop a detection / quantification method that achieves higher performance in terms of detection sensitivity than conventional technologies, such as ELISA, ECLIA, FIA or RIA, and does not present the disadvantages, particularly in terms of complexity and cost, of the ultrasensitive methods already proposed.

[0018] With regard to nucleic acid detection, the most common ultrasensitive techniques are PCR, qPCR, and LAMP, with qPCR offering the highest sensitivity. However, qPCR requires expensive equipment, skilled personnel, and a high cost per test. All these techniques use enzymes and amplification of the initial nucleic acid, which increases their sensitivity to impurities and their cost. Furthermore, PCR and LAMP are less expensive but do not allow for quantification of the nucleic acid concentration in the sample.

[0019] To avoid the drawbacks of these techniques, as mentioned in the article by Sakharov Y.

[14] , ELISA-type methods have been developed but suffer from low sensitivity. There are also more sensitive ELISA-type methods, as described in the article by Lorenzo et al.

[15] , but this increased sensitivity is achieved through complex and difficult-to-manufacture combinations of nanomaterials. Thus, there is currently no simple solution that combines ultra-sensitivity in nucleic acid detection with low cost and complexity.

[0020] Rare earth-based photoluminescent nanoparticles have already been proposed as luminescent probes in various applications

[16] .

[0021] For example, Dosev et al.

[17] exploit the luminescence properties of Eu:Gd2O3 luminescent nanoparticles by exciting the Gd2O3 matrix, for the detection of protein microstructures deposited on a substrate. As for Yi et al.

[18] , they use up-conversion photon phosphors of the NaYF4:Yb,Er nature, which absorb two near-IR photons to emit one photon in the visible range.

[0022] Rare-earth-based photoluminescent nanoparticles have already been implemented for single-particle detection and single-molecule tracking, taking advantage of the absence of blinking for single-particle detection, compared to semiconductor nanoparticles or Quantum Dots (

[19] and

[20] ). However, it was by no means foreseeable that these lanthanide-based nanoparticles could be used for the ultrasensitive detection and quantification of biomolecules, in the case of ensemble detection of biomolecules. Indeed, apart from the absence of blinking, the luminescence properties of rare-earth-based luminescent nanoparticles are considered inferior to those of Quantum Dots.In these particles, particularly those composed of a metal oxide matrix where some ions are replaced by rare-earth ions, luminescence can be excitatory either by matrix excitation followed by energy transfer to the luminescent rare-earth ions, or by direct excitation of the luminescent rare-earth ions in the visible spectrum. Regarding matrix excitation, its absorption band is generally located in the UV, which presents two drawbacks: few lasers are currently available at these wavelengths, and existing lasers are bulky and expensive; and, at these wavelengths, biomolecules absorb and emit strongly, which can produce a significant background noise signal that must be eliminated.Regarding the direct excitation of rare earth ions, the extinction coefficient for a nanoparticle is lower than that of Quantum Dots but can be comparable to that of an efficient organic fluorophore

[16] .

[0023] Yuan et al.

[21] , in a review article, present time-resolved biological luminescence assays using lanthanide-based nanoparticles. Lanthanide-based luminescent probes are particles comprising lanthanide complexes, which limits the number of lanthanide ions per particle for a given particle volume, or they are up-conversion nanoparticles. For example, in publications using nanoparticles comprising lanthanide complexes, small-diameter nanoparticles (8–9 nm) contain only between 3,000 and 5,000 ions

[28] . Only much larger nanoparticles, especially those larger than 100 nm, can contain on the order of 30,000 ions or more

[22] and

[23] .

[0024] Similarly, Corstjens et al.

[24] use up-conversion nanoparticles for the in vitro detection of ITFN-gamma in human peripheral blood mononuclear cells. Up-conversion nanoparticles are suitable for imaging deep tissues (generally, excitation in the near-IR of Yb3+ where tissues absorb little compared to the visible). On the other hand, because excitation requires the absorption of two photons, high excitation intensities are necessary. Moreover, the quantum efficiency of these systems is low, on the order of 1%. Thus, the number of photons emitted is relatively low.

[0025] Lanthanide complexes or chelates are also proposed as luminescent probes for immunoassays in publications

[23] ,

[25] ,

[26] and

[27] . However, these complexes or chelates typically contain only a single lanthanide ion or, at best, a few (less than ten) lanthanide ions.

[0026] Finally, we can also cite the publication Zhou et al.

[28] which proposes a method of improved sensitivity detection from inorganic nanoparticles doped with lanthanides, following a complex protocol of dissolution of the nanoparticles and detection of the emission of the micelles containing the lanthanides thus formed.

[0027] As for document EP 1 282 824, it describes the use of surface-modified, inorganic luminescent nanoparticles as probes for detecting a biological or other organic substance. The detection method proposed in this document is based on the ELISA detection principle. However, this document does not propose their use for ultrasensitive detection.

[0028] We can also cite US patent 7,550,201, which proposes the use of inorganic nanoparticles doped with lanthanide ions, particularly for diagnostic applications. However, this patent does not propose their use for an ultra-sensitive detection method.

[0029] Also, the publications by Son et al.

[29] and Nichkova et al.

[30] propose the use of Eu:Gd2O3 nanoparticles as an alternative to organic fluorophores as probes for DNA detection and for phenoxybenzoic acid detection, respectively. However, these documents do not suggest their use for ultrasensitive detection.

[0030] The Applicant proposed in application WO 2019 / 025618 an ultrasensitive detection method based on the direct excitation of rare-earth ions in luminescent nanoparticles by laser radiation. In this approach, the absorption of the nanoparticles remains low, and their excitation typically requires a 1W laser to achieve ultrasensitive detection. Such laser equipment is unfortunately expensive to implement. Description of the invention

[0031] The present invention aims precisely to propose a new ultrasensitive detection method, based on the use of particular luminescent nanoparticles doped with rare earth ions, (i) freeing itself from the constraints of implementing expensive laser equipment and (ii) improving detection sensitivity.

[0032] The invention relates more particularly, according to a first aspect, to an ultra-sensitive in vitro detection and / or quantification method for a substance of biological or chemical interest in a sample to be analyzed, in particular a biological sample, by detection of the luminescence emission emitted by photoluminescent inorganic nanoparticles, comprising at least the following steps: i. arrangement of photoluminescent particles formed in whole or in part from a photoluminescent inorganic nanoparticle with a vanadate or vanadate / phosphate matrix, of formula (I): A! JmxVO4(iy)(PO4)y (I) in which: • A is chosen from yttrium (Y), gadolinium (Gd), lanthanum (La) and their mixtures, in particular A represents Y; • Ln is chosen from europium (Eu), dysprosium (Dy), thulium (Tm), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb) and their mixtures, in particular Ln represents Eu; • 0 < x < 1; in particular 0.02 < x < 0.5, in particular 0.05 < x < 0.4 and more particularly x equals 0.4, 0.2, 0.1 or 0.05; and • 0 < y < 1, in particular y equals 0, the photoluminescent inorganic nanoparticles being coupled to a direct or indirect coupling agent to the substance of interest, ii. bringing said photoluminescent particles into contact with the substance of interest under conditions of direct or indirect coupling of the substance of interest with the coupling agent, iii. excitation of the matrix of photoluminescent inorganic nanoparticles of formula (I), by radiation with a wavelength between 240 nm and 330 nm; iv. detection of luminescence emission by photoluminescent inorganic nanoparticles, in particular time-resolved detection, and v. determination of the presence and / or concentration of the substance of interest by interpretation of said measurement of the emission of luminescence by the particles.

[0033] The coupling agent may be a targeting agent of the substance of interest coupling directly to the substance of interest in step (ii).

[0034] Alternatively, the coupling agent is a molecule that allows the attachment of a targeting agent to the substance of interest, in particular the targeting agent being attached to the substance of interest prior to the contact of the photoluminescent particles with the substance of interest. In this case, the process may involve contacting the substance of interest with the targeting agents under conditions of coupling of the targeting agents with the substance of interest between the steps (i) and (ii), the coupling of the substance of interest with the coupling agent is done indirectly by coupling the targeting agent with the molecule.

[0035] Preferably, the excitation of the matrix of photoluminescent inorganic nanoparticles of formula (I) is carried out by radiation with an emission wavelength between 260 and 330 nm, better between 260 nm and 310 nm, better between 270 and 290 nm.

[0036] Preferably, the excitation of the matrix of photoluminescent inorganic nanoparticles of formula (I) is carried out by radiation in the UV-B and / or ruv-c.

[0037] 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.

[0038] The method of the invention is thus based on the excitation of the vanadate or vanadate / phosphate matrix of the luminescent nanoparticles AbxLnxVO4(i_ y)(PO4)y by UV radiation, in particular UV-B and / or UV-C radiation, typically at a wavelength of about 280 nm, and the transfer of energy to the luminescent rare earth ions.

[0039] Contrary to what a person skilled in the art might think, i.e. that we will have too many spurious emissions by exciting in the UV, this latter disadvantage is counterbalanced by the fact that the absorption of the matrix is ​​much greater when we excite in the UV than when we directly excite the lanthanide ions, as is the case in international application WO 2019 / 025618.

[0040] As illustrated in the examples that follow, the inventors have shown that it is possible, despite the use of excitation wavelengths, to achieve ultrasensitive detection.

[0041] In fact, although it is known that the absorption at the peak excitation of the vanadate matrix of lanthanide-enriched vanadate nanoparticles is strong, excitation of these nanoparticles in UV wavelengths is typically contraindicated in ultrasensitive detection applications, due to the parasitic emission, excited at these wavelengths, of signals from the autofluorescent materials and molecules on the supports and in the analyzed media.

[0042] Preferably, the detection of luminescence emission by the particles is time-resolved. For the purposes of this invention, "time-resolved detection" means either detection that is sufficiently rapid to measure the rise and fall of the luminescence signal following the start and stop of the excitation, or delayed detection of the luminescence signal emitted by the photoluminescent particles implemented according to the invention, i.e., after the end of The parasitic emission of autofluorescent materials and molecules. A time-resolved luminescence measurement was described, for example, in document WO 03008974. This is possible because the parasitic emission of autofluorescent materials and molecules typically has a lifetime on the order of nanoseconds, while the lifetime of the emission of photoluminescent inorganic nanoparticles with a vanadate or vanadate / phosphate matrix is ​​on the order of a few hundred microseconds, particularly in the case of europium.

[0043] The invention notably enables "ultra-sensitive" detection; in particular, the method exhibits a detection sensitivity of the biological or chemical substance of interest in the sample of less than or equal to 10 pM, preferably less than or equal to 1 pM, or even less than or equal to 0.1 pM, or less than or equal to 0.01 pM (i.e., 10 fM), or even less than or equal to 1 fM, even better less than or equal to 0.1 fM (i.e., 100 aM), even better less than or equal to 0.01 fM (i.e., 10 aM). Such detection is possible without amplification and / or without the use of enzymes. It is possible to exploit the spectral width of the narrow emission of rare-earth ions, particularly less than 10 nm in the case of europium emission at 617 nm, to reject, with an efficient emission filter, the broadband contributions of spurious emissions.

[0044] Furthermore, the inventors have shown that the use of time-resolved detection makes it possible to circumvent the problem of spurious emissions at the wavelengths used in an even more effective way, and to access so-called "ultrasensitive" detection.

[0045] The invention thus takes advantage of the long emission time characteristic of nanoparticles of formula (I), in particular greater than 1 ps, or even greater than 10 ps, ​​or even greater than 100 ps, ​​unlike the short lifetimes, on the order of a few nanoseconds for fluorescent molecules, to overcome at least partially the parasitic background signal by carrying out a delayed detection of the luminescence signal.

[0046] Advantageously, time-resolved luminescence detection can be implemented using simple and inexpensive equipment, in particular modulation of the supply current to the excitation source, a conventional photomultiplier tube, and a 100 kHz A / D converter, as described later in the text. It is also possible to modulate the excitation source by a mechanical chopper or by any other method known to those skilled in the art.

[0047] The method of the invention thus advantageously makes it possible to achieve a performance in terms of detection sensitivity much higher than the performance of conventional detection techniques such as ELISA, ECLIA, FIA or RIA for the detection of proteins or peptides or organic molecules such as hormones. For nucleic acids, the method of the invention makes it possible to achieve a performance approaching the detection sensitivity of PCR without using amplification or the enzymes necessary for DNA amplification, while allowing quantification of the concentration.

[0048] Advantageously, the ultrasensitive method of the invention thus allows detection at least 10 times, in particular at least 50 times, better at least 100 times more sensitive than the ELISA type enzymatic immunodetection method using the same recognition and targeting antibodies.

[0049] The ultrasensitive method according to the invention thus allows the detection and / or quantification of a substance of interest present in a sample at a concentration strictly below 100 pM, or even below 10 pM, or even below 1 pM, better still below 0.1 pM, or even below 0.01 pM (10⁻¹¹ fM), or less than or equal to 1 fM, better still below 0.1 fM (i.e., 100 aM), better still below 0.01 fM (i.e., 10⁻¹¹ aM). These concentrations depend on the target molecule and, in particular, on the affinity of the recognition compound, or targeting compound, coupled to the probe, but are comparable to those detectable by ultrasensitive methods (Quanterix or Singulex).

