Ultra-high sensitivity detection method using photoluminescent particles

Abstract

The present invention relates to a method for ultra-sensitive in vitro detection and / or quantification of a biological or chemical target substance in a sample by detecting luminescence radiation emitted by inorganic photoluminescent nanoparticles, the method comprising at least the steps of: arranging inorganic photoluminescent nanoparticles comprising a vanadate or vanadate / phosphate matrix of a specific formula linked to a binder for linking to the target substance; contacting the target substance with the photoluminescent particles under conditions that link the target substance to the binder; exciting the matrix of the inorganic photoluminescent nanoparticles by irradiation at a wavelength of 240 nm to 330 nm; detecting luminescence radiation from the inorganic photoluminescent nanoparticles; and determining the presence and / or concentration of the target substance by interpreting the measurement of luminescence radiation from the particles.

Description

[Technical Field]

[0001] The present invention relates to the fields of research, bioanalysis, and in vitro diagnostics. More specifically, the subject of the present invention is a method for the ultra-sensitive in vitro detection and / or quantification of substances of biological or chemical interest, such as samples, particularly biomarkers, proteins, peptides, hormones, antibodies, DNA, RNA, and other compounds in biological samples, by detecting luminescence emission from photoluminescent inorganic nanoparticles having controlled optical and physicochemical properties. [Background technology]

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

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

[0004] These methods generally rely on the use of probes to detect and / or quantify concentrations in solution. These probes are linked to a recognition compound, or targeting agent, which allows the probe to bind to the molecular species to be analyzed. This recognition compound may be a molecule, DNA, aptamer, protein, or antibody. The probe, bound to the molecular species to be analyzed using the recognition compound, can be detected by one or more methods based, for example, their luminescence, absorbance, chemical reactivity, radioactivity, or others.

[0005] In particular, the most commonly used biochemical test for the detection of proteins is the enzyme-linked immunosorbent assay (ELISA), which generally relies on the use of horseradish peroxidase as an enzyme that causes a reaction with a substrate, and quantifies the resulting chemical reaction by measuring the absorbance of the reaction product in solution. The selection of molecular recognition compounds to be linked to probes is critical to 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. For example, the publications of references [1] and [2] discuss the characteristics of these mechanisms in detail.

[0006] Luminescent probes generally provide detection with higher sensitivity than probes detected by absorbance. This is firstly because the measurement of light intensity is performed against a black background, and secondly because fluctuations in light intensity are measured (measurement against a bright background).

[0007] Among other assay methods currently proposed, electrochemiluminescence immunoassay (ECLIA), fluorescence immunoassay (FIA), and radioimmunoassay (RIA) may also be mentioned.

[0008] However, these methods have various drawbacks that limit their ultimate detection sensitivity, particularly limitations related to the luminescence properties of the probes used (ECLIA, FIA), safety risks, expensive equipment, and the requirement for specialists to perform RIA-type tests (non-automated equipment), among others.

[0009] In particular, currently available luminescent probes have several drawbacks that prevent them from being used to fully realize their potential as diagnostic probes. These drawbacks include, for example, in the case of organic fluorescent dyes, the phenomenon of photobleaching, which is reflected by the disappearance of fluorescence after irreversible chemical changes induced by illumination, or the phenomenon of emission blinking in semiconductor nanocrystals, i.e., "quantum dots", where probes periodically stop emitting light, thus making them unsuitable for generating a constant signal. Other drawbacks arise, for example, from the width of the emission spectrum of luminescent probes. Specifically, if the emission spectrum is too broad, it becomes difficult to filter out background signals that may be present and affect the quality of the signal, especially the signal-to-noise ratio. In addition to the optical factors that contribute to the effectiveness of a probe in biological assays, the practicality and ease of use of the probe must also be considered. That is, certain particles, for example in the case of semiconductor nanocrystals, lose their luminescent properties after freezing, which is a drawback for the storage of bioconjugated agents. The ease of linking the probe to a molecular compound that enables targeting of the desired molecule is also an aspect to be considered when selecting an appropriate probe. Namely, some particles including semiconductor nanocrystals are synthesized in organic solvents. As a result, their use in biological applications requires an additional surface treatment step to disperse these particles in water, and this method is complex to implement and may become unstable over time[3]. The functionalization of these particles with chemical groups enabling linkage to a molecular compound that recognizes a target molecule is also based on weak chemical bonds, which limits their stability and impairs the reproducibility of detection assays.

[0010] Furthermore, the colloidal properties of particles / probes are critically important for carrying out biological assays. Specifically, solutions with good colloidal stability can provide a particularly homogeneous environment for assays and better reproducibility of the results of these assays.

[0011] Finally, complexity and cost are important factors in the selection of a diagnostic probe. For example, gold nanoparticles and their surface plasmon resonance properties have been proposed as diagnostic probes, but they have not become established as probes for in vitro diagnosis, probably due to the complexity of the detection method [4] or the high cost.

[0012] Furthermore, currently available in vitro detection methods are not entirely satisfactory in terms of the detection sensitivity that can be achieved to expand the applications of in vitro diagnostics, for example, by enabling early detection of disease or by enabling diagnosis of disease progression or the effectiveness of treatment.

[0013] To improve detection sensitivity, two commercially available ultra-high sensitivity immunoassay techniques have been developed. These are the methods developed by Quanterix and Singulex, described in U.S. Patent Application No. 7914734, among others. These are based on the use of functionalized magnetic beads as reactive surfaces to capture target molecules. Thus, the Quanterix technique proceeds by capturing individual beads functionalized with antigen-directed antibodies. Each bead is trapped in a well and analyzed using an ELISA-type test. Singulex, on the other hand, concentrates the analyte trapped with beads and determines the concentration by counting the fluorescence signal using a confocal detection device and an excitation laser that helically scans the sample.

[0014] While these two methods achieve higher detection sensitivity than the conventional detection techniques already discussed, they are extremely complex and expensive. They require the use of specialized equipment specifically for their detection methods, which is incompatible with current automated in vitro diagnostic devices.

[0015] Semiconductor nanocrystals, or "quantum dots," have also been proposed for ultra-high sensitivity detection tests ([5], [6], [7], [8], and [9]). However, the effectiveness of these tests is limited, as already discussed, by the drawbacks associated with this type of luminescent probe: the complexity and high cost of synthesis and functionalization, insufficient colloidal stability, and loss of luminescence properties after freezing.

[0016] Finally, while high detection sensitivity is possible through methods based on the use of gold nanoparticles that utilize surface plasmon detection, fluorescence quenching, silver deposition on gold nanoparticles, and other phenomena ([9]-

[13] ), these methods are generally extremely complex.

[0017] There is still a need to develop detection / quantification methods that achieve higher detection sensitivity than conventional technologies such as ELISA, ECLIA, FIA, or RIA, and that do not have the drawbacks of complexity and cost associated with already proposed ultra-high sensitivity methods.

[0018] Regarding nucleic acid detection, the most common ultra-sensitive detection methods are PCR, qPCR, and LAMP, with qPCR being the most sensitive. However, qPCR requires expensive equipment and skilled personnel, resulting in a high cost per test. All of these methods utilize enzyme and initial nucleic acid amplification, which contributes to their high sensitivity to impurities and high cost. Furthermore, while PCR and LAMP are relatively inexpensive, they cannot quantify the nucleic acid concentration in the sample.

[0019] To circumvent the shortcomings of these methods, ELISA-type methods have been developed, as described in the paper by Sakharov Y.

[14] , but they suffer from low sensitivity. There is also an ELISA-type method described in the paper by Lorenzo et al.

[15] , which is highly sensitive, but this increased sensitivity is achieved using a combination of composite nanomaterials that is difficult to develop. Therefore, currently, there is no simple solution that combines ultra-high sensitivity with low cost and complexity in nucleic acid detection.

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

[16] .

[0021] For example, Dosev et al.

[17] detected the microstructure of proteins deposited on a substrate by exciting a Gd2O3 matrix and utilizing the luminescence properties of Eu:Gd2O3 luminescent nanoparticles. Yi et al.

[18] used a NaYF4:Yb,Er type photon upconversion phosphor that absorbs two near-infrared photons and emits photons in the visible range.

[0022] Rare-earth metal-based photoluminescent nanoparticles have been used for single-particle detection and single-molecule tracking, taking advantage of the benefit of not having the flickering required for single-particle detection compared to semiconductor nanoparticles or quantum dots (

[19] and

[20] ). However, it was never foreseeable that these lanthanide ion-based nanoparticles would be used for ultra-sensitive detection and quantification of biomolecules in the case of bulk biomolecule detection. Specifically, apart from the absence of flickering, the luminescence properties of rare-earth metal-based luminescent nanoparticles are considered inferior to those of quantum dots. In these particles, particularly those formed from metal oxide matrices in which certain ions are substituted with rare-earth metal ions, luminescence excitation can occur by excitation of the matrix and subsequent energy transfer to the luminescent rare-earth metal ions, or by direct excitation of the luminescent rare-earth metal ions in the visible range. Regarding matrix excitation, its absorption band is generally in the UV range, which has two drawbacks: there are currently few lasers available at these wavelengths, and the lasers that do exist are bulky and expensive. Also, biomolecules strongly absorb and emit light at these wavelengths, which can result in a significant background signal that must be eliminated. Regarding the direct excitation of rare earth metal ions, the extinction coefficient of nanoparticles is smaller than that of quantum dots, but is considered to be about the same as that of efficient organic fluorescent dyes

[16] .

[0023] Yuan et al.

[21] presented a review article on time-resolved luminescence bioassays performed using lanthanide nanoparticles. Lanthanide luminescent probes are particles containing lanthanide complexes that limit the number of lanthanide ions per particle relative to a given particle volume, i.e., “upconversion” nanoparticles. For example, in publications using lanthanide complex-containing nanoparticles, small-sized nanoparticles (8–9 nm) contain only 3,000–5,000 ions.

[28] Only larger nanoparticles, particularly those larger than 100 nm, can contain approximately 30,000 or more ions (

[22] and

[23] ).

[0024] Similarly, Corstjens et al.

[24] used upconversion nanoparticles for in vitro detection of IFN-gamma in human peripheral blood mononuclear cells. Upconversion nanoparticles enhance deep tissue (Yb in the near-infrared, where tissues generally absorb very little light compared to the visible spectrum). 3+ This is suitable for excitation of [a specific element]. However, the fact that excitation requires the absorption of two photons necessitates a high excitation intensity. Furthermore, the quantum yield of these systems is low, about 1%. Therefore, the number of photons emitted is relatively small.

[0025] Lanthanide complexes or chelates have also been proposed in publications

[23] ,

[25] ,

[26] , and

[27] as luminescent probes for immunoassays. However, these complexes or chelates typically contain only a single lanthanide ion, or at most a few (less than 10) lanthanide ions.

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

[28] , which proposes an improved detection method using lanthanide-doped inorganic nanoparticles, based on the conclusion of a complex protocol for the dissolution of nanoparticles and the detection of the emission of micelles containing lanthanides thus formed.