[0050] Advantageously, the ultra-sensitive method of the invention, while enabling detection performance comparable to already proposed ultra-sensitive technologies, proves to be particularly advantageous in terms of ease of implementation and cost.

[0051] Preferably, the photoluminescent particle disposition step comprises the disposition of a plurality of photoluminescent particles, the excitation (iii) comprises, in particular, the simultaneous excitation of the matrices of a plurality of photoluminescent inorganic nanoparticles of formula (I) and the detection (iv) comprises, in particular, the simultaneous detection of the luminescence emission by the photoluminescent inorganic nanoparticles excited in the excitation step (iii).

[0052] Preferably, excitation and detection take place in an analysis space, preferably fixed during the analysis, comprising a plurality of photoluminescent particles and the determination of the presence and / or concentration of the substance of interest (v) is carried out by interpretation of the measurement carried out in said analysis space.

[0053] Preferably, the excitation, detection and determination of the presence and / or concentration of the substance of interest are carried out without scanning the sample during the analysis.

[0054] Thus, unlike the complex technologies developed by Quanterix and Singulex, the latter notably requiring a scan of the sample, the method The ultra-sensitive method of the invention employs a compact, low-cost detection apparatus, each component of which is readily available for purchase, for the excitation and measurement of luminescence emitted by nanoparticles, as detailed later in the text, and requires no apparatus components specifically dedicated to the ultra-sensitive detection method of the invention. Advantageously, it is thus compatible with integration into an automated analysis device, requiring only minor ergonomic adjustments.

[0055] Also, the excitation of the nanoparticle matrix, carried out in the wavelength range specified above, can be implemented using a light-emitting diode type source with reduced power which is much less expensive than laser illumination devices, such as a laser diode, used in the methods described previously.

[0056] Furthermore, the method of the invention is also suitable for multiplexed analyses. Thus, the process of the invention can be implemented for the simultaneous detection and / or quantification of at least two different substances in a sample, in particular according to a procedure described later.

[0057] The method of the invention can be implemented for the analysis of substance(s) of biological or chemical interest in various samples, also called "analytes." The sample may be, in particular, a biological sample, especially a human tissue sample, for example, chosen from blood, serum, plasma, saliva, urine, and cerebrospinal fluid. The sample may also be diluted fecal matter, a vaginal smear, a nasopharyngeal swab, or sputum. A diluent may be used with the sample to be analyzed, particularly when the liquid sample is plasma, serum, whole blood, a nasal or vaginal smear, or sputum, for example.

[0058] At least one nanoparticle can be coupled with a plurality of coupling agents, the process comprising a step of coupling the nanoparticles with the coupling agents comprising dissolving the nanoparticles with a proportion of coupling agent greater than the proportion of nanoparticles, in particular with a ratio of at least two coupling agents for one nanoparticle or even at least 10 coupling agents per nanoparticle, or even between 10 and 80 coupling agents.

[0059] It may also be a solution containing biological molecules.

[0060] The method of the invention can, for example, be implemented for the detection and / or quantification of biomarkers, antibodies, DNA, and / or RNA. It can be implemented to detect human, animal, bacterial, viral, or circulating DNA and / or RNA. It can also be used to provide a genotype in a sample. biological. This can include in particular any type of nucleic acid including tRNA, mRNA, miRNA, dsRNA, circRNA, ncRNA, IncRNA.

[0061] According to another aspect of the invention, the invention relates to the use of the method defined above for in vitro diagnostic purposes. Advantageously, the ability of the ultra-sensitive method to detect extremely low levels of certain substances in biological samples allows, for example, the use of the method of the invention for earlier disease detection, or for diagnosing the progression of a disease or the effect of a therapeutic treatment. Furthermore, this ultra-sensitive detection method allows the use, as biomarkers, of substances whose concentration is currently too low to be detected with conventional methods. It also makes them detectable in readily accessible biological media (saliva, urine, blood, etc.).) biomarkers whose concentration is too low to be detected with conventional methods and requires invasive methods such as cerebrospinal fluid sampling, for example.

[0062] According to yet another aspect, the invention relates to an in vitro diagnostic set, comprising • at least photoluminescent particles formed in whole or in part from a photoluminescent inorganic nanoparticle as defined above, said particles being coupled to a coupling agent, in particular the coupling agent being a surface functionalization with chemical groups, for example carboxyl, amino, thiol, aldehyde or epoxy groups, provided by molecules, for example citric acid or polyacrylic acid, and / or coupled to molecules, for example streptavidin, said chemical groups or molecules being capable of enabling the coupling of said particles with a targeting agent of the substance of interest; or a targeting agent of the substance of interest; and • a detection and / or quantification system comprising at least: • an illumination device, preferably of the light-emitting diode type, with an emission wavelength between 260 and 330 nm, better between 260 nm and 310 nm, better between 270 and 290 nm, which may have a power less than or equal to 500 mW, better less than or equal to 200 mW, for example between 50 and 150 mW; • a device for detecting the intensity of light emitted by the particles; the detection and / or quantification system comprising a device for collecting and spectrally filtering the emitted luminescence, a photomultiplier and / or a photodiode and an AD converter which may also include apparatus for implementing time-resolved detection of luminescence emission, in particular an electronic modulation system for the power of the excitation source, a mechanical chopper, a photomultiplier and an AD converter.

[0063] The illumination device can be in the UV-B or UV-C range.

[0064] Preferably, the detection and / or quantification system is fixed during the analysis.

[0065] Preferably, the detection device is devoid of a confocal detection device.

[0066] Such an in vitro diagnostic set allows the easy implementation of an ultra-sensitive detection and / or quantification method according to the invention, for example to measure biomarkers or antibodies in a biological sample.

[0067] The diseases that can be diagnosed with the in vitro diagnostic set of 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...), for which there is one or more specific binding partner(s) (ligand, antibody, complementary nucleic acids, aptamers, ...).

[0068] 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).

[0069] The ultra-sensitive detection method of the invention is not limited to the aforementioned applications. It can thus be implemented for the detection of GMO DNA in seeds, for example, or for the detection of a pollutant or pathogen in water or in food intended for consumption.

[0070] The applications of the ultrasensitive detection method according to 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 probe fragment bound to a nanoparticle, and then detect them by hybridization with complementary fragments from another region grafted onto a solid substrate, following an approach similar to DNA microarrays of the Affymetrix type. One advantage of the invention lies in the absence of the amplification step usually required for these approaches.

[0071] In the rest of the text, the photoluminescent inorganic nanoparticle of formula (I) of the invention will be referred to more simply as "nanoparticle".

[0072] Other features, variations and advantages of the method according to the invention will become clearer from the following description, examples and figures, given by way of illustration and not limitation of the invention.

[0073] In the following text, the expressions "between ... and ...", "ranging from ... to ..." and "varying from ... to ..." are equivalent and are meant to mean that the limits are included, unless otherwise stated.

[0074] Unless otherwise indicated, the expression "including one" should be understood as "comprising at least one". Luminescent particles of the invention

[0075] As previously stated, the ultra-sensitive detection method according to the invention is based on the detection of the luminescence emission of photoluminescent particles comprising, in particular, being formed of a vanadate or vanadate / phosphate matrix photoluminescent inorganic nanoparticle as described previously, and coupled to at least one direct or indirect coupling agent with the substance of interest. Photoluminescent inorganic nanoparticle

[0076] As previously stated, the photoluminescent nanoparticles implemented according to the invention have the formula (I): A! XnxVO4(iy)(PO4)y(I) in which: A is chosen from yttrium (Y), gadolinium (Gd), lanthanum (La) and their mixtures, in particular A represents Y; Ln is chosen from europium (Eu), dysprosium (Dy), thulium (Tm), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb) and their mixtures, in particular Ln represents Eu; 0 < x < 1; in particular 0.02 < x < 0.5, notably 0.05 < x < 0.4, and more specifically x equals 0.4, 0.2, 0.1, or 0.05; and 0 < y < 1, in particular y equals 0.

[0077] The properties of the nanoparticles used will determine the sensitivity that can be achieved by the method of the invention.

[0078] The inorganic nanoparticles of the invention are advantageously formed from a vanadate or vanadate / phosphate crystalline matrix having at least 103 rare earth ions.

[0079] The rare earth ions in the nanoparticles of the invention are not in the form of rare earth ion complexes or chelates, formed of rare earth ions in combination with suitable organic ligands, as for example described in the publication Yuan et al.

[21] .

[0080] Preferably, the nanoparticles of the invention comprise between 1,000 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.

[0081] The nanoparticles of the invention can be doped with rare earth ions of the same or different types. According to one embodiment, as detailed later in the text, the process of the invention uses at least two distinct types of nanoparticles, distinguished by the nature of the rare earth ions. The simultaneous use of nanoparticles enriched in different lanthanides allows for multiplexing the detection of different substances, for example, different biomarkers, within the same sample.

[0082] The photoluminescent nanoparticles of the invention may have an average size greater than or equal to 20 nm and strictly less than 1 pm.

[0083] In particular, they have an average size between 20 nm and 500 nm, in particular between 20 nm and 200 nm and especially between 20 nm and 100 nm.

[0084] In particular, the average size of the photoluminescent nanoparticles of the invention may be greater than or equal to 20 nm, in particular greater than or equal to 30 nm, in particular between 30 and 60 nm.

[0085] In particular, as illustrated in the examples that follow, the photoluminescent nanoparticles according to the invention can have an average size of the order of 20 to 50 nm.

[0086] Larger nanoparticles can, for example, be obtained by size sorting by centrifugation of the particles as exemplified, so as to retain, in the size distribution, only the largest particles, or by grinding the bulk material. Any other technique known to those skilled in the art can also be used.

[0087] The photoluminescent nanoparticles used in the process of the invention thus advantageously 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. For example, a spherical Y0.6Eu 0>4VO4 nanoparticle with a diameter of 30 nm contains 70,000 Eu3+ ions (calculation of the number of ions according to reference

[31] Casanova et al. APL 2006). Furthermore, the photoluminescent nanoparticles should not be too large to avoid steric interference during their association with the substance to be dosed immobilized for example on the surface of a support, as described later in the text.

[0088] The average size can be measured by transmission electron microscopy. Transmission electron microscopy images make it possible to determine the shape of the nanoparticles (spherical, ellipsoidal) and to deduce the average dimensions of the nanoparticles. In the case of particles that are generally spherical, the average size refers to the average diameter of the particles.

[0089] In the case of ellipsoidal particles, the average size is understood to be 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.

[0090] According to a particular embodiment, the nanoparticles of the invention are of an overall elongated ellipsoidal shape (“prolate” in English).

[0091] More specifically, they may have a major axis length, denoted a, of between 20 and 60 nm; and a minor axis length, denoted b, of between 10 and 30 nm. In particular, the nanoparticles of the invention may have an average major axis length, a, of 40 nm and an average minor axis length, b, of 20 nm.

[0092] Advantageously, the nanoparticles of the invention exhibit low polydispersity. The polydispersity index, which can be deduced from TEM measurements, can in particular be strictly less than 0.2.

[0093] Advantageously, the nanoparticles used in the process of the invention are capable of emitting more than 10⁸ photons before the emission stops, in particular more than 10⁹, or even more than 10¹⁰ photons. In many cases, particularly in the case of Eu-doped YVO₄ or GdVO₄ particles, no cessation of emission is observed.

[0094] Moreover, advantageously, the nanoparticles of the invention exhibit a long emission lifetime. In particular, they can exhibit an emission lifetime greater than or equal to 5 ps, in particular greater than or equal to 10 ps, ​​notably greater than or equal to 20 ps, ​​or even greater than or equal to 50 ps, ​​or even greater than or equal to 100 ps.

[0095] The emission lifetime is understood as the lifetime of the excited state of the emitting nanoparticle, and is determined in practice by the duration of the emission of luminescence photons after cessation of excitation, i.e. the characteristic time of the decline of luminescence after cessation of excitation.

[0096] As discussed previously, the ultra-sensitive method of the invention can take advantage of the long emission duration of the particles of the invention (a few hundred ps in the case of Yi xEuxVO4 particles, compared to the lifetimes of usual fluorophores on the order of nanoseconds), to perform time-resolved detection with sufficient temporal resolution, in particular less than 100 ps, ​​better less than 10 ps, ​​in particular delayed detection of emission.

[0097] Advantageously, the nanoparticles implemented according to the invention do not lose their luminescence after freezing.

[0098] In a particular embodiment, the nanoparticles used are of the aforementioned formula (I) in which y is 0. In other words, the nanoparticles used in the process of the invention can be of the formula Ai xLnxVO4 (!'), in which A, Ln and x are as defined above.

[0099] According to a particular embodiment, A in the formula (I) or (!') represents yttrium (Y).

[0100] According to another particular embodiment, Ln in formula (I) or (!') represents Eu.

[0101] Thus, according to one embodiment, the particles of the invention comprise a nanoparticle of formula Yi xEuxVO4 in which 0 < x < 1, in particular 0.02 <x<0,5, en particulier 0,05<x<0,4 et plus particulièrement x vaut 0,4, 0,2, 0,1 ou 0,05.

[0102] The nanoparticles according to the invention are predominantly crystalline and polycrystalline in nature, in particular of average crystallite size, deduced by X-ray diffraction, as detailed in Example 1 below, between 3 and 40 nm.

[0103] According to another particular embodiment, the nanoparticles are single-crystal with an olive shape and with sizes between 30 and 200 nm. Preparation of nanoparticles

[0104] The rare-earth ion-doped crystalline matrix nanoparticles used in the process of the invention can be prepared by any conventional method known to those skilled in the art. Rare-earth-doped yttrium vanadate-based nanoparticles have, for example, been described in detail in articles

[32] and

[33] .

[0105] Advantageously, the nanoparticles of the invention are easily synthesized in an aqueous medium, which has the advantage of eliminating any subsequent solvent transfer step.

[0106] In particular, the nanoparticles can be formed by coprecipitation reaction, in aqueous medium, from precursors of said elements A and Ln, and in the presence of orthovanadate ions (VO43) and possibly phosphate ions (PO43).

[0107] The precursors of elements A and Ln can conventionally be in the form of salts of said 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.

[0108] For example, the synthesis of nanoparticles of formula Yi _ xEuxVO4 can implement, as precursor compounds of yttrium and europium, yttrium nitrates (Y(NO3)3) and europium nitrates (Eu(NO3)3).

[0109] According to a particularly preferred embodiment, orthovanadate ions (VO43) are generated in situ from a metavanadate salt, preferably ammonium metavanadate (NH4VO3).

[0110] Orthovanadate ions can be more particularly formed in situ by reaction of said metavanadate salt with a base (more precisely with two or three equivalents of strong base) as described in the article Neouze et al

[34] .

[0111] In the case of phosphate ions, a phosphate salt is added, such as sodium phosphate or ammonium phosphate.

[0112] Thus, according to a particularly advantageous embodiment, the process of the invention comprises at least the steps of: (a) prepare an aqueous solution (1) by mixing, in aqueous medium, a metavanadate salt, in particular ammonium metavanadate (NH4VO3), and possibly a phosphate salt, and a base, possibly a source of tetraalkylammonium cations, in particular a tetraalkylammonium hydroxide; (b) add to the aqueous solution (1) an aqueous solution (2) comprising said precursors of elements A and Ln, in particular in the form of salts, especially nitrates; under conditions conducive to the formation by co-precipitation of said nanoparticles; and (c) recover the nanoparticles following the removal of counter-ions.

[0113] In a particular embodiment, the aqueous solution (2) containing the precursors of elements A and Ln may also include complexing agents of these elements, such as citrate, for example tetraalkylammonium citrate.

[0114] According to a particular embodiment, the addition in step (b) of solution (2) to solution (1) is carried out drop by drop.

[0115] According to another particularly preferred embodiment, the nanoparticles are prepared by colloidal conversion of rare earth hydroxycarbonate particles (see example 1.2).

[0116] Thus, according to one variant, the process of the invention comprises at least the steps of: (a) prepare an aqueous solution (1) by mixing, in aqueous medium, a metavanadate salt, in particular ammonium metavanadate (NH4VO3), and possibly a phosphate salt; (b) prepare hydroxycarbonate nanoparticles of formula Al xLnx3+CO3 OH from precursors of elements A and Ln, in particular in the form of salts, especially nitrates, and from a source of bicarbonate ions, especially in excess, especially urea, under conditions conducive to the formation by co-precipitation of said hydroxycarbonate nanoparticles, (b') add the aqueous solution (1) to the hydroxycarbonate nanoparticles in colloidal suspension under conditions conducive to the formation by co-precipitation of the nanoparticles according to formula I; and (c) recover the nanoparticles according to formula I.

[0117] According to a particular embodiment, the addition in step (b') of the solution (1) to the hydroxycarbonate nanoparticles is carried out drop by drop.

[0118] According to another embodiment, the solution (1) can be mixed with the solution (2) or the hydroxycarbonate nanoparticles all at once, and not drop by drop.

[0119] The aqueous medium of the solutions is more particularly formed of water and / or a mixture of water and ethylene glycol.

[0120] It is for a person skilled in the art to adjust the quantities of the different reagents, in particular the precursors of the metavanadate ions, possibly phosphate, of said elements A and Ln, and of urea, in view of the desired nature of the nanoparticle according to the invention.

[0121] In particular, the stoichiometric proportions of the different reactants according to the aforementioned formulas must be respected.

[0122] Advantageously, the nanoparticle preparation process does not require heating of the solution, unlike, in particular, the hydrothermal methods proposed in publications

[26] to

[29] . In particular, all steps (a) to (c) for the synthesis of the particles according to the invention can advantageously be carried out at room temperature (20-25°C).

[0123] Step (c) consists more particularly of purifying the solution of particles obtained, in particular to eliminate excess counter-ions.

[0124] The purification steps may more specifically include dialysis or centrifugation steps and redispersion of the particles in an aqueous medium, for example by sonication. The particles may be redispersed in an aqueous medium, in particular in water.

[0125] The synthesis of luminescent nanoparticles 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 the bulk material.

[0126] Preferably, the nanoparticles, in particular those recovered in step (c) above, are mixed with a protecting agent, and the resulting mixture is subjected to post-synthesis annealing at a temperature between 500°C and 1500°C, more particularly between 800°C and 1300°C. The protecting agent is then removed by a suitable method depending on the protecting agent, in particular by acid dissolution. This step (d) is preferably carried out before coupling the nanoparticle with the coupling agent, in particular before its binding with a targeting agent or before its functionalization. Alternatively, the nanoparticles are subjected to post-synthesis annealing under hydrothermal conditions, in particular at a temperature between 120°C and 300°C. Annealing reduces the photoreduction effect of the nanoparticles when they are subjected to excitation. It also makes it possible to increase the quantum yield of nanoparticle emission. Targeting agent

[0127] The particles used as luminescent probes according to the process of the invention can be coupled (or grafted) to at least one targeting agent coupling the substance to be measured in the sample to be analyzed.

[0128] By "targeting agent" is meant a compound enabling a bond with a substance of biological or chemical interest, the identification of which is sought.

[0129] The nature of the targeting agents used is of course chosen with regard to the substance of interest in the sample.

[0130] The particles used in the ultra-sensitive method according to the invention are perfectly suited to a wide variety of biological targets, the specificities being dependent on the nature of the targeting agent(s) grafted onto the surface of the nanoparticle.

[0131] The targeting agent may be more specifically selected from a polyclonal or monoclonal antibody, an antibody fragment, a nanobody, 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 or oligonucleotide, 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 may also be used.

[0132] According to a particular embodiment, it is an antibody or fragment of antibody or an oligonucleotide or fragment of oligonucleotide.

[0133] 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 VNAr obtained from cartilaginous fish antibodies).

[0134] The term “antibody” according to the invention includes chimeric antibodies; human or humanized antibodies, recombinant and modified antibodies, conjugated antibodies, and their fragments.

[0135] According to a particular embodiment, the antibodies or antibody fragments implemented according to the invention target specific markers of cancer cells.

[0136] The targeting agent may also be derived from a molecule known to bind a cell surface receptor. For example, the targeting fragment may 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 targeting agent

[0137] It is the responsibility of a person skilled in the art to implement the appropriate coupling / grafting methods to adequately prepare the particles coupled to one or more targeting agents. The quantity of targeting agent(s) used is adjusted according to the quantity of particles, or vice versa.

[0138] The targeting agent can be grafted directly, or via a spacer (also referred to as "linker" or "spacer"), to the nanoparticle.

[0139] The methods of coupling (also called grafting) particles to biomolecules are well known to those skilled in the art. These generally involve coupling by covalent bonding, surface complexation, electrostatic interactions, encapsulation, or adsorption.

[0140] 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 targeting agent to form a covalent bond.

[0141] Examples of chemical groups that may be present on the surface of nanoparticles include carboxyl, amino, thiol, aldehyde and epoxy groups.

[0142] A coating of the particles with silica can be used to facilitate the subsequent functionalization of the particles.

[0143] Amino groups can be provided by molecules such as amino organosilanes, such as aminotriethoxysilane (APTES). The advantage of APTES The key lies in the fact that it forms a capsule around the nanoparticle via covalent bonds. The amines delivered by APTES are thus very stable over time. The amino groups can be transformed into carboxyl groups by reaction with succinic anhydride.

[0144] Carboxyl groups can be provided by molecules such as citric acid or a polyacrylic acid (PAA).

[0145] The nanoparticles may further have polyethylene glycol (PEG) molecules on their surface to minimize non-specific adsorption of the nanoparticles onto the detection surface. Preferably, the PEG may have a molar mass of 500 to 20,000 g.mol1.

[0146] In other cases, the particles may be pre-coupled to molecules suitable for subsequent coupling with a targeting agent.

[0147] For example, the particles can be coupled to streptavidin suitable for coupling with a biotinylated targeting agent.

[0148] By way of example, example 1 illustrates the coupling of nanoparticles with biotinylated antibodies by coupling nanoparticles coupled with streptavidin with biotinylated antibodies.

[0149] In other cases, the coupling of nanoparticles with antibodies can be achieved directly by coupling antibodies to nanoparticles functionalized with APTES. The amino groups provided by APTES can first be transformed into carboxyl groups by reaction with succinic anhydride, as mentioned above. Then, the carboxyl groups can be activated by any technique known to those skilled in the art, in particular by reaction with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), to subsequently react with the amine functions on the surface of a polypeptide and form a covalent amide bond, when the targeting agent is a protein or an antibody.

[0150] The functionalization of nanoparticles by APTES can advantageously be carried out following the coating of the nanoparticles by a layer of silica.

[0151] Coupling of the targeting agent to the surface of the nanoparticles can also be done by any other method known to those skilled in the art.

[0152] It can also be advantageously carried out by coating the nanoparticles with a silica layer, followed by a coating reaction with APTES (3-aminopropyltriethoxysilane), whose amine groups are used to react with a bifunctional crosslinking agent having two NHS groups. Subsequently, the nanoparticles coupled to the crosslinking agents can react with the amine groups on the surface of a protein (antibody, streptavidin, etc.). This type of coupling process is described in particular in references

[35] and

[36] .

[0153] Advantageously, the particles used in the ultrasensitive detection method according to the invention exhibit low polydispersity. Preferably, the polydispersity index, which can be deduced from TEM measurements or dynamic light scattering (DLS) measurements, is 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.

[0154] As mentioned previously, the process according to the invention can further implement several means of amplifying the luminescence signal emitted by the photoluminescent particles per analyte, in particular by varying the characteristics of the coupling of the photoluminescent nanoparticles with one or more targeting agents.

[0155] At least one nanoparticle can be coupled with a plurality of coupling agents, in particular targeting agents or molecules capable of enabling further coupling with a targeting agent, the process comprising a step of coupling the nanoparticles with the coupling agents comprising dissolving the nanoparticles with a proportion of coupling agent greater than the proportion of nanoparticles, in particular with a ratio of at least two coupling agents for one nanoparticle or even at least 10 coupling agents per nanoparticle, or even between 10 and 80 coupling agents.

[0156] The photoluminescent particles can each comprise a plurality of nanoparticles linked together, in particular by coupling agents, to form an aggregate of nanoparticles, preferably the photoluminescent particles being in colloidal dispersion in the solution at step (ii) of contacting the sample to be analyzed.

[0157] Thus, in a particular embodiment, at least one nanoparticle can be coupled with a plurality of targeting agents, in particular identical ones, especially biotinylated ones, in order to advantageously lead to the formation of controlled aggregates of streptavidin-functionalized nanoparticles linked together via the targeting agents. These nanoparticle aggregates can be formed by using, during the coupling of the nanoparticles with the targeting agent, a proportion of targeting agent greater than the proportion of nanoparticles, in particular a ratio of at least two targeting agents to one nanoparticle. Preferably, the ratio is adjusted so as to lead to aggregate sizes that maintain good colloidal dispersion in the medium in which the photoluminescent particles are implemented, in particular in aqueous media.

[0158] Any other method of forming controlled aggregates of nanoparticles can be implemented. For example, it is possible to add to the surface of the nanoparticles complementary oligonucleotides in addition to coupling agents that will lead to the formation of nanoparticle aggregates.

[0159] The implementation of particles formed from aggregates of photoluminescent nanoparticles makes it possible to amplify the luminescence signal per analyte, and thus to further improve the sensitivity of the detection.

[0160] In another particular embodiment, the ultrasensitive detection method according to the invention employs at least two distinct types of photoluminescent particles, each comprising nanoparticles according to formula (I), which are coupled to distinct coupling agents capable of binding to distinct sites on the same analyte. For example, the particles can be coupled to distinct oligonuclides that recognize different regions of the nucleic acid-type analyte. The use of photoluminescent particles associated with different sites on the surface of the analyte advantageously allows two or more nanoparticles to be attached to a single analyte molecule, thereby amplifying the luminescence signal per analyte and further lowering the sensitivity threshold achievable by the method of the invention.

[0161] The nanoparticles can each be coupled to a plurality of coupling agents, each coupling to a region of a substance of interest, in particular a plurality of oligonucleotides configured to bind to different regions of a nucleic acid, in particular distinct regions, preferably separated by a distance less than or equal to the size of the particle. These coupling agents may be identical or different. When a single nanoparticle binds to the substance of interest with more than one coupling agent, this allows the use of the biochemical principle of avidity and improves the binding of the nanoparticle to the analyte, thereby limiting the risk of loss of bond between the two during washing. Furthermore, this allows several nanoparticles to bind to different sites on the same analyte, thereby increasing detection sensitivity, as before.