[0027] European Patent No. 1282824 describes the use of surface-modified inorganic luminescent nanoparticles as probes for detecting biological or other organic substances. The detection method proposed in the said document is based on the ELISA detection principle. However, the said document does not propose its use for ultra-high sensitivity detection.

[0028] U.S. Patent No. 7,550,201 can also be mentioned. This patent proposes the use of lanthanide ion-doped inorganic nanoparticles, particularly for diagnostic applications. However, the aforementioned patent does not propose its use for ultra-high-sensitivity detection methods.

[0029] Publications by Son et al.

[29] and Nichkova et al.

[30] also propose the use of Eu:Gd2O3 nanoparticles as a substitute for organic fluorescent dyes as probes for DNA detection and phenoxybenzoic acid detection, respectively. However, these publications do not suggest at all that they can be used for ultra-high-sensitivity detection.

[0030] A publication by Mousseau et al.

[39] proposes the detection of luminescence emitted by photoluminescent nanoparticles containing lanthanide ions in a vanadate matrix for the case of a membrane detection method. The vanadate matrix is ​​excited by UV irradiation at a wavelength of 278 nm, and the energy is transferred to the photoluminescent lanthanide ions, from which the radiation is detected by a smartphone camera. The matrix is ​​excited at a low intensity to limit unwanted radiation and prevent the camera from detecting residual excitation signals.

[0031] In international patent application 2019 / 025618, the applicant proposed an ultra-sensitive detection method based on the direct excitation of rare-earth metal ions in luminescent nanoparticles by laser irradiation. In this approach, the absorbance of the nanoparticles remains low, and their excitation typically requires a 1W laser to achieve ultra-sensitive detection. Unfortunately, using such laser equipment is expensive. [Prior art documents] [Patent Documents]

[0032] [Patent Document 1] U.S. Patent Application No. 7914734 [Patent Document 2] European Patent No. 1282824 [Patent Document 3] U.S. Patent No. 7550201 [Patent Document 4] International Patent Application No. 2019 / 025618 [Patent Document 5] International Publication No. 03 / 008974 [Overview of the project] [Problems that the invention aims to solve]

[0033] This invention aims to specifically propose a novel, ultra-high-sensitivity detection method based on the use of specific luminescent nanoparticles doped with rare-earth metal ions, thereby (i) freeing the detection method from the constraint of using expensive laser equipment, and (ii) improving detection sensitivity. [Means for solving the problem]

[0034] In the first aspect of the present invention, more specifically, the present invention provides a method for ultra-sensitive in vitro detection and / or quantification of a biological or chemical target substance in a sample to be analyzed, particularly a biological sample, by detecting luminescence radiation emitted by photoluminescent inorganic nanoparticles, wherein the method includes at least the following: (i) Equation (I): A 1-x Ln x VO 4(1-y) (PO4) y (I) A step of preparing photoluminescent particles formed entirely or partially from photoluminescent inorganic nanoparticles containing vanadate or a vanadate / phosphate matrix, 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, more specifically x is 0.4, 0.2, 0.1 or 0.05, 0≦y<1, in particular y is equal to 0, a step, wherein the photoluminescent inorganic nanoparticles are linked to an agent for direct or indirect linkage to a target substance, (ii) a step of contacting said photoluminescent particles with the target substance under conditions that directly or indirectly link the target substance and the linking agent, (iii) a step of exciting a matrix of photoluminescent inorganic nanoparticles of formula (I) by irradiation having a wavelength of from 240 nm to 330 nm, (iv) a step of detecting luminescence emission by the photoluminescent inorganic nanoparticles, in particular by time-resolved detection, and (v) a step of determining the presence and / or concentration of the target substance by interpreting the measurement of luminescence emission by said particles relates to a method comprising

[0035] The linking agent may be an agent targeting the target substance that is directly linked to the target substance in step (ii).

[0036] As a variant, the linking agent is a molecule that enables binding of an agent targeting the target substance, and the targeting agent is bound to the target substance inter alia before contacting the photoluminescent particles with the target substance. In this case, the method may comprise, between step (i) and step (ii), a step of contacting the target substance with the targeting agent under conditions for linking the targeting agent to the target substance, and the linkage of the target substance to the linking agent is carried out indirectly by linking the targeting agent to the molecule.

[0037] Preferably, excitation of the matrix of the photoluminescent inorganic nanoparticles of formula (I) is carried out by irradiation at an emission wavelength of 260 to 330 nm, more preferably 260 nm to 310 nm, and still more preferably 270 to 290 nm.

[0038] Preferably, excitation of the matrix of the photoluminescent inorganic nanoparticles of formula (I) is carried out by irradiation in the UV-B and / or UV-C range.

[0039] For the purposes of the present invention, "analysis" of a substance in a sample includes detection of the presence or absence of said substance or qualitative characterization, as well as assay of said substance or quantitative characterization.

[0040] That is, the method of the present invention relates to luminescent nanoparticles A by UV irradiation, particularly UV-B and / or UV-C irradiation, typically at a wavelength of about 280 nm 1-x Ln x VO 4(1-y) (PO4) y is based on excitation of the vanadate or vanadate / phosphate matrix and energy transfer to luminescent rare earth metal ions.

[0041] Contrary to what a person skilled in the art would think, i.e., that excitation in the UV range would generate a lot of unwanted radiation, this disadvantage is offset by the fact that, as in the case of International Patent Application No. 2019 / 025618, when excitation is in the UV range, the absorbance of the matrix is much greater than when lanthanide ions are directly excited.

[0042] As illustrated in the following examples, the present inventors have shown that it is possible to achieve ultrasensitive detection despite the use of said excitation wavelength.

[0043] In fact, it is known that lanthanide-enriched vanadate nanoparticles exhibit strong absorbance at the excitation peak of the vanadate matrix. However, excitation of these nanoparticles at UV wavelengths is typically not recommended for ultra-sensitive detection applications due to unwanted emission of signals from autofluorescent materials excited at these wavelengths, as well as molecules on the support and in the analytical medium.

[0044] Preferably, the detection of luminescence emission by particles is time-resolved detection. For the purposes of the present invention, the term “time-resolved detection” means detection that is fast enough to measure the rise and decay of the luminescence signal after the start and termination of excitation, or delayed detection of the luminescence signal emitted by photoluminescent particles used in accordance with the present invention, i.e., detection after the unwanted emission from autofluorescent materials and molecules has ceased. Time-resolved luminescence measurements are described, for example, in International Publication No. 03 / 008974. This is possible because the lifetime of emission from photoluminescent inorganic nanoparticles containing vanadate or vanadate / phosphate matrix is ​​several hundred microseconds, in the case of europium in particular, compared to the lifetime of unwanted emission from autofluorescent materials and molecules, which is typically on the order of nanoseconds.

[0045] The present invention enables ultra-high sensitivity detection, and this method has a detection sensitivity of 10 pM or less, better 1 pM or less, even 0.1 pM or less, even 0.01 pM (i.e., 10 fM) or less, or 1 fM or less, or even better 0.1 fM (i.e., 100 aM) or even better 0.01 fM (i.e., 10 aM) or less for biological or chemical target substances in a sample. Such detection is possible without amplification and / or the use of enzymes. By taking advantage of the fine emission spectral width of rare earth metal ions, particularly the less than 10 nm in the case of europium emission at 617 nm, it is possible to eliminate broadband contributions of unwanted emission by efficient emission filtering.

[0046] Furthermore, the inventors have shown that the use of time-resolved detection more efficiently avoids the problem of unwanted radiation at the wavelength used, providing access to "ultra-high sensitivity" detection.

[0047] In other words, unlike the short lifetime of fluorescent molecules, which is approximately a few nanoseconds, the present invention utilizes the long radiative lifetime unique to nanoparticles of formula (I), particularly exceeding 1 μs, 10 μs, or even 100 μs, to perform delayed detection of luminescence signals, thereby at least partially eliminating unwanted background signals.

[0048] Advantageously, time-resolved luminescence detection may be carried out using simple and inexpensive apparatus, particularly a modulator for the excitation light source supply current, a conventional photomultiplier tube, and a 100 kHz AD converter, as described below. The excitation light source can also be modulated using a mechanical chopper or any other method known to those skilled in the art.

[0049] The photomultiplier tube may be configured to detect only visible light and not UV light. This, in particular, allows for limiting interference originating from unwanted signals from residual excitation light. This improves detection sensitivity.

[0050] In other words, the method of the present invention advantageously achieves detection sensitivity far higher than that of conventional detection methods such as ELISA, ECLIA, FIA, or RIA for the detection of organic molecules such as proteins, peptides, or hormones. For nucleic acids, the method of the present invention achieves performance approaching that of PCR without using enzymes necessary for amplification or DNA amplification, while simultaneously enabling the quantification of concentration.

[0051] In other words, advantageously, the ultra-high sensitivity method of the present invention enables detection with at least 10 times, particularly at least 50 times, and better at at least 100 times higher sensitivity than ELISA-type enzyme immunoassays using the same recognition antibody and targeted antibody.

[0052] In other words, the ultra-high sensitivity method according to the present invention enables the detection and / or quantification of target substances present in a sample at concentrations strictly below 100 pM, or less than 10 pM, or less than 1 pM, better less than 0.1 pM, or less than 0.01 pM (10 fM), or 1 fM or less, better less than 0.1 fM (i.e., less than 100 aM), or better less than 0.01 fM (i.e., less than 10 aM). These concentrations depend on the target molecule linked to the probe, particularly the recognition compound or the affinity of the target compound, but are comparable to those detected by ultra-high sensitivity methods (Quanterix or Singulex).

[0053] Advantageously, the ultra-high sensitivity method of the present invention simultaneously enables detection performance comparable to that of previously proposed ultra-high sensitivity techniques, while proving particularly advantageous in terms of ease of implementation and cost.

[0054] Preferably, the step of preparing photoluminescent particles includes the step of preparing a plurality of photoluminescent particles, the excitation (iii) includes, in particular, the step of simultaneously exciting a matrix of a plurality of photoluminescent inorganic nanoparticles of formula (I), and the detection (iv) includes, in particular, the step of simultaneously detecting the luminescence emission from the photoluminescent inorganic nanoparticles excited in the excitation step (iii).

[0055] Preferably, excitation and detection are fixed during the analysis and carried out within an analytical space containing a plurality of photoluminescent particles, and the determination of the presence and / or concentration of the substance of interest (v) is carried out by interpreting the measurements obtained within the analytical space.

[0056] Preferably, the excitation, detection, and determination of the presence and / or concentration of the target substance are performed without scanning the sample during the analysis.

[0057] In other words, unlike the complex techniques developed by Quanterix and Singulex (the latter in particular requiring sample scanning), the ultra-sensitive method of the present invention, as detailed below, uses a compact and inexpensive detection device whose components are readily available on the market for the excitation and measurement of luminescence emitted by nanoparticles, and does not require any specially designed equipment. Therefore, advantageously, it is well-suited for integration into automated analytical devices with minimal ergonomic considerations.