[0162] It is understood that these particular embodiments can be combined to achieve maximum amplification of the luminescence signal per analyte in the sample to be analyzed.

[0163] Association of luminescent probes with the substance of interest

[0164] In a first step of the ultra-sensitive detection method according to the invention, the photoluminescent particles are associated with the substance of the sample to be analyzed.

[0165] This step can be carried out, in a manner similar to conventional methods, for example the enzyme immunoassay ELIS A, on the surface of a support, as schematically represented in [Fig. 1a].

[0166] This variant embodiment will be more particularly described later in the text.

[0167] It notably involves the prior immobilization, as described in example 2, of the substance of the sample to be analyzed, on the surface of a support.

[0168] In particular, the process may include functionalizing a support with additional coupling agents of the substance of interest, bringing the substance of interest into contact under recognition conditions with the additional coupling agents of the support, followed by bringing into contact a solution comprising the particles as described above in suspension, in particular in the form of a colloidal dispersion.

[0169] The support can be of various kinds. It can be coverslips, for example glass coverslips, multi-well plates such as those used in the examples, microplates, gel membranes, strips or microchannels. It can also be a plastic of good optical quality or any other material of sufficient optical quality.

[0170] Preferably, the surface of the support is pre-passivated so that the luminescent particles do not attach to it in the absence of the substance of interest.

[0171] Surface passivation can be carried out by any method known to a person skilled in the art.

[0172] This could involve, for example, passivating the glass surface using a molecule comprising polyethylene glycol (PEG), for example, using silane-PEG molecules. Preferably, the PEG has a molar mass of 500 to 20,000 g / mol. The longer the PEG, the better the resulting passivation. However, the PEG should advantageously not be too long so as not to cause steric hindrance to the binding of the substance of interest and to allow interaction between the targeting agent bound to the surface and the target. In particular, the smaller the coupling agent, the shorter the PEG should be.

[0173] The support is further functionalized on its surface by a first targeting agent for the substance to be detected / quantified. This may be, more specifically, an antibody, known as a capture antibody, as shown in phase 1 of [Fig. 1a], particularly when the substance of interest is a biomarker, protein, or polypeptide.

[0174] When the substance of interest is of the antibody type, the targeting agent may be of the antigen type specific to the antibody to be detected.

[0175] Surface functionalization can be carried out by any method known to those skilled in the art. For example, it can be carried out by adsorption following prolonged contact over several hours, by spotter printing (deposition of microdroplets of solutions containing the targeting molecules onto the surface using a robot), or by a contact printing technique allowing the transfer of molecules by contact between the topological motifs of a buffer (for example, a polydimethylsiloxane buffer). PDMS) and the surface of the substrate, or by other means known to those skilled in the art allowing the deposition of targeting agents on the surface of the support.

[0176] The sample to be analyzed is then brought into contact with the functionalized surface of said support so as to allow the association of the substance to be detected / measured with the targeting agent carried by the support (phases 2 and 3 of [Fig.la]).

[0177] This step involves, for example in a conventional ELISA test, an incubation step of the sample on the surface of the support, and a washing / rinsing of the support to remove the solution and unbound molecules. After rinsing, only the targeting agent / analytical substance complexes, for example antibody / antigen, remain attached to the surface of the support, with the exception of a few molecules that may have bound non-specifically.

[0178] Finally, the photoluminescent particles, as described above, formed in whole or in part from a photoluminescent nanoparticle and coupled to a coupling agent of the substance of interest, for example an antibody, are coupled with the substance of interest immobilized on the surface of the support (phase 4 of [Fig. 1a]).

[0179] This step involves incubating the photoluminescent particle solution on the surface of the functionalized support and then washing / rinsing the support to remove any particles not bound to it. After rinsing, only the targeting agent / analytical substance / particle complexes coupled to at least one coupling agent, for example monoclonal antibody / antigen / polyclonal antibody-nanoparticle, remain attached to the surface of the support, with the exception of a few nanoparticles that may have bound non-specifically. The incubation time can be adjusted by preliminary trials to maximize the luminescence emission signal. Generally, it can range from 30 minutes to 2 hours.

[0180] The coupling of particles coupled to a coupling agent with the substance of interest may, for example, involve recognition of a ligand / anti-ligand pair, for example biotin or biotinylated compounds / avidin or streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, such as digoxygenin (DIG) / anti-DIG antibody, sugar / lectin, polynucleotide / polynucleotide complement, etc., it being understood that one of the elements of these pairs constitutes the substance of interest, or the targeting agent or another element coupled to the substance of interest.

[0181] Thus, in one embodiment, step (i) comprises at least the following steps:

[0182] (a) have a support whose surface is previously passivated and functionalized with a capture agent for the substance of interest, for example a monoclonal antibody, called a capture antibody;

[0183] (b) bring said sample to be analyzed into contact with the support of step (a) in conditions conducive to the association of said substance of interest with the capture agent; and

[0184] (c) bringing the photoluminescent particles coupled to at least one coupling agent with said support from step (b) to associate directly or indirectly the particles with said substance immobilized on the surface of the support.

[0185] The association may be direct or indirect; in particular, the coupling agent may be a targeting agent for the substance of interest or a molecule capable of binding to a targeting agent coupled to the substance of interest. In the latter case, the process may involve contacting the substance of interest with targeting agents prior to contacting the particles. This contacting may occur before or after the capture of the substance of interest on the support. Preferably, the targeting agent is different from the capturing agent.

[0186] It is understood that the substance of interest can be immobilized in several predefined and distinct areas of the surface of the support.

[0187] This can be achieved, in particular, by implementing localized functionalization of the support surface with said targeting agent (for example, a capture antibody), as is the case when functionalizing multiple wells of a multiwell plate. This can involve deposition of the same targeting agent in several predefined areas. In this case, these multiple areas serve to detect the same substance in several different samples.

[0188] Such an implementation is more particularly carried out when using the ultra-sensitive detection method according to the invention for multiplexed analysis.

[0189] In the context of multiplex analysis, which allows the simultaneous detection and / or quantification of at least two different substances in a sample, the different substances to be analyzed in the sample can be immobilized in predefined and distinct areas on the surface of the support, for example by locally functionalizing the surface of the support with targeting agents specific to each of the substances to be analyzed. The surface is pre-passivated so that the photoluminescent particles do not adhere to it in the absence of the substances to be analyzed.

[0190] In this case, the particles used must have multiple different targeting agents on their surface, and, more specifically, at least one targeting agent specific to each of the substances to be analyzed. In this way, the substances to be analyzed will be quantified by the emission intensity of the particle on each area, the spatial location of the area in question indicating the nature of the substance.

[0191] It is also possible to combine multiplexed approaches for several samples and for several substances to be analyzed by locally functionalizing Predefined and distinct zones on the surface of the support are targeted by specific agents for each of the substances to be analyzed, and this is repeated as many times as there are samples to be analyzed. In this case, the emission intensity of the particle in each zone indicates the presence and / or concentration of each substance of interest in each sample, with the spatial location of the zone indicating both the nature of the substance and the number of samples.

[0192] It is also possible for multiplexed detection to implement at least two types of nanoparticles, doped with distinct rare-earth ions, exhibiting distinct emission wavelengths, for example YV04:Eu and YAG:Ce, and coupled to coupling agents, each to one of two different substances of interest. Detecting the luminescence signal using two different emission filters allows for the detection and / or quantification of each of the substances to be analyzed.

[0193] Preferably, in the context of such a variant of multiplexed detection, at least two types of nanoparticles having distinct emission wavelengths, and each coupled to targeting agents of each of the substances to be analyzed, can be used, so as to separate the luminescence signals obtained.

[0194] The combination of the two approaches can also be used (analysis of several different samples and several different substances in each sample), in particular for the comparison of the concentrations of target molecules between at least two samples, this being carried out by comparing the intensities of the emission colours of each of the nanoparticles, each coupled to the specific targeting agent of each substance of interest, and this for several deposition areas each corresponding to a different sample.

[0195] Alternatively, the combination of the two approaches can also be used—analysis in several different samples (sampling from different sources, or from the same source at different times, under different conditions, under different stimuli, etc.) of several different substances in each sample—particularly for comparing the concentrations of target molecules between at least two samples. This is done by comparing the intensities of the emission colors of each of the nanoparticles, each coupled to the specific targeting agents of each substance of interest, for several deposition areas corresponding to different target molecules. In this case, comparing the emission colors to a reference deposition area provides a comparison of the concentrations of a molecule between the two samples.

[0196] Various other combinations of these approaches can be envisaged: for example, analyzing four substances using two types of nanoparticles with two different emission colors, each coupled to two of the four different coupling agents necessary for the recognition of the four substances to be analyzed, and two distinct areas of the surface of the support locally functionalized by two of the four coupling agents specific to each of the substances to be analyzed.

[0197] By way of example, multiplexing can be implemented for the identification of viral variants in the case of virus genome detection.

[0198] The invention is of course not limited to the embodiment variant described below in which the substance of interest is immobilized on the surface of a support (such as glass strips or multi-well plates for example).

[0199] Other configurations are conceivable for the association of the photoluminescent particles of the invention with the substance of interest.

[0200] Alternative embodiments may implement, for example, a gel to separate biological molecules according to their size and / or charge followed by a transfer to a membrane where the molecules are specifically detected by nanoparticles coupled to the targeting agent, similar to the "Western blot" method.

[0201] In other variants, the reaction surface is not of the solid support type, but can be, for example, another magnetic nanoparticle, a magnetic microbead, etc. A magnetic field can then capture the luminescent nanoparticles associated with the analyte and the magnetic nanoparticles or beads in the vicinity of a surface before proceeding to measure the luminescence.

[0202] The measurement can, for example, be carried out directly within the sample to be analyzed. In the case of a gaseous sample, this sample holder can take the form of a closed volume to prevent dispersion of the sample being tested. The sample holder can also take the form of a cuvette or cuvette, particularly in the case of a solution.

[0203] The ultrasensitive method of the invention can also be adapted for use in flow cytometry technologies (in English, "Fluorescence-activated cell sorting" or FACS). In this case, particles of the invention, coupled with targeting agents for the recognition of molecules specific to the cell type to be analyzed, are brought into contact with the cells, and the cytometry system must be adapted to include an excitation source, preferably a UV laser diode, at a wavelength suitable for exciting the nanoparticle matrix.

[0204] The ultrasensitive method of the invention can also be adapted for use in immunocytochemistry and immunohistochemistry technologies. Luminescence measurement

[0205] As previously stated, the ultra-sensitive method according to the invention more particularly implements a step (iii) of excitation by UV-B radiation and / or UV-C of photoluminescent particles coupled to the substance of interest and a step (iv) of detection of the luminescence emitted by the particles. Detection assembly

[0206] The ultra-sensitive method according to the invention is advantageously implemented in with the help of simple and inexpensive equipment.

[0207] More specifically, it generally comprises: - an illumination device with a wavelength between 240 nm and 330 nm, preferably between 260 and 330 nm, preferably between 260 nm and 310 nm, preferably between 270 and 290 nm, preferably of the light-emitting diode type, preferably with a power rating of 500 mW or less, preferably 200 mW or less, for example between 50 and 150 mW; and - a device for detecting the light intensity emitted by the nanoparticles in step (iii).

[0208] Preferably, the illumination device emits in the UV-B and / or UV-C range.

[0209] Preferably, the detection device is devoid of a confocal system.

[0210] Reference will be made, in the remainder of the text, to the attached Figures 2 and 3, which represent, schematically and partially, installations adapted to the implementation of the ultra-sensitive process of the invention.

[0211] The apparatus may further include a suitable support for immobilizing the substance of interest of said sample during the process, as described above.

[0212] According to one embodiment, the apparatus according to the invention comprises a translation system for the support or the illumination device, allowing different localized areas of the support, for example different wells of a multi-well plate, to be illuminated successively. Such a variant is notably used for implementing the detection method of the invention for spatial multiplexing and / or for measuring several samples, the translation system allowing the successive illumination of each of the predefined areas containing the substances to be analyzed in the same sample, or even in several different samples.

[0213] The illumination device may also include an optical assembly, in particular a system of at least one lens, disposed on the path of the excitation beam so as to control the beam size at the level of the area of ​​the support presenting the particles associated with the substance of interest.

[0214] The optical beam-shaping assembly may conventionally include a collimation and beam-size reduction system, for example using lenses, in particular two lenses. This allows control of the illuminated area at the level of the region of the support presenting the particles associated with the substance of interest in order to obtain an appropriate intensity (by example 10 W / cm2) and an illumination whose dimensions are close to or smaller than those of the deposition spot (for example 1 mm in diameter).

[0215] Time-resolved detection can be achieved by mechanical or electronic chopping of the excitation beam. To enable such time-resolved detection, the apparatus, in particular the illumination device, may include a mechanical chopper placed in the path of the excitation beam. Chopping of the illumination can also be achieved electronically by modulating the current supplying the illumination source or by any other method known to those skilled in the art, such as by using an acousto-optic crystal.

[0216] Such time-resolved detection thus advantageously makes it possible to limit the contribution to the luminescence signal of parasitic species present in the sample (serum, blood, etc.) or in the solid substrates used (glass, plastic, etc.), in particular biomolecules, by temporally separating parasitic luminescence signals from the signal emitted by the nanoparticles, since these parasitic signals generally have characteristic lifetimes of less than 1 ps, or even less than 100 ns, or even less than 10 ns.

[0217] The detection of luminescence emission can be carried out by measuring the light intensity emitted at a luminescence wavelength of the photoluminescent particles used. For example, in the case of the use of Yi xEuxVO4 nanoparticles, the light intensity emitted can be measured at the luminescence wavelength of Eu3+, namely 617 nm.

[0218] The detection device may include a single detector, in particular a photomultiplier, photodiode, avalanche photodiode, or a detector array of photosensitive devices consisting of a 2D surface of detection pixels such as a CCD or EM-CCD camera or a CMOS camera. A 2D detection device makes it possible to simultaneously measure the emission signal of nanoparticles from different areas corresponding to different samples and / or different substances to be analyzed on the surface of the support and does not require movement of the support or the excitation beam.

[0219] Preferably, the light intensity detection device includes a single detector, in particular a photomultiplier, which makes it possible to make a less expensive detection device.

[0220] It may also include an optical assembly for collecting the emitted luminescence, in particular a system of at least one lens with a large numerical aperture, for focusing the luminescence emission towards the detector, in particular towards the photomultiplier.

[0221] Interference filters can also be placed in the path of the emitted beam to spectrally eliminate spurious signals.

[0222] Detection can be performed by reflection, that is, on the side of the support receiving the excitation beam, in particular by an epifluorescence device. Such detection makes it possible to reduce the background noise of the measurement, which is a particularly sensitive parameter, and thus improves the sensitivity of the measurement.

[0223] Alternatively, it can be operated in transmission. Luminescence measurement analysis

[0224] The process of the invention finally includes a step (v) of determining the presence and / or concentration of the substance by interpretation of the luminescence measurement.

[0225] It is understood that the detection device according to the invention may further include any means of analyzing the luminescence emission, for example a converter for recording and exploiting the luminescence signal.

[0226] The interpretation of the luminescence measurement can be carried out by reference to a pre-established standard or calibration.

[0227] More specifically, the quantity of the substance of interest in the sample can be determined by reference to a pre-established calibration curve by means of measurements carried out with samples of known quantity of said substance, preferably under conditions identical to those of the study of the sample, these identical conditions including in particular the solvent and the pH of the medium.

[0228] Advantageously, as illustrated in example 3, the ultra-sensitive method according to the invention makes it possible to detect and quantify a substance of interest in a sample at a content strictly less than 10 pM, in particular less than 1 pM, or even less than 0.1 pM, or even less than 0.01 pM (i.e. 10 fM), or even less than 1 fM, even better less than 0.1 fM (i.e. 100 aM), even better less than 0.01 fM (i.e. 10 aM).

[0229] In fact, it allows detection at least ten times, in particular at least 100 times, or even 1000 times more sensitive than the ELISA type enzymatic immunodetection method, using the same recognition and targeting antibodies. In vitro diagnostic set

[0230] The invention further relates, according to another aspect, to an in vitro diagnostic system, particularly for implementing the process as described above, comprising at least: - luminescent particles formed in whole or in part from a photoluminescent inorganic nanoparticle as defined above, said particles being coupled to one or more coupling agents, in particular being functionalized on the surface with chemical groups, for example carboxyl, amino, thiol, aldehyde or epoxy groups, provided by molecules, for example APTES, and / or coupled to molecules, for example streptavidin, said chemical groups or molecules being capable of enabling the coupling of said particles with a targeting agent of the substance of interest; or being already coupled to at least one targeting agent of the substance of interest; • a detection and / or quantification system comprising at least: • an illumination device as described above, • a device for detecting the intensity of light emitted by the particles, the detection and / or quantification system may include apparatus enabling time-resolved detection of luminescence emission, in particular a current modulation system supplying the illumination source, a photomultiplier and an AD converter.

[0231] A coating of the particles with silica can be used to facilitate the subsequent functionalization of the particles.

[0232] The in vitro diagnostic assembly according to the invention may further include a suitable support for immobilizing the substance of interest of said sample, as described above.

[0233] This may be a support whose surface has been passivated and functionalized with a capture agent, in particular a targeting agent, of the substance to be detected / quantified, for example with a first antibody, as described previously.

[0234] In a first embodiment, the in vitro diagnostic assembly according to the invention may include photoluminescent particles according to the invention already coupled to a targeting agent, in particular to antibodies, called "revealing antibodies" to distinguish them from capture antibodies immobilized at the level of the support.

[0235] The particles coupled to the targeting agent can be obtained as described above. In another embodiment, the in vitro diagnostic assembly may include particles that are not coupled to a targeting agent, from which the user can prepare particles coupled to one or more targeting agents for implementation in the ultrasensitive method according to the invention.

[0236] According to a particular embodiment, the in vitro diagnostic assembly according to the invention can thus comprise several containers comprising, in isolation, said uncoupled particles on the one hand and, on the other hand, one or more targeting agents.

[0237] Alternatively, the in vitro diagnostic set according to the invention does not include a targeting agent, the user being able to obtain separately the targeting agent, for example biotinylated, of his choice, from any competent supplier.

[0238] The preparation of particles coupled to targeting agents involves, in this variant of the embodiment, mixing the uncoupled particles according to the invention with the targeting agent, in concentration ratios predetermined by the contents of the containers in the case of the presence of the targeting agent within the in vitro diagnostic assembly according to the invention, or determined by the user, for example as described in Example 1.

[0239] The particles not coupled to a targeting agent implemented in an in vitro diagnostic set according to the invention may include particles coupled to molecules, for example streptavidin, capable of enabling the coupling of said particles with a targeting agent, for example biotinylated, for example a biotinylated antibody.

[0240] Other pairs of molecules can be considered for this type of coupling, for example hapten / antibody, antigen / antibody, peptide / antibody, such as digoxygenin (DIG) / anti-DIG antibody, sugar / lectin and polynucleotide / polynucleotide complement.

[0241] Alternatively, the particles not coupled to a targeting agent implemented in an in vitro diagnostic set according to the invention may include particles functionalized on the surface with chemical groups suitable for coupling said particles with a targeting agent of the substance of interest, for example carboxyl, amino, thiol, aldehyde or epoxy groups, provided by molecules, such as APTES.

[0242] It is understood that the functionalization of the surface of the nanoparticles may comprise more than one layer. For example, as mentioned above, the particles of the invention may comprise a layer for preparing or stabilizing the surface of the nanoparticles, such as a silica layer, followed by a functionalization layer with active chemical groups, such as a layer made of APTES (aminopropyltriethoxysilane).

[0243] The examples and figures shown below are given solely as illustrative and not as a limitation of the invention. Brief description of the drawings

[0244] [Fig. la] schematically represents a principle of detection and quantification of biomolecules: surface functionalized with a capture antibody (55) (phase 1); contacting the sample to be analyzed (phase 2), washing (phase 3) and association of the photoluminescent particles coupled to a targeting agent (42) (here an antibody) with the substance of interest (40), here immobilized on the surface of the support followed by a washing to remove the non-immobilized particles (phase 4);

[0245] [Fig. 1b] schematically represents a principle for detecting DNA / RNA nucleic acids using the particles according to the invention: single-stranded DNA (55), partially complementary to the strand to be detected, is fixed to a support. The single-stranded DNA constitutes a capture agent for the DNA or RNA to be detected. Next, the sample containing the DNA or RNA to be detected (40) is incubated with the functionalized support (phase A). After rinsing, the nanoparticles (38) coupled to single-stranded DNA (42), forming a targeting agent for the DNA or RNA to be detected, partially complementary to at least one unpaired portion of the DNA or RNA to be detected, are incubated with the support (phase B). After rinsing, only the nanoparticles (38) immobilized on the surface of the support after pairing with the DNA or RNA to be detected are present (phase C). They can be detected and quantified as described in the text.Contrary to what is schematically shown in the figure, it is preferable to have spacers between the surface and the complementary area to the substance of interest of the capture agent and between the surface of the nanoparticle and the complementary area of ​​the targeting agent.

[0246] [Fig.2] schematically represents a simple device for UV excitation of nanoparticles and detection of their transmission luminescence according to the invention.

[0247] [Fig.3a] schematically represents a UV excitation device;

[0248] [Fig.3b] schematically represents a reflection detection device according to the invention;

[0249] [Fig. 3c] schematically represents an assembly of the UV excitation device of [Fig. 3a] and the detection device of [Fig. 3b]. The additional lenses compared to the transmission device of [Fig. 2] allow better control of the collimation of the UV LED, the size and angles of the excitation beam at the sample level, as well as the size and angles of the luminescence collected when focused on the photomultiplier.

[0250] [Fig. 4] shows the detection of recombinant insulin in solution up to concentrations of 834 aM (10 fg / mL) with the detection device of [Fig. 2] and synthesized annealed YVO4:Eu (20%) nanoparticles as shown in Example 1 below. The nanoparticles are coupled to streptavidin in a 40:1 ratio to nanoparticles and coupled to biotinylated antibodies in a 60:1 ratio to nanoparticles. The same antibodies as those in ELIS Kit A were used. The black line indicates the average signal of the "white" samples. The grey line indicates the signal value corresponding to the average signal of the "white" samples plus 3 times the standard deviation of the "white" samples.

[0251] [Fig.5] represents the detection of gamma interferon in buffer solution (Figure 5a) or serum (Figure 5b) up to concentrations of 2.6 fM (50 fg / mL) with the detection device [Fig. 2] and synthesized annealed YVO4:Eu (20%) nanoparticles as shown in Example 1 below. The capture antibody is the same as that in Thermo Fisher Kit 13-7319-81; the detection antibody is the full antibody, while the kit contains a Fab fragment of the same antibody. The nanoparticles are coupled to streptavidin in a 40:1 ratio to the nanoparticles and coupled to biotinylated antibodies in a 30:1 ratio to the nanoparticles. The black line indicates the mean signal of the "blank" samples. The gray line indicates the signal value corresponding to the mean signal of the "blank" samples plus 3 times the standard deviation of the "blank" samples.

[0252] [Fig.6] represents the detection of gamma interferon in buffer solution (Figure 6a) or serum (Figure 6b) using a Thermofischer 88-7316-88 ELISA kit. The detection limit of the kit indicated by the supplier is 260 fM (5 pg / mL);

[0253] [Fig. 7] depicts the detection of HIV-p24 in solution by antibodies from a QC221 Quantikine Immunoassay Control Set 896 HIV-1 Gag p24 Biotechne kit as a capture and coupling agent. Detection is performed with the detection device [Fig. 2] and synthesized annealed YVO4:Eu (20%) nanoparticles as shown in Example 1 below; the detection antibody from the kit was biotinylated upstream to form the coupling antibody coupled to the nanoparticles in order to target the substance of interest during detection.

[0254] [Fig.8] schematically represents a detection principle optimized for the nucleic acids: surface functionalized with a capture oligonucleotide (55) (phase 1); contacting the sample to be analyzed, after denaturation, with detection oligonucleotides forming targeting agents (42) to couple the nucleic acid of interest (40) of the sample with the detection oligonucleotide (42) (phase 2), association of the nucleic acid-detection oligonucleotide conjugates with the functionalized surface (phase 3) and association of the photoluminescent particles (38) coupled to molecules allowing the fixation of the targeting agent (here streptavidin molecules) (44) with the detection oligonucleotide (42) in solution and the nucleic acid-detection oligonucleotide conjugates (phase 4), followed by a wash to remove the non-immobilized particles, i.e. not coupled to the nucleic acid-detection oligonucleotide conjugates;detection of luminescence emission by immobilized nanoparticles after excitation in UV-B light and / ; or UV-C at a wavelength of 280 nm with an intensity of 4.5mW / cm2 (phase 5).

[0255] [Fig. 9] represents the detection of the SARS-CoV-2 double-stranded ni gene in solution obtained by PCR up to concentrations of 50 fM with the detection device of [Fig. 2] and annealed YVO4:Eu(20%) nanoparticles synthesized as shown in Example 1 (Fig. 9a). Detection of the synthesized SARS-CoV-2 double-stranded ni gene (detection up to concentrations of 5 fM, Fig. 9b) and single-stranded (detection up to concentrations of 0.5 fM, [Fig. 9c]). The nanoparticles are coupled to streptavidin in a 40:1 ratio and coated with biotinylated detection oligonucleotides complementary to the DNA to be detected in a 1:180 ratio (streptavidin in excess). Here, the average signal of the "blank" samples was subtracted from all measured values. Thus, the average signal of the "blank" samples appears as equal to 0.The grey line indicates the signal value corresponding to the average signal of the "white" samples plus 3 times the standard deviation of the "white" samples.

[0256] [Fig. 10] represents the detection of the single-stranded SARS-CoV-2 ni gene with the device of [Fig. 2] and YVO4:Eu(5%) nanoparticles annealed at 1000°C synthesized as described in Example 1.2, detecting up to concentrations of 10 aM. The nanoparticles were coupled to streptavidin in a 40:1 ratio to the nanoparticles and coated with biotinylated detection oligonucleotides complementary to the DNA to be detected in a 1:180 ratio to the number of streptavidin molecules (excess streptavidin). Thus, the average signal of the "blank" samples is indicated by a black line. The gray line indicates the signal value corresponding to the average signal of the "blank" samples plus 3 times the standard deviation of the "blank" samples.