[0058] Furthermore, the excitation of the nanoparticle matrix in the wavelength range specified above may be carried out using a low-power light-emitting diode source, which is significantly cheaper than laser illumination devices such as laser diodes used in the methods already described.

[0059] Furthermore, the method of the present invention is also suitable for multiplex analysis. That is, the method of the present invention can be used to simultaneously detect and / or quantify at least two different substances in a sample, particularly by following the procedure presented below.

[0060] The method of the present invention may be carried out for the analysis of biological or chemical target substances in various samples, also known as “analytical substances.” The sample may be a biological sample, particularly a human sample selected from, for example, blood, serum, plasma, saliva, urine, and cerebrospinal fluid. The sample may be a diluted feces, vaginal smear, nasopharyngeal swab, or sputum. A diluent may be used with the sample to be analyzed, particularly when the liquid sample is, for example, plasma, serum, whole blood, nasal or vaginal smear, or sputum.

[0061] At least one nanoparticle may be linked with multiple binders, and this method includes a step of linking nanoparticles with binders, which includes dissolving nanoparticles in a proportion of binders greater than the proportion of nanoparticles, particularly at least two binders per nanoparticle, or at least ten binders per nanoparticle, or 10 to 80 binders.

[0062] This may be a solution containing biological molecules.

[0063] The method of the present invention may be used, for example, for the detection and / or quantification of biomarkers, antibodies, DNA, and / or RNA. It may be used to detect human, animal, bacterial, viral, or circulating DNA and / or RNA. This may also enable the provision of genotypes in biological samples. This may include any type of nucleic acid, particularly tRNA, mRNA, miRNA, dsRNA, circRNA, ncRNA, and lncRNA.

[0064] In another aspect, the present invention relates to the use of a method already defined for in vitro diagnostic purposes. Advantageously, the ability of the ultra-sensitive method to detect certain substances at very low levels in biological samples enables the use of the present invention for, for example, early detection of disease or diagnosis of disease progression or the effectiveness of treatment. Furthermore, this ultra-sensitive detection method makes it possible to use substances as biomarkers that are currently undetectable by conventional methods due to their low concentrations. It also makes it possible to detect biomarkers in easily accessible biological media (saliva, urine, blood, etc.) that are undetectable by conventional methods due to their low concentrations, requiring, for example, invasive methods such as cerebrospinal fluid sampling.

[0065] In yet another embodiment, the present invention is Photoluminescent particles, at least, which are formed whole or partially from photoluminescent inorganic nanoparticles as already defined, wherein the particles are linked to a binder, and the binder is surface-functionalized with chemical groups supplied by molecules such as citric acid or polyacrylic acid, for example carboxyl, amino, thiol, aldehyde, or epoxy groups, and / or linked to molecules such as streptavidin, and the chemical groups or molecules are suitable for enabling the linking of the particles to an agent for targeting a target substance, or are an agent for targeting a target substance, and at least, A lighting device, preferably a light-emitting diode type, having an emission wavelength of 260-330 nm, more preferably 260-310 nm, and even more preferably 270-290 nm, and capable of having an output of 500 mW or less, more preferably 200 mW or less, for example 50-150 mW. A device for detecting the intensity of light emitted by particles. Apparatus for performing time-resolved detection of luminescence radiation, including a device for collecting and spectrally filtering emitted luminescence, a photomultiplier tube, and / or a photodiode and an AD converter, and a detection and / or quantification system which may include a system for modulating the output of an excitation light source, a mechanical chopper, a photomultiplier tube, and an AD converter. Regarding in vitro diagnostic kits, including those mentioned.

[0066] The lighting device may be a device that emits UV-B or UV-C light.

[0067] The photomultiplier tube may be configured to detect only visible light and not UV light. This makes it possible to limit interference from unwanted signals induced by residual excitation light, thereby improving detection sensitivity.

[0068] Preferably, the detection and / or quantification system is fixed during the analysis.

[0069] Preferably, the detection device does not include a confocal detection device.

[0070] Such in vitro diagnostic kits enable easy implementation of the ultra-sensitive detection and / or quantification methods according to the present invention for assaying biomarkers or antibodies in biological samples, for example.

[0071] The diseases that can be diagnosed by the in vitro diagnostic kit of the present invention are not limited to any diseases that are revealed by the presence of disease-specific markers, such as a molecule of biological interest (protein, nucleic acid, etc.) that has one or more specific binding partners (ligand, antibody, complementary nucleic acid, aptamer, etc.).

[0072] Examples may include infectious diseases (bacterial, parasitic, or viral, e.g., AIDS), inflammatory diseases, and autoimmune diseases, as well as diseases of cardiology, neurology, or oncology (e.g., solid tumors such as breast cancer or prostate cancer).

[0073] The ultra-high sensitivity detection method of the present invention is not limited to the applications described above. That is, it may also be used, for example, to detect GMO DNA in seeds, or to detect contaminants or pathogens in water or food intended for consumption.

[0074] In other words, the applications of the ultra-high-sensitivity detection method according to the present invention can be extended from immunology to molecular genetics or the detection of DNA and RNA. This may be used to label one or more RNA strands derived from a biological sample with partially complementary probe fragments conjugated to nanoparticles, and then to detect them by hybridization to complementary fragments derived from another region grafted onto a solid substrate using an approach similar to that of an Affymetrix-type DNA chip. One advantage of the present invention is that these approaches do not require the amplification step that is usually necessary.

[0075] In the following text, the photoluminescent inorganic nanoparticles of formula (I) of the present invention will be simply referred to as "nanoparticles".

[0076] Other features, variations, and advantages of the method according to the present invention will become clearer by reading the following specification, examples, and drawings, which are given as a non-limiting description of the present invention.

[0077] In the following description, unless otherwise stated, the expressions "between ~", "range of ~" and "vary to ~" are equivalent and mean that the limit values are included.

[0078] Unless otherwise indicated, the expression "comprising ~" should be understood as "comprising at least one ~".

[0079] Luminescent particles of the present invention As already indicated, the ultra-sensitive detection method according to the present invention comprises photoluminescent inorganic nanoparticles containing the previously described vanadate or vanadate / phosphate matrix, and is based in particular on the detection of luminescence emission of photoluminescent particles formed therefrom and linked to at least one agent for direct or indirect linking to a target substance.

[0080] Photoluminescent inorganic nanoparticles As already indicated, the photoluminescent nanoparticles used according to the present invention have the formula (I): A 1-x Ln x VO 4(1-y) (PO4) y (I) wherein A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), and mixtures thereof, and 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, and in particular Ln represents Eu, 0 < x < 1, particularly 0.02 ≦ x ≦ 0.5, especially 0.05 ≦ x ≦ 0.4, more specifically x is 0.4, 0.2, 0.1, or 0.05, 0 ≦ y < 1, particularly y is 0.

[0081] The properties of the nanoparticles used will determine the sensitivity that can be achieved by the method of the present invention.

[0082] The inorganic nanoparticles of the present invention are advantageously at least 10 3 It is formed from a vanadate or vanadate / phosphate crystal matrix containing a number of rare earth metal ions.

[0083] The rare earth metal ions in the nanoparticles of the present invention are not in the form of rare earth metal ion complexes or chelates formed from rare earth metal ions combined with suitable organic ligands, as described, for example, in the publication by Yuan et al.

[21] .

[0084] Preferably, the nanoparticles of the present invention contain 1,000 to 6,000,000 rare earth metal ions, particularly 5,000 to 50,000, and more specifically 20,000 to 100,000 rare earth metal ions.

[0085] The nanoparticles of the present invention may be doped with rare earth metal ions of the same or different properties. According to a modification of one embodiment, as will be described in detail below, the method of the present invention uses at least two different types of nanoparticles that differ in the properties of the rare earth metal ions. By using nanoparticles rich in different lanthanides simultaneously, it is possible to multiplex the detection of different substances in the same sample, such as different biomarkers.

[0086] The photoluminescent nanoparticles of the present invention may have an average particle size of 20 nm or more and strictly less than 1 μm.

[0087] In particular, these have an average particle size of 20nm to 500nm, especially 20nm to 200nm, and especially 20nm to 100nm.

[0088] In particular, the average particle size of the photoluminescent nanoparticles of the present invention may be 20 nm or more, especially 30 nm or more, and most notably 30 to 60 nm.

[0089] In particular, as described in the following examples, the photoluminescent nanoparticles according to the present invention may have an average particle size of about 20 to 50 nm.

[0090] Large nanoparticles can be obtained, for example, by centrifuging particles and separating them by particle size, as illustrated, and retaining only the largest particles in the particle size distribution, or by grinding bulk material. Any other method known to those skilled in the art may be used.

[0091] That is, the photoluminescent nanoparticles used in the method of the present invention are advantageous in that they have a sufficient volume to contain a large number of rare earth metal ions and therefore emit a sufficient luminescent signal to enable detection of low concentrations. For example, spherical nanoparticles Y with a diameter of 30 nm 0.6 EU 0.4 VO4 is 70,000 Eu 3+ It contains ions (see reference

[31] Casanova et al. APL 2006 for calculation of the number of ions). Furthermore, as described in the text below, photoluminescent nanoparticles should not be too large to avoid steric hindrance when combined with substances to be assayed, for example, immobilized on the surface of a support.

[0092] The average particle size may be measured by a transmission electron microscope. Transmission electron microscope images allow for the determination of the nanoparticle shape (spherical, elliptical) and estimation of the average size of the nanoparticles. Generally, for spherical particles, the term "average particle size" refers to the average diameter of the particles.

[0093] In the case of elliptical particles, the term "average particle size" refers to the average particle size of spheres having the same volume as the ellipsoid. It is generally assumed that the length of the third axis of the ellipsoid, which is not visible in a two-dimensional projection (transmission image), is equal to the length of the smallest axis.

[0094] According to certain embodiments, the nanoparticles of the present invention generally have an elongated elliptical shape ("flattened body").

[0095] More specifically, these may have a major axis length referred to as a of 20-60 nm and a minor axis length referred to as b of 10-30 nm. In particular, the nanoparticles of the present invention may have an average major axis length a of 40 nm and an average minor axis length b of 20 nm.

[0096] Advantageously, the nanoparticles of the present invention have low polydispersity. The polydispersity index can be estimated from TEM measurements and may be less than 0.2 in particular.

[0097] Advantageously, the nanoparticles used in connection with the method of the present invention are 10 before the emission stops. 8 More than 10 photons, especially 10 9 pieces or 10 10 It is suitable for emitting more than one photon. In many cases, especially in the case of Eu-doped YVO4 or GdVO4 particles, no cessation of emission is observed.

[0098] Furthermore, advantageously, the nanoparticles of the present invention have a long radiative lifetime. In particular, they may have a radiative lifetime of 5 μs or more, especially 10 μs or more, particularly 20 μs or more, or 50 μs or more, or 100 μs or more.

[0099] Radiation lifetime is understood to be the lifetime of the excited state of a radiating nanoparticle, and is practically determined by the duration of luminescence photon emission after excitation ceases, i.e., the characteristic time of luminescence decay after excitation ceases.

[0100] As already discussed, the ultra-high sensitivity method of the present invention provides a long radiative lifetime for the particles of the present invention (compared to the lifetime of conventional phosphors, which is about a few nanoseconds, Y 1-x EU x In the case of VO4 particles, several hundred microseconds can be utilized to achieve sufficient temporal resolution, particularly time-resolved detection at less than 100 microseconds, or even better, less than 10 microseconds, especially delayed emission detection.

[0101] Advantageously, the nanoparticles used according to the present invention do not lose luminescence even after freezing.

[0102] In certain embodiments, the nanoparticles used are those of formula (I) above, wherein y is 0. In other words, the nanoparticles used in the method of the present invention are of formula A 1-x Ln x VO4(I'), wherein A, Ln, and x are as defined above.

[0103] According to a specific embodiment, A in formula (I) or (I') represents yttrium (Y).

[0104] According to another specific embodiment, Ln in formula (I) or (I') represents Eu.

[0105] That is, according to a variant of one embodiment, the particles of the present invention are of formula Y 1-x Eu x VO4 nanoparticles, wherein 0<x<1, in particular 0.02≦x≦0.5, in particular 0.05≦x≦0.4, more specifically x is 0.4, 0.2, 0.1, or 0.05.

[0106] The nanoparticles according to the present invention are predominantly crystalline, substantially polycrystalline, and in particular have an average crystallite size, determined by X-ray diffraction, of 3 to 40 nm, as detailed in Example 1 below.

[0107] According to another specific embodiment, the nanoparticles are olive-shaped single crystals with a particle size of 30 to 200 nm.

[0108] Preparation of Nanoparticles The nanoparticles comprising a rare earth metal ion-doped crystalline matrix used in the method of the present invention may be prepared by any conventional method known to those skilled in the art. For example, nanoparticles based on rare earth metal-doped yttrium vanadate are described in detail in papers

[32] and

[33] .

[0109] Advantageously, the nanoparticles of the present invention are readily synthesized in an aqueous medium, which has the advantage of eliminating a subsequent solvent transfer step.

[0110] In particular, nanoparticles are derived from precursors of elements A and Ln, as well as orthovanadate ions (VO4 3- ) and optionally phosphate ions (PO4 3- It may be formed by a coprecipitation reaction in an aqueous medium in the presence of ).

[0111] Precursors of elements A and Ln may exist in the conventional form of salts of the elements, such as nitrates, chlorides, perchlorates, or acetates, particularly nitrates. The precursors of elements A and Ln, and their amounts, are of course appropriately selected in relation to the desired properties of the nanoparticles.

[0112] For example, equation Y 1-x EU x For the synthesis of VO4 nanoparticles, yttrium nitrate (Y(NO3)3) and europium nitrate (Eu(NO3)3) may be used as precursor compounds for yttrium and europium, respectively.

[0113] In a particularly preferred embodiment, orthovanadate ion (VO4 3- ) is produced in situ from a metavanadate salt, preferably ammonium metavanadate (NH4VO3).

[0114] Orthovanadate ions may be formed in situ by reacting the metavanadate salt with a base (more precisely, two or three equivalents of a strong base), as described in the paper by Neouze et al.

[34] .

[0115] In the case of phosphate ions, phosphates such as sodium phosphate or ammonium phosphate are added.

[0116] That is, according to a particularly advantageous embodiment, the method of the present invention provides at least, (a) A step of preparing an aqueous solution (1) by mixing a metavanadate salt, particularly ammonium metavanadate (NH4VO3), and optionally a phosphate, and optionally a base, particularly tetraalkylammonium hydroxide, which is a source of tetraalkylammonium cations, in an aqueous medium. (b) Adding an aqueous solution (2) containing precursors of elements A and Ln, particularly in the form of salts, especially nitrates, to aqueous solution (1) under conditions that promote the formation of nanoparticles by coprecipitation, and (c) Steps to recover nanoparticles after removing counterions The process includes steps consisting of the following:

[0117] In certain embodiments, the aqueous solution (2) containing precursors of elements A and Ln may also contain a compounding agent for these elements, such as a citrate, such as tetraalkylammonium citrate.

[0118] According to a particular embodiment, the addition of solution (2) to solution (1) in step (b) is carried out by dropwise addition.

[0119] According to another particularly preferred embodiment, the nanoparticles are prepared by colloidal conversion of rare-earth metal hydroxycarbonate particles (see Example 1.2).

[0120] That is, according to one modification, the method of the present invention provides at least, (a) A step of preparing an aqueous solution (1) by mixing a metavanadate salt, particularly ammonium metavanadate (NH4VO3), and optionally a phosphate in an aqueous medium. (b) Under conditions that promote the formation of hydroxycarbonate nanoparticles by coprecipitation, a precursor of elements A and Ln in particular in the form of salts, especially nitrates, and a source of excess bicarbonate ions, especially urea, of formula A 1-x Ln x 3+ A process for preparing CO3OH hydroxycarbonate nanoparticles, (b') A step of adding an aqueous solution (1) to hydroxycarbonate nanoparticles in a colloidal suspension under conditions that promote the formation of nanoparticles of formula I by coprecipitation, and (c) Step to recover nanoparticles of formula I Includes.

[0121] According to a particular embodiment, the addition of solution (1) to the hydroxycarbonate nanoparticles in step (b') is carried out by dropwise addition.

[0122] According to a variation of another embodiment, solution (1) may be mixed with solution (2) or hydroxycarbonate nanoparticles in a single step rather than by dropwise addition.

[0123] The aqueous medium of the solution is more specifically formed from water and / or a mixture of water and ethylene glycol.

[0124] Adjusting the amounts of various agents, particularly metavanadate ions, optionally phosphates, precursors of elements A and Ln, and urea in a manner appropriate to the desired properties of the nanoparticles according to the present invention is within the scope of the skills of those skilled in the art.

[0125] In particular, the stoichiometric proportions of various drugs calculated using the above formula must be adhered to.

[0126] Advantageously, the method for preparing the nanoparticles does not require heating of the solution, unlike the hydrothermal method proposed in publications

[26] -

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

[0127] The essence of step (c) is, more specifically, the purification of the resulting particle solution, particularly the removal of excess counterions.

[0128] The purification process more specifically consists of dialysis or centrifugation of the particles in an aqueous medium and redispersion by, for example, ultrasound. The particles may be redispersed in an aqueous medium, particularly water.

[0129] The synthesis of luminescent nanoparticles according to the present invention, particularly nanoparticles having a particle size of several tens of nanometers or more, can be carried out by any other approach known to those skilled in the art, such as grinding of bulk materials.

[0130] Preferably, the nanoparticles, particularly those recovered in step (c) above, are mixed with a protective agent, and the resulting mixture is subjected to post-synthesis annealing at a temperature of 500°C to 1500°C, more specifically 800°C to 1300°C, and the protective agent is removed by a method suitable for the protective agent, particularly by acid dissolution. This step (d) is preferably carried out before linking the nanoparticles with a binder, particularly before linking them with a targeting agent, or before their functionalization. As a variation, the nanoparticles are subjected to post-synthesis annealing under hydrothermal conditions, particularly at a temperature of 120°C to 300°C. Annealing reduces the photoreduction effect of the nanoparticles when they are subjected to excitation. Annealing also increases the quantum yield of the radiation of the nanoparticles.

[0131] Targeting agent The particles used as luminescent probes according to the method of the present invention may be linked (or grafted) to at least one targeting agent linked to the substance to be assayed in the sample to be analyzed.

[0132] The term "targeting agent" refers to a compound that enables binding to a biological or chemical target substance whose identification is required.

[0133] Needless to say, the properties of the targeting agent used are selected in relation to the target substance in the sample.

[0134] The particles used in the ultra-high sensitivity method according to the present invention are perfectly suited to a wide variety of biological targets, and their specificity depends on the properties of the targeting agent grafted onto the surface of the nanoparticles.

[0135] The targeting agent may be selected more specifically from polyclonal or monoclonal antibodies, antibody fragments, nanobodies, oligonucleotides, peptides, hormones, ligands, cytokines, peptide mimes, proteins, carbohydrates, chemically modified proteins, chemically modified nucleic acids or oligonucleotides, chemically modified carbohydrates targeting known cell surface proteins, aptamers, protein-DNA / RNA assemblies, or chloroalkanes used by HaloTag-type labeling. SNAP-Tag or CLIP-Tag-type approaches may also be used.

[0136] According to a particular embodiment, this is an antibody or antibody fragment, or an oligonucleotide or a fragment of an oligonucleotide.

[0137] Suitable antibody fragments include at least one variable domain of immunoglobulin, such as a single variable domain Fv, scFv, Fab, (Fab')2, and other proteolytic fragments or "nanobodies" (single-domain antibodies, such as V obtained from camelid antibodies). H V obtained from H fragments or antibodies from cartilaginous fish NAR ) includes.

[0138] The term "antibody" in this invention includes chimeric antibodies, human or humanized antibodies, recombinant and modified antibodies, conjugated antibodies, and fragments thereof.

[0139] According to certain embodiments, the antibody or antibody fragment used in the present invention targets a specific marker of cancer cells.

[0140] Targeting agents may also be derived from molecules known as cell surface receptor binders. For example, targeting fragments may be derived from low-density lipoproteins, transferrin, EGF, insulin, PDGF, fibrinolytic enzymes, anti-HER2, anti-HER3, anti-HER4, annexin, interleukin, interferon, erythropoietin, or colony-stimulating factors.

[0141] Linking of particles and targeting agents Preparing particles linked to one or more targeting agents in an appropriate manner using a suitable linking / grafting method is within the skill of a person skilled in the art. The amount of targeting agent used is adjusted according to the amount of particles, and vice versa.

[0142] The targeting agent may be grafted directly onto the nanoparticles or via a spacer (also called a "linker").

[0143] Methods for linking particles to biomolecules (also known as grafting) are well known to those skilled in the art. These methods generally include linking by covalent bonding, surface complex formation, electrostatic interaction, encapsulation, or adsorption.

[0144] In certain cases, such as with covalent bonding, particles can be pre-functionalized with chemical groups, which then react with other chemical groups supported by the targeting agent to form covalent bonds.

[0145] Examples of chemical groups that may be present on the surface of nanoparticles include carboxyl, amino, thiol, aldehyde, and epoxy groups.

[0146] The particle coating with silica may be used to facilitate subsequent particle functionalization.

[0147] The amino group may be provided by aminoorganicsilanes, such as aminotriethoxysilane (APTES). The advantage of APTES is that it forms a covalent capsule around the nanoparticles. Therefore, the amine provided by APTES is extremely stable over time. The amino group may be converted to a carboxyl group by reaction with succinic anhydride.

[0148] The carboxyl group may be provided by molecules such as citric acid or polyacrylic acid (PAA).

[0149] The nanoparticles may contain polyethylene glycol (PEG) molecules on their surface to minimize nonspecific adsorption of the nanoparticles onto the detection surface. Preferably, the PEG may have a molecular weight of 500 to 20,000 g / mol.