[0257] [Fig. 11] schematically represents a coupling of nanoparticles (38) coupled with streptavidin (44) which binds biotinylated antibodies (40) (42) in a proportion at least twice greater than that of the nanoparticles to form aggregates, some biotinylated antibodies coupling to at least two nanoparticles;

[0258] [Fig. 12] schematically represents a coupling of nanoparticles 38 coupled with streptavidin (44) binding biotinylated carbon chains (46) (42) comprising an arm linked to detection DNA (complementary to the target DNA) (48) and at least two arms linked to biotin (42), the carbon chains being in proportion at least twice greater than that of the nanoparticles to form aggregates, some carbon chains (46) coupling to at least two nanoparticles via biotin;

[0259] [Fig. 13] schematically represents a coupling of nanoparticles 38, each coupled with detection oligonucleotides (42i to 425) different from the others nanoparticles, complementary detection oligonucleotides at different regions of a target nucleic acid (40) fixed in a well functionalized by capture agents (55), for example oligonucleotides complementary to the target nucleic acid; and

[0260] [Fig. 14] schematically represents a coupling of a nanoparticle (38) coupled with different detection oligonucleotides (42i to 424) complementary oligonucleotides of a target nucleic acid (40) fixed in a well functionalized by capture agents (55i to 553), for example oligonucleotides complementary to the target nucleic acid, at least two different oligonucleotides of the nanoparticle binding to different but close areas of the same nucleic acid and at least two targeting agents binding to different areas of the target nucleic acid. Detailed description

[0261] Preparation of luminescent particles of formula Y 0>6 Eu 0.4 VO 4

[0262] YVO4 nanoparticles doped with 20% Europium are prepared by a known method, in particular by the method as described in detail in application WO 2019 / 025618 or by the method described below.

[0263] A freshly prepared 10 mL aqueous solution of 0.1 M NH4VO3 and 0.3 M N(CH3)4OH (solution 1) is prepared. A 10 mL volume of another solution of 0.1 M Y(NO3)3 and Eu(NO3)3 (Y3+ + Eu3+ ions) is added dropwise using a syringe pump to solution 1 at a flow rate of 1 mL / min. The molar concentration ratio of Y(NO3)3 to Eu(NO3)3 is chosen according to the desired Y3+ to Eu3+ ion ratio in the nanoparticle; typically, the Y3+:Eu3+ molar ratio is 0.8:0.2.

[0264] Upon addition of the Y(NO3)2 / Eu(NO3)3 solution, the solution becomes diffusive and appears white / milky. The synthesis continues until the Y(NO3)2 / Eu(NO3)3 solution is completely added. The solution is then left under stirring for 15 days at room temperature. The final 20 mL solution must now be purified to remove excess counterions. To do this, centrifugations (typically three) at 11,000 g (Sigma 3K10, Bioblock Scientific) for 80 minutes, each followed by redispersion by sonication (Branson Sonifier 450 operating at 50% with a power of 540 W), are used until a conductivity strictly below 100 pS·cm⁻¹ is achieved.

[0265] The nanoparticles obtained are annealed under hydrothermal conditions at 220°C for 2 hours in an autoclave. Such annealing improves crystallinity and thus reduces the photoreduction effect of the nanoparticles when excited in UV-B and / or ruv-c.

[0266] Preparation of luminescent particles of formula Y o>95 Eu 0,s VO 4

[0267] Vanadate nanoparticles of formula Y 0.95 Eu 0.5 VO 4 were prepared via the Colloidal conversion of rare-earth hydroxycarbonate particles in two steps. In a general procedure, 20 mL of a 0.1 mol L⁻¹ RE(NO₃)₃ solution [RE = (Y₀ 0.95Eu 0.05)], 100 mL of Milli-Q water, 80 mL of ethylene glycol (EG), and 15 g of urea were added to a 500 mL three-necked flask. The final H₂O / EG volume ratio was 3:2 (i.e., a 40% EG volume fraction in water). The mixture was homogenized with vigorous stirring at room temperature for 30 minutes. The flask was then connected to a condenser and heated to 95 °C for 2 hours. The resulting suspension was centrifuged at 26,323 g for 25 minutes. The pellet was redispersed in Milli-Q water and centrifuged again (final conductivity less than 100 pS cm⁻¹). Finally, the RECO3OH nanoparticles were redispersed in 200 mL of Milli-Q water and heated to 100 °C.After 20 minutes, a 0.1 mol L⁻¹ NH₄VO₃ solution (2 mmol, 20 mL, Sigma-Aldrich) was rapidly added to the colloid while stirring. The system was maintained at 100 °C for 2 hours, and the particles were collected by centrifugation (26323 g, 30 minutes) and purified by dialysis against Milli-Q water for 24 hours.

[0268] Observation of the particles by electron microscopy shows that they have an olive shape with average dimensions of 153 nm in length and 67 nm in width.

[0269] In a typical protected annealing procedure, a silica polymeric sol was prepared under acidic conditions by mixing TEOS (Si(OC2H5)4), water (pH 1.25), and ethanol in a molar ratio of 1:5:3.8, and allowing it to age for 1 h at 60 °C. To facilitate the dissolution of this silica matrix, a porous structure was prepared by gelling the silica around self-organizing micellar assemblies of a surfactant copolymer. The copolymer Pluronic PE6800 (EO73PO28EO73), with a molar weight of 8080 g / mol (BASF Europe), was dissolved in ethanol at 40.4 g / L. The final solution was obtained by mixing the colloidal solution of the nanoparticles, the silica solution and the PE6800 solution.For YVO4:Eu particles, the basic nature of the colloidal solution necessitates the use of surfactant concentrations in the soil that do not consistently lead to an organized silica matrix (typical molar ratios V:Si:PE6800 are 1:5:0.05). The gel was dried at 90 °C for over 6 hours, and the resulting powder was air-annealed at 1000 °C in two stages. The first annealing was carried out at 500 °C at a rate of 100 °C / h with a final stage of 1 hour, and the second annealing at 1000 °C at a rate of 100 °C / h with a final stage of 2 hours at 500 °C and 10 minutes. at 1000°C. The first annealing is necessary for the complete removal of organic matter (PAA and PE6800 polymers).

[0270] The silica powder containing the particles was dissolved in excess 2% hydrofluoric acid for 3 h in a Si:HF molar ratio of 1:9. The hydrofluoric acid and dissolved silica were then removed by two centrifugations at 14,000 g, the first for 1 min and the second for 10 min. The precipitate was diluted in pure water, and a few drops of sodium hydroxide were added to fix the pH to 10⁻¹¹. The final solution was stabilized by the addition of PAA (V:PAA 1:0.05) and by sonication in a cold bath for 5 min.

[0271] Coupling of nanoparticles with the streptavidin protein

[0272] Following nanoparticle synthesis and annealing, the nanoparticles are functionalized as described in international application WO2019 / 025618 by a silicate step, an amination step using APTES, and then a step transforming the amines into carboxylic acids using an anhydride. For greater versatility, the nanoparticles are coupled with a 40:1 streptavidin:nanoparticles ratio by the method detailed below.

[0273] The appropriate volume is pipetted to obtain 150 pL of -COOH-coated nanoparticles at a concentration of 200 nM. The sample is then centrifuged for 15 minutes at 13700 g and the supernatant is discarded. 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) are solubilized at a concentration of 50 mg / mL each in a 50 mM buffer of 2-(N-morpholino)ethanesulfonic acid (MES) at pH 5.5. This solution is rapidly added to the nanoparticle pellet and the sample is sonicated for 15 seconds at 50% amplitude (GEX130 ultrasonic processor, tip ref. 423-A). After incubation at room temperature for 25 minutes with stirring, the sample is centrifuged for 15 minutes at 15000 g and the nanoparticle pellet is redispersed in a 50-mM phosphate buffer pH 7.4 by sonication for 15 seconds at 50%.40 equivalents of streptavidin (Sigma, s4762-10MG) are then added and the sample is incubated at 25 °C with stirring at 800 rpm (Eppendorf Thermomixer C) for 2.5 hours. The sample is centrifuged for 15 minutes at 13700 g and the pellet is resuspended in 500 pL of blocking buffer (50 mM phosphate buffer pH 7.4 containing 2% mPEG-NH2 500 (MF001005-500 Biochempeg)) by pulsed sonication 1 s on / ls off for 10 s at 20% amplitude on ice. The sample is then incubated at room temperature for 1 hour with shaking, centrifuged for 15 minutes at 13700 g, and the pellet, corresponding to the streptavidin-nanoparticle conjugates, is redispersed in a storage buffer (Tris 20 mM, pH 8, 1% BSA) and stored at -80°C.

[0274] Coupling of streptavidin-coupled nanoparticles (Nanoparticles-SA) with biotinylated antibodies

[0275] The nanoparticles coupled with streptavidin are then coupled to a biotinylated antibody, specific to the substance to be measured, by a known method, in particular as described in the protocols below by adapting the ratio of biotinylated antibody and nanoparticles according in particular to the target substance to be measured (insulin, interferon gamma (IFN-gamma) or HIV-I-p24 protein as described below).

[0276] Preferably, as illustrated in [Fig. 10], the amount of biotin 42 is greater than that of the nanoparticles 38 and antibodies 40, so that the nanoparticles 38 aggregate. Such an aggregate improves detection sensitivity by increasing the number of nanoparticles bound to each molecule of the substance of interest. Indeed, it is no longer a single nanoparticle that binds to a substance of interest, but an aggregate of nanoparticles, which exhibits a much stronger luminescence signal.

[0277] Coupling of streptavidin-coupled nanoparticles with biotinylated oligonucleotides

[0278] Alternatively, when it comes to detecting nucleic acids, as illustrated in [Fig. 11], the nanoparticles 38 coupled with streptavidin 44 can couple with carbon chains 46 having at least two biotinylated arms 42 and one arm bearing a detection oligonucleotide. The fact that the carbon chains have two or more biotinylated arms allows them to bind to two streptividines, in particular from different nanoparticles, thus enabling the formation of a complex.

[0279] As illustrated in [Fig. 12], the nanoparticles 38 can be coupled with different targeting agents 42i to 425, in particular different complementary oligonucleotides, to target different areas of a substance of interest 40 fixed to a support, in particular a nucleic acid. This improves sensitivity by allowing the attachment of several nanoparticles to the same molecule of the substance of interest.

[0280] As illustrated in [Fig. 13], each nanoparticle 38 can carry several different targeting agents 42i to 424, in particular different complementary oligonucleotides, to target different areas sufficiently close to a substance of interest 40, in particular a nucleic acid. This improves the coupling strength of the nanoparticle 38 with the substance of interest 40 by reducing the dissociation rate. The capture surface can also be functionalized with targeting agents 55i to 553, in particular different complementary oligonucleotides, to bind to different areas of the substance of interest 52.

[0281] Nanoparticles can be added to a support container, in particular the micro-wells of a plate, and then the sample to be analyzed is incubated in the same micro-well.

[0282] Preparation of the capture surface: antibody fixation.

[0283] 100 pL of capture antibodies are taken from the commercial kit as indicated in The kit protocol is applied to the kit's fixation buffer and incubated in each well of a multiwell plate (655097, Greiner) for 16 h at 4 °C with shaking at 300 rpm. The wells are then washed twice with the commercial diluent provided with the kit or with 0.01% PBS Tween if it is not included. Next, 200 pL of blocking agent is added to each well to reduce nonspecific interactions for 2 h at 4 °C with shaking at 300 rpm.

[0284] Preparation of the capture surface: fixation of oligonucleotides.

[0285] The anti-digoxigenin antibody (Ab64509, Abcam) is applied to the bottom of multiwell plates at 10 pg / mL in 100 pL of PBS buffer pH 7.2 and incubated overnight at 4°C with shaking. The plate is washed three times with 200 pL of modified 5X SSC (SSC: 750 mM NaCl, 75 mM sodium citrate, 0.05% SDS, 0.05% lauroylsarcosine). The wells are blocked with 3% PBS buffer pH 7.2 for 2 hours at 4°C with shaking and washed once with 200 pL of 5X SSC. 100 µl of capture oligo Digoxigenin-5' (5'-capture oligonucleotide) 16.5 pM are added and the plates are maintained at 4°C under stirring.

[0286] Experimental setup for measuring luminescence

[0287] The detection setup includes an illumination device and a luminescence detection device shown in [Fig.2]. This is the setup that was used for all the results presented below.

[0288] In the detection setup of [Fig. 2], the illumination device consists of a light-emitting diode (LED) (1) coupled to a 1 mm diaphragm positioned 3 mm from the diode (1). The LED (Hex-S6060-DR250-W275-P100-V6.5, Laser Components) emits in the UV-C range at a wavelength of 275 nm and with a power of 100 mW. This allows for the illumination of a single well in the multi-well plate, the bottom of each well being positioned 20 mm from the diode. The detection device consists of a collimating lens (20) of the light emitted by the nanoparticles in the sample, by a converging lens (26) which focuses the emitted light onto a photomultiplier type detection module (28) (PMM02, Thorlabs) equipped with an interference filter which filters the emitted light at 617 nm.