[0150] In other examples, the particles may be pre-attached to a suitable molecule to allow for subsequent attachment to a targeting agent.

[0151] For example, the particles may be linked to a streptavidin suitable for enabling linkage with a biotinylated targeting agent.

[0152] For example, Example 1 describes the linkage of streptavidin-linked nanoparticles to biotinylated antibodies by linking streptavidin-linked nanoparticles to biotinylated antibodies.

[0153] In other cases, the linkage of nanoparticles to antibodies may be carried out by directly linking the antibodies to nanoparticles functionalized by APTES. As described above, the amino groups provided by APTES can first be converted to carboxyl groups by reaction with succinic anhydride. The carboxyl groups can then be activated by any method known to those skilled in the art, particularly by reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and react with amine functional groups on the surface of polypeptides to form covalent amide bonds when the targeting agent is a protein or antibody.

[0154] Functionalization of nanoparticles with APTES may, advantageously, be carried out after the nanoparticles have been coated with a silica layer.

[0155] The binding of the targeting agent to the surface of the nanoparticles may be carried out by any other method known to those skilled in the art.

[0156] This may be favorably carried out by a coating reaction with APTES (3-aminopropyltriethoxysilane), in which nanoparticles are coated with a silica layer, and then their amine functional groups react with a bifunctional crosslinking agent containing two NHS functional groups. Subsequently, the crosslinked nanoparticles can react with amine functional groups on the surface of proteins (antibodies, streptavidin, etc.). This type of crosslinking method is described in references

[35] and

[36] , among others.

[0157] Advantageously, the particles used in the ultra-high sensitivity detection method according to the present invention have low polydispersity. Preferably, the polydispersity index, which can be estimated by MET measurement or dynamic light scattering (DLS) measurement, is strictly less than 0.2. If this is not the case after particle synthesis or functionalization, low polydispersity can be achieved by particle size sorting by centrifugation or any other method known to those skilled in the art.

[0158] As already stated, the method according to the present invention may also include several means for amplifying the luminescence signal emitted by photoluminescent particles per analyte, in particular by changing the coupling properties of the photoluminescent nanoparticles with one or more targeting agents.

[0159] At least one nanoparticle can be linked with multiple binders, particularly a targeting agent or a molecule suitable for enabling subsequent linkage with a targeting agent. This method includes a step of linking nanoparticles with binders, which involves dissolving nanoparticles in a proportion of binders greater than the proportion of nanoparticles, particularly at least two binders per nanoparticle, or at least ten binders per nanoparticle, or in a ratio of 10 to 80 binders.

[0160] Each photoluminescent particle may contain multiple nanoparticles linked together by a binder to form a nanoparticle aggregate, and the photoluminescent particles are preferably colloidally dispersed in a solution in step (ii) of contacting the sample to be analyzed.

[0161] That is, in certain embodiments, at least one nanoparticle may be linked to multiple targeting agents, particularly identical targeting agents, particularly biotinylated targeting agents, thereby advantageously leading to the formation of controlled aggregates of streptavidin-functionalized nanoparticles linked to each other via the targeting agents. These nanoparticle aggregates may be formed by using a proportion of targeting agent greater than the proportion of nanoparticles, particularly at a ratio of at least two targeting agents per nanoparticle, when linking the nanoparticles with the targeting agents. Preferably, this ratio is adjusted to result in an aggregate size that allows for the maintenance of a good colloidal dispersion in the medium in which the photoluminescent particles are used, particularly in aqueous media.

[0162] Any other method for forming controlled nanoparticle aggregates may be employed. For example, complementary oligonucleotides may be added to the surface of the nanoparticles in addition to the binder, thereby forming nanoparticle aggregates.

[0163] By using particles formed from photoluminescent nanoparticle aggregates, it becomes possible to amplify the luminescence signal per analyte, thereby further improving detection sensitivity.

[0164] In another specific embodiment, the ultra-high sensitivity detection method according to the present invention uses at least two different types of photoluminescent particles, each comprising nanoparticles of formula (I) linked to different binders suitable for binding to different sites on the same analyte. For example, the particles may be linked to different oligonucleotides that recognize different regions of nucleic acid-type analytes. By using photoluminescent particles combined with different sites on the surface of the analyte, one or more nanoparticles can bind to a single analyte molecule, thereby amplifying the luminescence signal per analyte and further lowering the sensitivity threshold that can be achieved through the method of the present invention.

[0165] Each nanoparticle is linked to multiple binders, each of which may be linked to multiple oligonucleotides configured to bind to different zones of the target substance, particularly different zones of nucleic acids, and especially preferably different zones separated from each other by a distance less than or equal to the particle diameter. The binders may be the same or different. When a single nanoparticle is linked to the target substance by two or more binders, the principle of biochemical affinity can be utilized to improve the binding of the nanoparticle to the analyte, thereby limiting the risk of loss of binding during washing. Furthermore, this allows several nanoparticles to bind to different sites on the same analyte, improving detection sensitivity as before.

[0166] It is understood that these particular embodiments may be combined to maximize the amplification of the luminescence signal per analyte in the sample to be analyzed.

[0167] Combination of luminescent probe and target substance In the first step of the ultra-high sensitivity detection method according to the present invention, photoluminescent particles are combined with a substance in the sample to be analyzed.

[0168] This process may be carried out on the surface of the support in a manner similar to conventional methods, such as ELISA immunoassay, as schematically shown in Figure 1a.

[0169] The ultra-high sensitivity detection method is preferably different from the membrane transfer method.

[0170] Variations of this embodiment are disclosed in more detail in the following text.

[0171] In particular, this embodiment includes pre-immobilizing the material of the sample to be analyzed onto the surface of a support, as described in Example 2.

[0172] In particular, this method may include the steps of: functionalizing a support with an additional agent for linking the target substance; contacting the target substance with the additional linking agent on the support under recognition conditions; and subsequently contacting a solution containing the particles already described in the form of a suspension, in particular a colloidal dispersion.

[0173] The support can be of various types. This may be a slide, such as a glass slide, a multiwell plate, a microplate, a membrane gel, a strip, or a microchannel, as used in the examples. It may be a plastic with good optical quality, or any other material with sufficient optical quality.

[0174] Preferably, the support is different from a strip-type support having a filtration membrane.

[0175] Preferably, the surface of the support is passivated so that luminescent particles do not adhere to the surface of the support when the target substance is not present.

[0176] Surface passivation may be carried out by any method known to those skilled in the art.

[0177] This may include passivation of glass surfaces using molecules containing, for example, polyethylene glycol (PEG), such as silane-PEG molecules. Preferably, the PEG may have a molecular weight of 500 to 20,000 g / mol. Longer PEG molecules result in better passivation. However, it is advantageous for the PEG to be relatively short to avoid steric hindrance to the binding of the target substance and to enable interaction between the targeting agent bound to the surface and the target. In particular, shorter PEG is preferable when the binder is small.

[0178] The support is also functionalized on its surface with a first agent to target the substance to be detected / quantified. More specifically, this agent may be an antibody known as a capture antibody, as shown in Phase 1 of Figure 1a, particularly when the target substance is a biomarker, protein, or polypeptide.

[0179] If the target substance is an antibody, the targeting agent may be an antigen specific to the antibody to be detected.

[0180] Surface functionalization may be carried out by any method known to those skilled in the art. This may be carried out by adsorption following prolonged contact for several hours, spot printing (depositing microdroplets of a solution containing target molecules onto the surface using a robot), contact printing techniques that enable molecular transfer by contact between a topological pattern of a pad (e.g., a polydimethylsiloxane PDMS pad) and the substrate surface, or by other means known to those skilled in the art that enable the deposition of a targeting agent onto the support surface.

[0181] The sample to be analyzed is then brought into contact with the functionalized surface of the support in order to enable the combination of the substance to be detected / assayed with the targeting agent supported on the support (Phase 2 and 3 in Figure 1a).

[0182] This process includes incubation of the sample on the surface of the support, as well as washing / rinsing the support to remove the solution and unbound molecules, similar to a conventional ELISA test. After rinsing, only the complex of the targeting agent and the substance to be assayed, such as the antibody / antigen, remains bound to the surface of the support, with the exception of some molecules that may have bound in a nonspecific manner.

[0183] Finally, as already described, the photoluminescent particles, formed entirely or partially from photoluminescent nanoparticles and linked to a target substance, such as a drug for linking antibodies, are linked to the target substance immobilized on the surface of a support (Phase 4 in Figure 1a).

[0184] This process includes incubating a solution of photoluminescent particles on the functionalized surface of a support, and washing / rinsing the support to remove particles not bound to the support. After rinsing, only targeting agents / substances / particles to be assayed, such as monoclonal antibody / antigen / polyclonal antibody-nanoparticles, bound to at least one binder, remain bound to the surface of the support, apart from some nanoparticles that may have bound in a nonspecific manner. The incubation time may be adjusted by preliminary testing to maximize the luminescence emission signal. Generally, the incubation time may be 30 minutes to 2 hours.

[0185] The linkage between particles linked to a linker and the target substance includes, for example, the recognition of ligand-antiligand pairs, such as biotin or biotinylated compounds and avidin or streptavidin, haptens and antibodies, antigens and antibodies, peptides and antibodies, such as digoxigenin (DIG) and anti-DIG antibodies, sugars and lectins, and polynucleotides and complementary polynucleotides. It is understood that one of the elements of these pairs constitutes the target substance, or the targeting agent, or another element linked to the target substance.

[0186] That is, in one embodiment, step (i) is at least the following: (a) A step of preparing a support whose surface has already been passivated and functionalized with a drug for capturing a target substance, such as a monoclonal antibody known as a capture antibody, (b) A step of bringing the sample to be analyzed into contact with the support from step (a) under conditions that promote the combination of the target substance and the capture agent, and (c) A step of bringing photoluminescent particles linked to at least one binder into contact with the support obtained in step (b), thereby directly or indirectly combining the particles with the material fixed on the surface of the support. Includes.

[0187] The combination may be direct or indirect, and in particular, the binder may be a molecule suitable for binding to a drug for targeting the target substance or a targeting agent bound to the target substance. In the latter case, the method may include a step of contacting the target substance with the targeting agent before contacting the target substance with particles. This contact may occur before the target substance is captured on the support or after the target substance has been captured. Preferably, the targeting agent is different from the capture agent.

[0188] It is understood that the target material may be fixed to several predefined different zones on the surface of the support.

[0189] This may be carried out, in particular, by using localized functionalization of the support surface with the targeting agent (e.g., capture antibody), similar to the functionalization of multiple wells in a multiwell plate. This includes the deposition of the same targeting agent in several predefined zones. In this case, these multiple zones are used to detect the same substance in several different samples.

[0190] Such embodiments are implemented more specifically when the ultra-high sensitivity detection method according to the present invention is used for multiplexed analysis.

[0191] In multiplex analysis, which enables the simultaneous detection and / or quantification of at least two different substances in a sample, the different substances to be analyzed in the sample may be immobilized in predefined zones on the surface of a support, for example, by locally functionalizing the surface of the support with a targeting agent specific to each of the substances to be analyzed. The surface is passivated beforehand to prevent photoluminescent particles from binding to the surface when the substance to be analyzed is not present.

[0192] In this case, the particles used must contain a number of different targeting agents on their surface, more specifically, at least one targeting agent specific to each of the substances to be analyzed. In this way, the substances to be analyzed are quantified using the radiation intensity of the particles in each zone, and the spatial location of the zones considered indicates the properties of the substance.

[0193] It is also possible to combine multiplexing approaches for several samples and several substances by locally functionalizing predefined zones on the surface of a support with a targeting agent specific to each of the substances to be analyzed, and repeating this process as many times as there are samples to be analyzed. In this case, the radiation intensity of particles in each zone provides information about the presence and / or concentration of each target substance in each sample, and the spatial location of the zones considered indicates both the properties of the substance and the number of samples.

[0194] For multiplexed detection, it is also possible to use at least two types of nanoparticles doped with different rare-earth metals having different emission wavelengths, such as YVO4:Eu or YAG:Ce, and each nanoparticle linked to a binder that links to one of two different target substances. By detecting luminescence signals using two different emission filters, it becomes possible to detect and / or assay each of the substances to be analyzed.

[0195] Preferably, in such a variation of multiplexed detection, at least two nanoparticles having different emission wavelengths may be used to separate the obtained luminescence signals, each linked to a drug that targets the respective substance to be analyzed.

[0196] In particular, to compare the concentrations of target molecules between at least two samples, a combination of two approaches (analysis of several different samples and several different substances within each sample) may be used, which is carried out by comparing the intensity of the respective emission colors of nanoparticles linked to a specific targeting agent for each target substance for several deposition zones, each corresponding to a different sample.

[0197] Alternatively, a combination of the two approaches may be used to analyze several different substances in each sample in several different samples (samples from different origins, or samples from the same origin at different time points, samples under different conditions, samples under different stimuli, etc.), and in particular to compare the concentrations of target molecules between at least two samples. This is done by comparing the intensity of the respective emission colors of nanoparticles linked to a specific drug targeting each of the target substances in several deposition zones, each corresponding to a different target molecule. In this case, the comparison of emission colors in the reference deposition zones provides a comparison of the molecular concentrations between the two samples.

[0198] Various other combinations of these approaches are considered. For example, four substances can be analyzed by using two nanoparticles with two different emission colors, each linked to two of four different binders necessary to recognize the four substances to be analyzed, and two different zones on a support surface locally functionalized with two of the four binders specific to each of the substances to be analyzed.

[0199] For example, multiplexing may be performed to identify viral variants in the case of detecting viral genomes.

[0200] Needless to say, the present invention is not limited to the variations of the embodiments described below in which the substance of interest is immobilized on the surface of a support (e.g., a glass slide or a multiwell plate).

[0201] Other configurations are possible for the combination of photoluminescent particles and target substances of the present invention.

[0202] For example, in a variation of the embodiment, a gel may be used to separate biological molecules according to their size and / or charge, and then transfer them to a membrane where the molecules are specifically detected by nanoparticles linked to a targeting agent, as in the "Western blotting" method.

[0203] In other variations, the reaction surface may not be a solid support but, for example, another magnetic nanoparticle, magnetic microbead, or the like. In that case, the magnetic field can capture the analyte and luminescent nanoparticles combined with the magnetic nanoparticles or beads near the surface, and luminescence can be measured.

[0204] Measurements can be performed, for example, directly within the sample to be analyzed. If the sample is in gaseous form, the sample support may be in the form of a closed volume to prevent dispersion of the test sample. The sample support may be in the form of a tank or cuvette, especially if the sample is in the form of a solution.

[0205] The ultra-sensitive method of the present invention may be adapted to be carried out using flow cytometry techniques (fluorescence-activated cell fractionation or FACS). In this case, particles of the present invention linked to a targeting agent intended to recognize specific molecules of the cell type to be analyzed are brought into contact with the cells, and the cytometry system must be adapted to include an excitation light source of a suitable wavelength for exciting the nanoparticle matrix, preferably a UV laser diode.

[0206] The ultra-high sensitivity method of the present invention may be adapted for use in immunocytochemistry and immunohistochemistry techniques.

[0207] Luminescence measurement As already shown, the ultra-high sensitivity method according to the present invention more specifically includes the steps of (iii) exciting photoluminescent particles linked to a target substance by UV-B and / or UV-C irradiation and (iv) detecting the luminescence emitted by the particles.

[0208] Detection device The ultra-high sensitivity method according to the present invention is advantageously carried out using simple and inexpensive equipment.

[0209] More specifically, the ultra-high sensitivity method according to the present invention is generally, - A lighting device having a wavelength of 240nm to 330nm, more preferably 260 to 330nm, more preferably 260 to 310nm, preferably 270 to 290nm, preferably of the light-emitting diode type, preferably with an output of 500mW or less, more preferably 200mW or less, for example 50 to 500mW, more preferably 50 to 150mW, and - A device for detecting the intensity of light emitted by nanoparticles in step (iii). Includes.

[0210] Preferably, the lighting device emits in the UV-B and / or UV-C range.

[0211] Preferably, the detection device does not include a confocal system.

[0212] Refer to Figures 2 and 3 attached to the following text. These schematically and partially represent suitable equipment for carrying out the ultra-high sensitivity method of the present invention.

[0213] As described above, the apparatus may include a suitable support for immobilizing the target substance of the sample during the process.

[0214] According to a modification of one embodiment, the apparatus according to the present invention comprises a mobile system for a support or illumination device that can continuously illuminate different localized zones of the support, for example, different wells of a multiwell plate. Such modifications are used, in particular, to carry out the detection method of the present invention for spatial multiplexing and / or for measuring several samples, and the mobile system allows for the continuous illumination of each of the predefined zones containing the substance to be analyzed in the same sample or in several different samples.

[0215] The illumination device may consist only of a light-emitting diode positioned as close as possible to the fixed surface and an aperture that ensures local excitation only on the fixed surface.

[0216] The illumination device may include a system of at least one lens positioned in the optical path of the excitation beam to control the size and / or angle of the beam aperture in the zone of the optical assembly, particularly in the zone of the support having particles combined with the material of interest.

[0217] An optical assembly for shaping a beam into a predetermined form may include, conventionally, a system for aligning the beam and reducing its size, for example, using lenses, particularly two lenses. This allows for an appropriate intensity (e.g., 10 W / cm²). 2 ) and in order to obtain illumination whose dimensions are the same as or smaller than the deposition spot (e.g., 1 mm in diameter), it becomes possible to control the illuminated zone in a support zone having particles combined with the target substance.

[0218] Time-resolved detection may be obtained 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 installed in the optical path of the excitation beam. Illumination may be electronically chopped by modulating the current supplied to the illumination source, or by any other method known to those skilled in the art, such as using an acousto-optic crystal.

[0219] Therefore, such time-resolved detection advantageously allows for limiting the contribution of parasitic species, particularly biomolecules, present in the sample (serum, blood, etc.) or the solid substrate used (glass, plastic, etc.) to the luminescence signal by transiently separating unwanted luminescence signals from the signal emitted by nanoparticles. This is because these unwanted signals generally have characteristic lifetimes of less than 1 μs, less than 100 ns, or less than 10 ns.

[0220] Luminescence emission may be detected by measuring the intensity of light emitted at the luminescence wavelength of the photoluminescent particles used. For example, Y 1-x EU x When using VO4 nanoparticles, the intensity of the emitted light is Eu 3+ The measurement may be performed at the luminescence wavelength, i.e., 617 nm.

[0221] The detection device may include a single detector of the photomultiplier tube, photodiode, or avalanche photodiode type, or a photosensitive device array type detector consisting of a two-dimensional surface of detection pixels such as a CCD or EM-CCD camera or CMOS camera. The two-dimensional detection device enables simultaneous measurement of radiation signals from nanoparticles originating from different zones corresponding to different substances to be analyzed on the surface of different samples and / or supports, without requiring movement of the support or excitation beam.

[0222] Preferably, the light intensity detection device includes a single detector, particularly a photomultiplier tube, which makes it possible to produce a relatively inexpensive detection device. The photomultiplier tube may be configured to detect only visible light and not UV light. This, in particular, makes it possible to limit interference from unwanted signals due to residual excitation light. This improves detection sensitivity.

[0223] Preferably, the detection device does not detect the excitation wavelength.

[0224] The detection device may include an optical assembly for collecting emitted luminescence, in particular a system of at least one lens having a large numerical aperture for focusing the luminescence emission on a detector, in particular a photomultiplier tube.

[0225] To spectrally remove unwanted signals, an interference filter may be provided in the optical path of the radiation beam.

[0226] Detection may be performed in the reflection, in other words, on the side of the support surface receiving the excitation beam, particularly by an epifluorescence device. Such detection reduces the background noise of the measurement, which is a particularly sensitive parameter, and makes the measurement more sensitive.

[0227] Alternatively, the measurement may be performed by transmission.

[0228] Analysis of Luminescence Measurement The method of the present invention finally includes step (v) determining the presence and / or concentration of a substance by interpreting a luminescence measurement.

[0229] It is understood that the detection device according to the present invention may include any means for analyzing luminescence radiation, such as a converter for recording and utilizing the luminescence signal.

[0230] The interpretation of luminescence measurements may be carried out by referring to established standards or calibrations.

[0231] More specifically, the amount of the target substance in the sample may be determined by referring to an already established calibration curve, preferably using measurements performed on a sample containing a known amount of the substance under the same conditions as those used in the study, and these same conditions include, in particular, the solvent and pH of the medium.

[0232] Advantageously, as described in Example 3, the ultra-high sensitivity method according to the present invention makes it possible to detect and quantify the target substance in a sample at a content of exactly less than 10 pM, particularly less than 1 pM, or less than 0.1 pM, or less than 0.01 pM (i.e., 10 fM), or less than 1 fM, or better less than 0.1 fM (i.e., 100 aM), or better less than 0.01 fM (i.e., 10 aM).

[0233] Specifically, the ultra-high sensitivity method according to the present invention enables detection with at least 10 times, and especially at least 100 times, or even 1000 times, higher sensitivity than ELISA-type enzyme immunoassays, using the same recognition antibody and target antibody.

[0234] In vitro diagnostic kit In another aspect, the present invention also includes at least, Luminescent particles formed entirely or partially from the previously defined photoluminescent inorganic nanoparticles, which are linked to one or more binders, and are surface-functionalized with chemical groups provided by molecules such as APTES, for example, carboxyl, amino, thiol, aldehyde, or epoxy groups, and / or linked to molecules such as streptavidin, wherein the chemical groups or molecules are suitable for linking the particles to an agent for targeting a target substance, or are already linked to at least one agent for targeting a target substance. at least, The lighting devices already described, A device for detecting the intensity of light emitted by particles. Includes, A detection and / or quantification system that, in some cases, enables time-resolved detection of luminescence radiation, particularly a system for modulating the current supplying the illumination source, a photomultiplier tube, and an AD converter. This includes, in particular, in vitro diagnostic kits for implementing the methods already described.