[0289] An alternative version of the detection setup is shown in [Fig. 3a]-c. The detection setup is explained below. The illumination device ([Fig. 3a]) consists of a UV-C emitting light-emitting diode (1) at a wavelength of 275 nm and with a power of 100 mW and a collimation system and reduction of the size of the laser beam (2). The collimation and beam size reduction system (2) shown consists, from the diode to the sample, of a collimating lens (3), a set of diaphragms (4) (two iris diaphragms, a field diaphragm and a diaphragm allowing total beam stopping), a converging lens (6), an iris diaphragm, called an aperture diaphragm (8) at the focal point of the converging lens, a collimating lens (10), a focusing lens (12), a dichroic mirror adapted to reflect the illumination light and transmit the light emitted by the sample (35), a large numerical aperture objective (16), for example with a numerical aperture of 0.79, and finally an illumination diaphragm (18).The illumination device could, however, consist of only the diode and a suitable diaphragm to limit the so-called "cross talk" effect, as illustrated in [Fig.2], to avoid simultaneously illuminating several wells of the multi-well plate.

[0290] The detection device ([Fig. 3b]) comprises, following the path of light from the sample, the objective (16) for collecting and collimating the luminescence emitted by the nanoparticles in the sample, a converging lens (22), a spectral filter (27) filtering the light at 617 nm comprising a collimating lens, a 617 nm interference filter and a converging lens, and a photomultiplier-type detection module (28) (PMM02, Thorlabs). [Fig. 3c] shows the complete detection setup comprising the illumination device and the detection device.

[0291] An analog-to-digital converter (NI9215, National Instrument) allows the signal to be recorded using Labview software.

[0292] All detection elements may be located on the same axis or not (through the appropriate use of one or more mirrors). A slide support translation system (Z8253, KCH301 Thorlabs) was implemented to allow for the successive observation of several biological samples by scanning.

[0293] Time-resolved detection for measuring the quantity of nanoparticles in the presence of spurious signals

[0294] The light-emitting diode can be powered by voltage pulses via an NL9215 input module, National Instruments, to create UV excitation pulses in order to eliminate spurious signals through time-resolved detection. Indeed, when the biological sample is illuminated by the diode, molecules other than the nanoparticles of interest emit fluorescence. The use of UV excitation pulses and a signal detection frequency of 100 kHz by the photomultiplier tube (signal acquisition every 10 ps) makes it possible to overcome this parasitic fluorescence. It is therefore possible, due to the long emission time of the particles of the invention, to perform time-resolved detection of the emission, in particular delayed detection of the emission, as described in detail below.

[0295] Time-modulated illumination makes it possible to limit the contribution to the luminescence signal of parasitic species present in the sample (serum, blood, etc.) or in the solid substrates used (glass, plastic, etc.). Indeed, the nanoparticles used (YVO4:Eu or GdVO4:Eu, for example) can be placed in an excited state with a long lifetime, on the order of a few hundred ps, compared to the lifetimes of conventional fluorophores, which are in the nanosecond range. This allows the temporal separation of parasitic luminescence signals from the signal emitted by the nanoparticles.

[0296] The modulated signal obtained is the alternation of a decay phase (illumination shutdown) and a luminescence recovery phase (illumination start-up) of all the emitters present in the sample. The decay / recovery of the luminescence signal is determined by two distinct parameters: (i) the lifetimes of the excited states of the emitters, and (ii) the dynamics of the ignition and shutdown times of the excitation beam. Variations of the experimental setup

[0297] The luminescence emission of the nanoparticles is collected: . either in transmitted light ([Fig.2]), . either in reflected light (figures 3a to 3c) using a dichroic mirror (30) at 347 nm.

[0298] For each sample concentration to be detected, several wells N of a multiwell plate can be used, typically 3. The measurement points for each concentration are then presented as the mean and standard deviation of the N values ​​obtained for each well. The measured value for each well is the average of 60,000 values ​​recorded over 600 ms with an acquisition rate of 100 kHz (1 voltage value recorded every 10 ps).

[0299] Typically, the limit of detection (LOD) is considered to be determined by the concentration generating a signal equal to or greater than the sum of the signal obtained at zero concentration ("blank") and 3 times the standard deviation of the "blank" signal. The limit of quantification can be considered to be the concentration generating a signal 10 times greater than this standard deviation. However, an experimental determination of the limit of quantification is preferable. Calibration of the detection device

[0300] Prior to the measurements, the detection device was calibrated with known concentrations of the substance to be detected.

[0301] Detection and quantification of a substance in a sample

[0302] The concentration of a substance in a sample is considered detectable when the signal obtained is at least equal to or greater than the sum of the signal for a sample of the same composition containing a zero concentration of the substance, called a "blank" sample, and three times the standard deviation of the signal of the "blank" sample.

[0303] To quantify the substance of interest (i.e., determine its concentration), the following protocol must be implemented: i. Perform a series of calibration measurements with the substance of interest at different known concentrations, for example from commercially available substances or from purification. Where possible, the calibration samples should be prepared with the same composition as the samples to be measured or with a composition as close as possible. Adjust the points obtained (signal in mV versus concentration of the substance of interest); ii. perform measurements of the samples to be analyzed (obtaining the signal value in mV); iii. assign to each measured sample a concentration value of the substance from the measured signal (in mV) and from the calibration curve carried out in step i) and its adjustment.

[0304] To determine from what concentration the substance in a sample is quantifiable with a given coefficient of variation (CV), e.g. 25%, 20%, 15% or 10%, the following protocol must be implemented: i. Carry out a sufficient number of measurements for samples of different known increasing concentrations located above the limit of detection (LOD); ii. Determine the standard deviation and coefficient of variation related to the variability of the measurement for each of these concentrations as well as the concentration from the calibration curve; iii. Determine the coefficient of variation related to the bias, i.e. the standard deviation between the nominal concentration and the concentration determined from the measurement, normalized to the value of the concentration; iv. If either of these two determined coefficients of variation is greater than the predetermined coefficient of variation, repeat the measurements at higher concentrations of the substance.

[0305] The limit of quantification for a given CV corresponds to the concentration of the substance for which the coefficient of variation related to the variability of the measurement and that related to the bias are equal or less than or predetermined CV.

[0306] Comparison of detection sensitivity between commercial ELISA tests and the method of the invention

[0307] For each analyte to be detected, the same 96-well plates, the same antibodies and substantially the same buffer solutions as those of commercial ELISA tests are used, with the difference that the HRP (HorseRadish Peroxidase) enzyme is replaced by nanoparticles of formula Yo,6Euo,4V04, obtained by the manufacturing process described above, functionalized on the surface with streptavidin molecules and coupled with biotinylated antibodies of each of the substances to be analyzed, prepared in example 1. Insulin detection

[0308] The sample analyzed is recombinant insulin (Abcam ELISA kit abl00578).

[0309] For nanoparticle-based detection, we use the same multiwell plates coated with commercial capture antibodies, the same biotinylated detection antibodies, insulin and diluent A, B and wash buffer solutions as those supplied in the Abcam kit above.

[0310] An insulin solution is prepared in diluent A to obtain a concentration of 10 ng.mL-1. Then, the solution is cascade diluted by a factor of 10 (triplicate for each concentration and fifty "blank" samples) and the microwell plate is incubated for 2h30 at 25 °C under stirring at 300 rpm (Eppedorf Thermomixer C).

[0311] In parallel, the streptavidin-nanoparticle conjugates are incubated with 60 eq of biotinylated detection antibodies in phosphate-buffered saline (PBS) pH 7.4 for 1.5 hours at 25°C. The solution is then centrifuged for 15 minutes at 13,000 g. The pellet is resuspended in diluent B by pulsed sonication (10 s on / 1 s off) in ice to obtain a final nanoparticle concentration of 4.5 nM.

[0312] The microwell plate is washed three times with the wash buffer. Then, 100 µL of nanoparticle-antibody conjugate solution is added and the plate is incubated for 1.5 hours at 25°C with shaking at 300 rpm. The plate is washed three times with the wash buffer and once with PBS pH 6.6. Then, 200 µL of PBS pH 6.6 is added to each well and the plate is read.

[0313] The standard deviation for the zero concentration is 28 mV (see [Fig.4]). The signal value measured for the concentration of 10 fg / mL (or 834 aM) is 409 mV, above the limit value of 289 mV equal to the mean of the "blank" samples plus 3 times the standard deviation determined for the "blank" samples.

[0314] For comparison, the lowest detectable concentration indicated by the ELISA kit supplier is 50 pg / mL (or 4.17 pM).

[0315] The minimum concentration that can be detected with the ultra-sensitive detection method according to the invention is thus 5000 times lower than the concentration detectable by ELISA using the same antibodies as the ELISA kit. Detection of gamma interferon

[0316] The sample analyzed is interferon gamma (Thermofisher ELISA kit 88-7316-88).

[0317] For nanoparticle-based detection, 100 pL of capture antibody from the commercial kit are diluted 250-fold in the kit's fixation buffer and incubated in each well of the multiwell plate for 16 h at 4 °C with shaking at 300 rpm. The wells are then washed twice with the commercial ELISA / Elispot diluent provided with the kit. Next, 200 pL of the ELISA / Elispot diluent are added to each well for a blocking step for 2 h at 4 °C with shaking at 300 rpm.

[0318] A range of interferon-gamma concentrations is prepared in commercial diluent (250 pg.mL-1 to 25 fg.mL-1). 100 pL is added to each well in triplicate, and the plate is incubated for 16 hours at 4°C with shaking at 300 rpm. The wells are then washed three times with PBS. In parallel, YVOVEu 20%:streptavidin 1:40 nanoparticles are mixed with 60 equivalents of detection antibody (4S.B3 Thermofischer 13-7319-85) in PBS pH 7.4 for 1 h at room temperature with gentle shaking. The mixture is centrifuged for 15 minutes at 13,700 g. The pellet is suspended in PBS by pulsed sonication (10 s on / 1 s off) in ice to obtain a nanoparticle concentration of 4.5 nM. Then, 100 µL of nanoparticle / 4S.B3 solution is added to each well, and the plate is incubated for 1.5 hours at 25°C at 300 rpm. The plate is washed three times with PBS pH 7.4 and read with the in-house reader ([Fig. 2]).

[0319] The results are illustrated in [Fig. 5]. In buffer (Figure 5a), the signal value measured for the concentration 160 fg / mL was 160.8 mV, above the detection limit of 149.1 mV, which is equal to the mean of the blank samples (133.8 mV) plus 3 times the standard deviation determined for the blank samples. In serum (Figure 5b), the signal value measured for the concentration 800 fg / mL was 149.7 mV, above the detection limit of 148.3 mV, which is equal to the mean of the blank samples (138.5 mV) plus 3 times the standard deviation determined for the blank samples.

[0320] For comparison, the detection of interferon gamma was performed according to the conditions of the ELISA kit. The experimental conditions followed were those indicated by the ELISA kit supplier. The results are shown in Figure 6a for measurements in buffer and in Figure 6b for measurements in serum. The lowest concentration measured was 5 pg / mL in buffer and 62.5 pg / mL in serum.

[0321] The minimum concentration that can be detected with the ultrasensitive detection method according to the invention is thus 31 times lower than the concentration detectable by ELIS A when measurements are made in buffer and 78 times lower than the concentration detectable by ELISA when measurements are made in serum. Detection of HIV-p24

[0322] The sample analyzed is HIV-p24 (QC221 Quantikine Immunoassay Control Set 896 HIV-1 Gag p24 Biotechne).

[0323] For nanoparticle-based detection, 100 pL of Biotechne capture antibody, MAB73602-100 pL at 10 pg / pL, are diluted in PBS and incubated in each well of the multiwell plate for 16 h at 4 °C with shaking at 300 rpm. In parallel, the streptavidin-nanoparticle conjugates are incubated with 30 eq of biotinylated detection antibody (Biotechne, NBP3-06466-100 pL) in PBS pH 7.4 for 1.5 h at 25 °C. The solution is then centrifuged for 15 minutes at 13,000 g. The pellet is resuspended in PBS by pulsed sonication 10 s 1s on / ls off in ice to obtain a final nanoparticle concentration of 4.5 nM.

[0324] The microwell plate is washed three times with PBS 7.4, Tween 20 0.01% wash buffer recommended by the antibody supplier. Then, 100 pL of nanoparticle-antibody conjugate solution is added and the plate is incubated for 1.5 hours at 25°C with shaking at 300 rpm. The plate is washed three times with the wash buffer and once with PBS pH 6.6. Then, 200 pL of PBS pH 6.6 is added to each well and the plate is read with the reader in [Fig. 2].

[0325] The standard deviation for the zero concentration is 5.4 mV. The signal value measured for the concentration 500 fg / mL (or 20.8 fM) is 251 mV, above the limit value of 246 mV equal to the mean of the "blank" samples plus 2 times the standard deviation determined for the "blank" samples in accordance with the definition of the commercial test (see [Fig.7]).

[0326] For comparison, the lowest detectable concentration reported by the antibody supplier is 15.6 pg / mL (or 650 fM), Biotechne HIV-1 Gag P-24 DuoSet ELISA kit. This limit of detection was defined as the concentration giving a signal equal to or greater than the mean signal of the "blank" samples plus 2 times the standard deviation of the "blank" samples.