[0235] To facilitate subsequent functionalization, particle coating with silica may be used.

[0236] The photomultiplier tube may be configured to detect only visible light and not UV light.

[0237] As previously described, the in vitro diagnostic kit according to the present invention may include a support suitable for immobilizing the target substance of the sample.

[0238] As already described, this may be a support whose surface is passivated and functionalized with a capture agent, in particular a drug to target the substance to be detected / quantified, such as a first antibody.

[0239] In a variation of the first embodiment, the in vitro diagnostic kit according to the present invention may include photoluminescent particles according to the present invention linked to an antibody, which is referred to as a “detection antibody” to distinguish it from a targeting agent, particularly a capture antibody immobilized on a support.

[0240] Particles linked to the targeting agent can be obtained as already described. In a variation of another embodiment, the in vitro diagnostic kit may contain particles not linked to the targeting agent, from which the user can prepare one or more particles linked to the targeting agent for use in the ultra-sensitive method according to the present invention.

[0241] Accordingly, according to a particular embodiment, the in vitro diagnostic kit according to the present invention may include several containers that separately contain, on the one hand, the unlinked particles and on the other hand, one or more targeting agents.

[0242] Alternatively, the in vitro diagnostic kit according to the present invention does not contain a targeting agent, and the user can obtain a selected targeting agent, such as a biotinylated targeting agent, separately from a qualified supplier.

[0243] In relation to a variation of this embodiment, the preparation of particles linked to a targeting agent includes, if the targeting agent is included in the in vitro diagnostic kit according to the present invention, the step of mixing unlinked particles according to the present invention with the targeting agent at a concentration ratio predetermined by the volume of the container, or at a concentration ratio determined by the user, as described in Example 1, for example.

[0244] In the in vitro diagnostic kit according to the present invention, particles that are not linked to a targeting agent may, in particular, be particles linked to a molecule suitable for enabling linkage between the particles and a targeting agent, such as a biotinylated targeting agent, such as a biotinylated antibody, such as streptavidin.

[0245] Other molecular pairs, such as hapten / antibody, antigen / antibody, peptide / antibody, such as digoxigenin (DIG) / anti-DIG antibody, sugar / lectin, and polynucleotide / complementary polynucleotide, may also be considered for this type of linkage.

[0246] Alternatively, particles not linked to a targeting agent used in the in vitro diagnostic kit according to the present invention may be particles functionalized on their surface with chemical groups suitable for linking the particles to an agent for targeting a target substance, such as chemical groups provided by molecules such as APTES, such as carboxyl, amino, thiol, aldehyde, or epoxy groups.

[0247] It is understood that the functionalization of the surface of nanoparticles may involve two or more layers. For example, as already stated, the particles of the present invention may include a layer for preparing or stabilizing the surface of the nanoparticles, such as a layer of silica, and a subsequent layer for functionalization with an activating chemical group, such as a layer of APTES (aminopropyltriethoxysilane).

[0248] The embodiments and drawings presented below are provided solely as a non-limiting description of the present invention. [Brief explanation of the drawing]

[0249] [Figure 1a] It is a diagram schematically representing the principle for detecting and quantifying biomolecules. Combination of surface functionalization with a capture antibody (55) (phase 1), contact with a sample to be analyzed (phase 2), washing (phase 3), and photoluminescent particles linked to a targeting agent (42), which is an antibody herein, and a target substance (40) immobilized on the surface of a support herein, followed by washing to remove unimmobilized particles (phase 4). [Figure 1b] It is a diagram schematically representing the principle for detecting DNA / RNA nucleic acids using the particles according to the present invention. A single-stranded DNA (55) partially complementary to the strand to be detected is bound to a support. The single-stranded DNA constitutes a capture agent for the DNA or RNA to be detected. Next, a sample (40) containing the DNA or RNA to be detected is incubated with the functionalized support (phase A). After rinsing, nanoparticles (38) that form an agent targeting the DNA or RNA to be detected and are linked to a single-stranded DNA (42) partially complementary to at least the unpaired portion of the DNA or RNA to be detected are incubated with the support (phase B). After rinsing, only nanoparticles (38) immobilized on the surface of the support after pairing with the DNA or RNA to be detected remain present (phase C). These may be detected and quantified as described herein. Unlike what is schematically illustrated, it is preferable to provide spacers between the surface and the zone complementary to the target substance of the capture agent, and between the surface of the nanoparticles and the zone complementary to the targeting agent. [Figure 2] It is a diagram schematically representing a simple device for luminescence detection by UV excitation and transmission of nanoparticles according to the present invention. [Figure 3a] It is a diagram schematically representing a UV excitation device. [Figure 3b] It is a diagram schematically representing a reflection detection device according to the present invention. [Figure 3c]It is a diagram schematically representing the combination of the UV excitation device shown in FIG. 3a and the detection device shown in FIG. 3b. The additional lens to the transmission device in FIG. 2 enables better control of the collimation of the UV LED, the size and angle of the excitation beam at the sample, and the size and angle of the collected luminescence when focused on the photomultiplier tube. [Figure 4] It is a diagram representing the detection of recombinant insulin in solution at concentrations up to 834 aM (10 fg / mL) using the detection device shown in FIG. 2 and YVO4:Eu (20%) nanoparticles synthesized and annealed as described in Example 1 below. The nanoparticles are conjugated to streptavidin at a ratio of 40 / 1 relative to the nanoparticles, and conjugated to biotinylated antibodies at a ratio of 60 / 1 relative to the nanoparticles. The same antibody as that in the ELISA kit was used. The black line indicates the average signal of the "blank" sample. The gray line indicates the signal value corresponding to the average signal of the "blank" sample plus three times the standard deviation of the "blank" sample. [Figure 5] It is a diagram representing the detection of interferon-gamma in buffer (FIG. 5a) or serum (FIG. 5b) at concentrations up to 2.6 fM (50 fg / mL) using the detection device shown in FIG. 2 and YVO4:Eu (20%) nanoparticles synthesized and annealed as described in Example 1 below. The capture antibody is the same as that in the Thermofischer 13-7319-81 kit. The detection antibody is an intact antibody, whereas the kit contains Fab fragments of the same antibody. The nanoparticles are conjugated to streptavidin at a ratio of 40 / 1 relative to the nanoparticles, and conjugated to biotinylated antibodies at a ratio of 30 / 1 relative to the nanoparticles. The black line indicates the average signal of the "blank" sample. The gray line indicates the signal value corresponding to the average signal of the "blank" sample plus three times the standard deviation of the "blank" sample. [Figure 6] It is a diagram representing the detection of interferon-gamma in buffer (FIG. 6a) and serum (FIG. 6b) using the Thermofischer ELISA kit 88-7316-88. The detection limit of the kit stated by the manufacturer is 260 fM (5 pg / mL). [Figure 7]This figure illustrates the detection of HIV-p24 in solution using antibodies from the QC221 Quantikine Immunoassay Control Set 896 HIV-1 Gag p24 Biotechne kit as a scavenger and conjugate. Detection is performed using the detection device shown in Figure 2 and synthesized and annealed YVO4:Eu(20%) nanoparticles as shown in Example 1 below. The detection antibodies in the kit are biotinylated upstream to form conjugated antibodies linked to the nanoparticles, so as to target the target substance during detection. [Figure 8] This diagram schematically illustrates an optimized detection principle for nucleic acids. It involves: surface functionalization with a capture oligonucleotide (55) (Phase 1); contact between the denatured sample to be analyzed and the detection oligonucleotide forming the targeting agent (42) to link the target nucleic acid (40) in the sample with the detection oligonucleotide (42) (Phase 2); combination of the nucleic acid detection oligonucleotide conjugate with the functionalized surface (Phase 3); and combination of the nucleic acid detection oligonucleotide conjugate with photoluminescent particles (38) linked to a molecule that enables the binding of the targeting agent (here, a streptavidin molecule) (44) to the detection oligonucleotide (42) in solution (Phase 4); subsequent washing to remove unimmobilized particles, i.e., particles not linked to the nucleic acid detection oligonucleotide conjugate; and detection of luminescence emission by immobilized nanoparticles after excitation with UV-B and / or UV-C light at a wavelength of 280 nm and an intensity of 4.5 mW / cm2 (Phase 5). [Figure 9]Figure 9a shows the detection of double-stranded SARS-CoV-2 n1 genes in solution obtained by PCR up to a concentration of 50 fM using the detection device shown in Figure 2 and synthesized and annealed YVO4:Eu(20%) nanoparticles as shown in Example 1. Detection of double-stranded synthetic SARS-CoV-2 n1 genes (detection up to a concentration of 5 fM, Figure 9b) and single-stranded genes (detection up to a concentration of 0.5 fM, Figure 9c). The nanoparticles were ligated to streptavidin at a ratio of 40 / 1 to the nanoparticles and coated with biotinylated detection oligonucleotides complementary to the DNA to be detected at a ratio of 1 / 180 to the number of streptavidin molecules (streptavidin excess). Here, the average signal of the "blank" sample was subtracted from all measurements. That is, the average signal of the "blank" sample is considered to be equal to 0. The gray line shows the signal value corresponding to the average signal of the "blank" sample plus 3 times the standard deviation of the "blank" sample. [Figure 10] This figure shows the detection of single-stranded SARS-CoV-2 n1 genes up to a concentration of 10 aM using the device shown in Figure 2 and YVO4:Eu (5%) nanoparticles synthesized as shown in Example 1.2 and annealed at 1000°C. The nanoparticles were ligated to streptavidin at a ratio of 40:1 to the nanoparticles and coated with biotinylated detection oligonucleotides complementary to the DNA to be detected at a ratio of 1 / 180 to the number of streptavidin molecules (streptavidin excess). That is, the average signal of the "blank" sample is shown by the black line. The gray line shows the signal value corresponding to the average signal of the "blank" sample plus three times the standard deviation of the "blank" sample. [Figure 11] This diagram schematically illustrates the linkage that forms aggregates of nanoparticles (38) linked to streptavidin (44) that bind to biotinylated (42) antibodies (40) at a ratio of at least twice that of nanoparticles. Certain biotinylated antibodies are linked to at least two nanoparticles. [Figure 12]This diagram schematically illustrates the linkage of nanoparticles 38 linked to streptavidin (44) which is bound to a biotinylated (42) carbon chain (46) containing an arm linked to detected DNA (complementary to target DNA) (48) and at least two arms bound to biotin (42). The carbon chains are proportionally at least twice as numerous as the nanoparticles, forming aggregates, and certain carbon chains (46) are linked to at least two nanoparticles via biotin. [Figure 13] This diagram schematically illustrates the linkage of nanoparticles 38, each linked to a detection oligonucleotide (421-425) that is different from other nanoparticles. The detection oligonucleotides are oligonucleotides complementary to different zones of the target nucleic acid (40) bound to wells functionalized by the capture agent (55), for example, oligonucleotides complementary to the target nucleic acid. [Figure 14] This diagram schematically illustrates the linkage of different detection oligonucleotides (421-424) complementary to the target nucleic acid (40) bound to the well functionalized by the capture agent (551-553), for example, oligonucleotides complementary to the target nucleic acid, at least two different oligonucleotides of nanoparticles that bind to different but adjacent zones of the same nucleic acid, and nanoparticles (38) linked to at least two types of targeting agents that bind to different zones of the target nucleic acid. [Figure 15] Figure 2 shows the detection signals of modified phagemids (DNAphagemids) modified to introduce the SARS-CoV-2 n1 gene, at different concentrations in solution, using the detection device shown in Figure 2 and YVO4:Eu (20%) nanoparticles synthesized and annealed as shown in Example 1 (Figure 15a), and YVO4:E (5%) nanoparticles synthesized and annealed at 1000°C as shown in Example 1.2 (Figure 15a). Error bars represent the standard deviation of measurements performed over three trials. The black line shows the signal value corresponding to the average signal of the "blank" sample, and the red line shows the signal value corresponding to the average of the "blank" sample plus three times the standard deviation of the "blank" sample. That is, the detection limits are 50 fM (Figure 15a) and 5 fM (Figure 15b). [Modes for carrying out the invention]

[0250] Formula Y 0.6 EU 0.4 Preparation of luminescent particles containing VO4 YVO4 nanoparticles doped with 20% europium are prepared by known methods, particularly the method described in detail in International Patent Application No. 2019 / 025618 or the method described below.