[0327] With the ultra-sensitive detection method according to the invention, the lowest measurable concentration being 500 fg / mL (or 20.8 fM), with the device of [Fig.2], the minimum concentration that can be detected is thus 32 times lower than the concentration detectable by ELISA using the same antibodies. Detection of the SARS-CoV-2 gene

[0328] The optimal scheme used for detection is shown in [Fig.8]. The sample analyzed is the PCR product of the SARS-CoV-2 ni gene. The anti-digoxigenin antibody (Ab64509, Abcam) is applied to the bottom of multiwell plates at 10 pg / mL in 100 pL of PBS buffer, pH 7.2, and incubated overnight at 4°C with shaking. The plate is washed three times with 200 pL of modified 5X SSC (750 mM NaCl, 75 mM sodium citrate, 0.05% SDS, 0.05% lauroylsarcosine). The wells are blocked with 3% PBS buffer, pH 7.2, for 2 hours at 4°C with shaking and washed once with 200 pL of modified 5X SSC. 100 pL of the capture oligo Digoxigenin-5' (5'-GACCCCAAAATCAGCGAAAT) 16.5 pM are added and the plates are maintained at 4°C with shaking. Concurrently, 60 pL of the target DNA at 43 nM are denatured for each concentration at room temperature for 15 minutes in a [0.1 M NaOH, 0.01% Tween 20] solution, then 540 pL of the detection oligo 3'-biotin (5'-CAGATTCAACTGGCAGTAACCAGA) at 40 nM in modified 5X SSC are added.After washing the wells with 200 pL of modified 5X SSC, 100 pL of this target-oligo detection DNA conjugate solution are added to the plate wells. The plate is incubated at 4°C with shaking for 30 minutes. 100 pL of 4.5 nM YVO4:Eu 20%-streptavidin nanoparticle conjugates are added, and the samples are incubated for 1 hour at 4°C with shaking. The wells are then washed 5 times with modified 5X SSC and 2 times with PBS before being read with the homemade UV reader [Fig. 2].

[0329] The standard deviation for the "blank" samples is 22.8 mV. The signal value measured for the 50 fM concentration is 348 mV, just above the limit value of 335 mV, which is equal to the mean of the "blank" samples plus three times the standard deviation determined for the "blank" samples. Thus, the limit of detection for the SARS-CoV-2 non-double-stranded gene is 50 fM (Figure 9a). When the substance of interest is the synthesized SARS-CoV-2 non-double-stranded gene (Eurogentec), the lowest detectable concentration is 5 fM (Figure 9b). When the substance of interest is the synthesized SARS-CoV-2 non-single-stranded gene (Eurogentec), the lowest detectable concentration is 0.5 fM (Figure 9c). This detection limit compares favourably with literature results obtained without enzyme and without nucleic acid amplification

[14] .

[0330] Detection of the SARS-CoV-2 ni gene with nanoparticles annealed at 1000°C

[0331] Nanoparticles of formula Yo.95Euo.5VO4 obtained according to the process described above were functionalized and coupled to streptavidin as described above, then coupled to biotinylated oligonucleotides as described above. The same protocol as in the previous example concerning the detection of the SARS-CoV-2 ni gene was then implemented. The signal value measured for the 10 aM concentration was 524.6 mV, well above the limit value. The 508 mV value was equal to the average of the "blank" samples plus three times the standard deviation determined for the "blank" samples. In this case, thanks to the higher number of photons emitted per unit time by these nanoparticles, linked to the greater number of ions (i.e., the larger size of the nanoparticles) and their higher quantum yield, a sensitivity limit of <10 aM was obtained, which is comparable to the detection limit of classical PCR (approximately 1 aM, corresponding to 30,000 nucleic acid molecules / mL). This result was therefore obtained without amplification and without the use of enzymes. List of documents cited

[0332] [1] Hermanson, Bioconjugate Techniques, Academie Press, 1996;

[0333] [2] Mason, Fluorescent and Luminescent Probes for Biological Activity. A Practical Guide to Technology for Quantitative Real-Time Analysis, Second Edition, Academie Press, 1999;

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[0335] [4] Nam et al., Science, 2003, 301, pp. 1884-1886;

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[0338] [7] Pisanic et al., Analyst, 2014, 139(12), pp. 2968-2981;

[0339] [8] Geissler et al., Angewandte Chemie International Edition, 2010, 49(8), pp. 1396-1401;

[0340] [9] Howes et al., Bionanotechnology, 2014, 346(6205), pp.53-63;

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[12] Zhu et al., Anal. Chem., 2013, 85(2), pp 1058-1064;

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[13] Cordeiro et al., Diagnostics, 2016, 6(4):43;

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[14] Sakharov Y., Sensors 2018, Microplate Chemiluminescent Assay for DNA Detection Using Apoperoxidase-Oligonucleotide as Conjugal Capture and HRP-Streptavidin Signaling System;

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[15] Lorenzo E., Guttierez-Galvez L., Talanta 2022,_Electrochemiluminescent nanostructured DNA biosensor for SARS-CoV-2 detection;

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[16] Bouzigues et al., ACS Nano, 2011, 5(11) pp. 8488-8505;

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[22] Tang et al., Clin Vaccine Immunol, 2009, 16(3), pp. 408-413;

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Claims

Demands

1. Method for the ultra-sensitive in vitro detection and / or quantification of a substance of biological or chemical interest (40) in a sample (35), in particular a biological sample, by detecting the luminescence emission emitted by photoluminescent inorganic nanoparticles, comprising at least the following steps: i. arrangement of photoluminescent particles (38) formed in whole or in part from a vanadate or vanadate / phosphate matrix photoluminescent inorganic nanoparticle of formula (I): A! xLnxVO4(i _y)(PO4)y(I) in which: . A is chosen from yttrium (Y), gadolinium (Gd), lanthanum (La) and their mixtures, in particular A represents Y; . Ln is chosen from europium (Eu), dysprosium (Dy), thulium (Tm), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb) and their mixtures, in particular Ln represents Eu; 0 < x < 1; in particular 0.02 < x < 0.5, in particular 0.05 < x < 0.4, and more particularly x equals 0.4, 0.2, 0.1, or 0.05; and . 0 < y < 1, in particular y equals 0, the photoluminescent inorganic nanoparticles being coupled to a direct (42) or indirect (44) coupling agent to the substance of interest, ii. bringing said photoluminescent particles (38) into contact with the substance of interest (40) under conditions of coupling of the substance of interest (40) with the coupling agent (42, 44), iii. excitation of the matrix of photoluminescent inorganic nanoparticles of formula (I), by radiation with a wavelength between 240 nm and 330 nm, the radiation being emitted by an illumination device with a power between 50 mW and 500 mW; iv. detection of luminescence emission by photoluminescent inorganic nanoparticles, in particular time-resolved detection, and v. determination of the presence and / or concentration of the substance of interest by interpretation of said measurement of luminescence emission by the particles, the process having a detection sensitivity of the biological or chemical substance of interest in the sample less than or equal to 10 pM.

2. A method according to claim 1, the sensitivity of detection of the biological or chemical substance of interest in the sample being less than 1 pM, or even less than 0.1 pM, or even less than 0.01 pM, or even less than or equal to 1 fM, even better less than or equal to 0.1 fM (i.e. 100 aM), even better less than or equal to 0.01 fM (i.e. 10 aM), in particular without amplification and without the use of enzymes.

3. A method according to any one of the preceding claims, wherein the coupling agent is a targeting agent (42) of the substance of interest coupling directly to the substance of interest in step (ii) or a molecule (44) enabling the attachment of a targeting agent of the substance of interest, in particular attached to the substance of interest prior to the contact of the photoluminescent particles with the substance of interest.

4. A method according to any one of the preceding claims, wherein at least one nanoparticle is coupled with a plurality of coupling agents, the method comprising a step of coupling the nanoparticles with the coupling agents comprising dissolving the nanoparticles with a proportion of coupling agent greater than the proportion of nanoparticles, in particular with a ratio of at least two coupling agents for one nanoparticle or even at least 10 coupling agents per nanoparticle, or even between 10 and 80 coupling agents.

5. A method according to claim 4, wherein each photoluminescent particle comprises a plurality of nanoparticles linked together, in particular by coupling agents, to form an aggregate of nanoparticles, preferably the photoluminescent particles being in colloidal dispersion in the solution at step (ii) of contacting the substance of interest.

6. A method according to any one of the preceding claims, employing at least two distinct types of photoluminescent particles, each comprising nanoparticles according to formula (I), which are coupled to distinct coupling agents, capable of binding to distinct sites of the same analyte, in particular coupled to distinct oligonucleotides recognizing different regions of the nucleic acid-type analyte.

7. A method according to any one of the preceding claims, wherein the nanoparticles are each coupled to a plurality of coupling agents to several regions of an analyte, in particular a plurality of oligonucleotides selected to bind to different regions of a nucleic acid.

8. A method according to any one of the preceding claims, wherein the substance of interest of said sample in step (i) is previously immobilized on the surface of a support, said surface being passivated so that said luminescent particles do not adhere to it in the absence of the substance of interest, in particular step (i) comprises at least the following steps: (a) having a support whose surface is previously passivated and functionalized with a capture agent for the substance to be detected / quantified, for example a monoclonal antibody, referred to as a capture antibody; (b) bringing said sample to be analyzed into contact with the support of step (a) under conditions conducive to the association of said substance with the capture agent;and (c) bring the photoluminescent particles coupled to at least one coupling agent into contact with said support obtained in step (b) in order to associate the particles directly or indirectly with said substance immobilized on the surface of the support.

9. A method according to any one of the preceding claims, employing at least two types of nanoparticles, doped with distinct rare earth ions, having distinct emission wavelengths and coupled to distinct direct or indirect coupling agents, each to one of two different substances of interest.

10. A method according to any one of the preceding claims, implemented using apparatus comprising - the illumination device at a wavelength between 240 nm and 330 nm, preferably between 260 and 330 nm, preferably between 260 nm and 310 nm, preferably between 270 and 290 nm, preferably of the light-emitting diode type (1), having a power of between 50 and 500 mW; and - a device for detecting the light intensity emitted by the nanoparticles in step (iii), in particular a single detector, for example of the photomultiplier type, photodiode, avalanche photodiode, or a detector of the type of array of photosensitive devices consisting of a 2D surface of detection pixels such as a CCD or EM-CCD camera or CMOS camera.

11. A method according to any one of the preceding claims, wherein the detection is time-resolved, the time-resolved detection being achieved by electronic or mechanical chopping of the incident UV-B and / or UV-C beam

12. A process according to any one of the preceding claims, the nanoparticles being prepared by colloidal conversion of rare earth hydroxycarbonate particles, in particular by at least the steps of: (a) preparing an aqueous solution (1) by mixing, in aqueous medium, a metavanadate salt, in particular ammonium metavanadate (NH4VO3), and optionally a phosphate salt;(b) prepare hydroxycarbonate nanoparticles of formula Al xLnx3+CO3OH from precursors of elements A and Ln, in particular in the form of salts, in particular nitrates, and a source of bicarbonate ions, in particular in excess, in particular urea, under conditions conducive to the formation by co-precipitation of said hydroxycarbonate nanoparticles, (b') add the aqueous solution (1) to the hydroxycarbonate nanoparticles in colloidal suspension under conditions conducive to the formation by co-precipitation of the nanoparticles according to formula I; and (c) recover the nanoparticles according to formula I.;

13. A process according to any one of the preceding claims, comprising in step (i), preferably before coupling the nanoparticle with the coupling agent, mixing the nanoparticles with a protecting agent and then post-synthesis annealing at a temperature between 500°C and 1500°C, more particularly between 800°C and 1300°C, and then removing the protecting agent by a suitable method depending on the protecting agent, in particular by acid dissolution.

14. Use of the method as defined according to any one of the preceding claims, for in vitro diagnostic purposes.

15. An in vitro diagnostic assembly, in particular for carrying out the method according to any one of claims 1 to 13, comprising at least: - photoluminescent particles formed in whole or in part from a vanadate or vanadate / phosphate matrix photoluminescent inorganic nanoparticle of formula (I): AlxLnxVO4(1. y)(PO4)y(I) in which: . A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La) and mixtures thereof, in particular A represents Y; . Ln is selected from europium (Eu), dysprosium (Dy), thulium (Tm), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb) and mixtures thereof, in particular Ln represents Eu; . 0 < x < 1; in particular 0.02 < x < 0.5, in particular 0.05 < x < 0.4 and more particularly x equals 0.4, 0.2 or 0.1 or 0.05; and .0 < y < 1, in particular y equals 0, said particles being surface functionalized with chemical groups, for example carboxyl, amino, thiol, aldehyde or epoxy groups, supplied by molecules, for example citric acid or polyacrylic acid, and / or coupled to molecules, for example streptavidin, said chemical groups or molecules being capable of enabling the coupling of said particles with a targeting agent of the substance of interest; or said particles being already coupled to at least one targeting agent of the substance of interest; and - a detection and / or quantification system comprising at least:. - an illumination device with a wavelength between 240 nm and 330 nm, preferably between 260 and 330 nm, preferably between 260 nm and 310 nm, preferably between 270 and 290 nm, preferably of the light-emitting diode type (1), with a power output between 50 and 500 mW, and - a time-resolved detection device for the light intensity emitted by particles with a detection sensitivity of the substance of biological or chemical interest in the sample less than or equal to 10 pM, and - optionally a suitable support for immobilizing the substance of interest of said sample.

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