[0251] Prepare 10 mL of fresh aqueous solution (Solution 1) containing 0.1 M NH4VO3 and 0.3 M N(CH3)4OH. Using a syringe pump, add 0.1 M ions (Y 3+ +Eu 3+ Add 10 mL of another solution containing Y(NO3)3 and Eu(NO3)3, which includes ) to solution 1 by dropwise addition at a flow rate of 1 mL / min. The molar ratio of Y(NO3)3 to Eu(NO3)3 is as follows: 3+ and Eu 3+ Select as a function of the desired ratio of ions. Typically Y 3+ and Eu 3+ The molar ratio is 0.8 / 0.2.

[0252] Upon addition of the Y(NO3)2 / Eu(NO3)3 solution, the solution diffuses and takes on a white / milky appearance. The synthesis is continued until all of the Y(NO3)2 / Eu(NO3)3 solution has been added. The solution is then stirred at room temperature for 15 days. At this point, the final 20 mL of solution must be purified to remove excess counterions. To do this, the solution is centrifuged at 11,000 × g (Sigma 3K10, Bioblock Scientific) for 80 minutes (typically three times), followed by redispersion with ultrasound (Branson Sonifier 450, 540 W output, operating at 50%) until a conductivity of exactly less than 100 μS / cm is achieved.

[0253] The obtained nanoparticles are annealed in an autoclave under hydrothermal conditions at 220°C for 2 hours. This annealing treatment improves crystallinity, and therefore reduces the photoreduction effect of the nanoparticles when excited with UV-B and / or UV-C.

[0254] Formula Y 0.95 EU 0.5 Preparation of luminescent particles containing VO4 Formula Y 0.95 EU 0.5 Vanadate nanoparticles containing VO4 were prepared in two steps via colloidal conversion of rare earth metal hydroxycarbonate particles. In a typical procedure, 0.1 mol / L of RE(NO3)3[RE = (Y 0.95 EU 0.05 20 mL of the solution of ), 100 mL of Milli-Q water, 80 mL of ethylene glycol (EG), and 15 g of urea were added to a three-necked flask (500 mL). The final volume ratio of H2O / EG was 3 / 2 (i.e., 40% of EG in water). The mixture was heated vigorously at room temperature for 30 minutes to homogenize it. The round-bottom flask was then connected to a condenser and heated at 95 °C for 2 hours. The resulting suspension was subjected to centrifugation at 26 323 × g for 25 minutes. The pellet was redispersed in Milli-Q water and subjected to further centrifugation (final conductivity less than 100 μS / cm). 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 NH4VO3 solution (2 mmol, 20 mL, Sigma-Aldrich) was rapidly added to the colloid with stirring. The system was maintained at 100°C for 2 hours, and particles were collected by centrifugation (26 323 × g, 30 minutes). The particles were then purified by 24 hours of dialysis in Milli-Q water.

[0255] Observation of the particles using an electron microscope reveals that they have an olive shape with average dimensions of 153 nm in length and 67 nm in width.

[0256] In a typical protected annealing procedure, a polymeric silica sol was prepared by mixing TEOS(Si(OC2H5)3)4), water (pH 1.25), and ethanol in a molar ratio of 1:5 / 3.8 under acidic conditions and aging at 60°C for 1 hour. To allow this silica matrix to dissolve more easily, a porous structure was prepared by the gelation of silica around a self-assembled micelle assembly of a surfactant copolymer. The copolymer Pluronic PE6800 (EO) has a molecular weight of 8080 g / mol. 73 PO 28 EO 73 ) (BASF Europe) was dissolved in ethanol at 40.4 g / L. The final solution was obtained by mixing the colloidal solution of nanoparticles, the silica solution, and the PE6800 solution. For YVO4:Eu particles, due to the basic properties of the colloidal solution, it is necessary to use a concentration of surfactant in the sol that does not systematically bring about an organized silica matrix (the usual V / Si / PE6800 molar ratio is 1 / 5 / 0.05). The gel was dried at 90°C for more than 6 hours, and the resulting powder was annealed in air at 1000°C in two steps. The first annealing was carried out at 500°C at a rate of 100°C / hour, with the final step carried out for 1 hour. The second annealing was carried out at 1000°C at a rate of 100°C / hour, including 2 hours at 500°C and 10 minutes at 1000°C. The first annealing is necessary to completely remove organic matter (PAA and PE6800 polymer).

[0257] Silica powder containing particles was dissolved in excess 2% hydrofluoric acid for 3 hours at a Si / HF molar ratio of 1 / 9. The hydrofluoric acid and dissolved silica were then removed by two centrifugations at 14,000 × g (1 minute, 10 minutes). The precipitate was diluted with pure water, and a few drops of sodium hydroxide were added to adjust the pH to 10-11. The final solution was stabilized by sonication in a cold bath for 5 minutes with the addition of PAA (V / PAA 1 / 0.05).

[0258] Linking of nanoparticles to streptavidin protein Following the synthesis and annealing of the nanoparticles, the nanoparticles are functionalized by a silicification step, an amination step using APTES, and an amine-to-carboxylic acid conversion step using an acid anhydride, as described in International Patent Application No. 2019 / 025618. To expand applications, the nanoparticles are linked to streptavidin in a 40 / 1 ratio using the method described in detail below.

[0259] To obtain 150 μL of nanoparticles coated with 200 nM -COOH, an appropriate volume is pipetteed. The sample is then centrifuged at 13,700 × g for 15 minutes, and the supernatant is removed. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are dissolved at concentrations of 50 mg / mL each in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 5.5. This solution is rapidly added to the nanoparticle pellet, and the sample is sonicated at 50% amplitude for 15 seconds (GEX130 ultrasonic processor, tip ref. 423-A). After incubation at room temperature with stirring for 25 minutes, the sample is centrifuged at 15,000 × g for 15 minutes, and the nanoparticle pellet is redispersed in 50 mM phosphate buffer at pH 7.4 by sonication at 50% for 15 seconds. Next, 40 equivalents of streptavidin (Sigma, s4762-10MG) are added, and the sample is incubated at 800 rpm (Eppendorf, Thermomixer C) at 25°C for 2.5 hours. The sample is centrifuged at 13,700 × g for 15 minutes, and the pellet is resuspended in 500 μL of blocking buffer (50 mM phosphate buffer, pH 7.4, containing 2% mPEG-NH2 500 (MF001005-500 Biochempeg)) by 10-second pulsed sonication with 20% amplitude on ice. The sample is then incubated at room temperature for 1 hour with stirring, centrifuged at 13,700 × g for 15 minutes, and the pellet corresponding to the streptavidin-nanoparticle conjugate is redispersed in storage buffer (20 mM Tris, pH 8, 1% BSA) and stored at -80°C.

[0260] Conjugation of streptavidin-linked nanoparticles (nanoparticle-SA) and biotinylated antibodies Next, streptavidin-linked nanoparticles are conjugated with biotinylated antibodies specific to the substance to be assayed, by using known methods, particularly the methods described in the protocols below, and adapting the ratio of biotinylated antibody to nanoparticles as a function specifically of the target substance to be measured (insulin, interferon gamma (IFN-gamma), or HIV-I-p24 protein described below).

[0261] Preferably, as shown in FIG. 10, the amount of biotin 42 is greater than the amount of nanoparticles 38 and antibodies 40, whereby nanoparticles 38 bind as aggregates. Such aggregates increase the amount of nanoparticles bound to each molecule of the target substance, thus enabling improvement in detection sensitivity. Specifically, it is no longer a single nanoparticle that binds to the target substance, but an aggregate of nanoparticles having a stronger luminescence signal.

[0262] Conjugation of streptavidin-linked nanoparticles and biotinylated oligonucleotides As a variant, when detecting nucleic acids, as shown in FIG. 11, the nanoparticles 38 linked to streptavidin 44 may be linked to a carbon chain 46 that includes at least two biotinylated arms 42 and one arm carrying a detection oligonucleotide. Due to the fact that the carbon chain contains two or more biotinylated arms, these can bind to two streptavidins that originate particularly from different nanoparticles, enabling the formation of a complex.

[0263] As shown in FIG. 12, the nanoparticles 38 are linked to different targeting agents 421 to 425, particularly different complementary oligonucleotides, and can target different zones of the target substance 40 bound to a support, particularly different zones of a nucleic acid. This allows several nanoparticles to bind to the same molecule of the target substance, thereby improving sensitivity.

[0264] As shown in Figure 13, each nanoparticle 38 contains several different targeting agents 421-424, particularly different complementary oligonucleotides, and can target zones of the target substance 40 that are different but sufficiently close, especially nucleic acids. This makes it possible to improve the binding strength between the nanoparticle 38 and the target substance 40 by reducing the dissociation rate. In order to bind to different zones of the target substance 52, the capture surface may also be functionalized with targeting agents 551-553, particularly different complementary oligonucleotides.

[0265] The nanoparticles are often added to the microwells of a support container, particularly a plate, and the sample to be analyzed is then incubated in the same microwells.

[0266] Preparation of the capture surface: Antibody binding As indicated in the kit protocol, collect 100 μL of capture antibody from the kit's binding buffer in a commercially available kit and incubate it in each well of a multi-well plate (655097, Greiner) at 4°C for 16 hours with shaking at 300 rpm. Then wash the wells twice with the commercially available diluent provided with the kit, or 0.01% Tween PBS if this is not available. Next, add 200 μL of blocking solution per well and reduce nonspecific interactions at 4°C for 2 hours with shaking at 300 rpm.

[0267] Preparation of the capture surface: Binding of oligonucleotides Add 10 μg / mL of anti-digoxigenin antibody (Ab64509, Abcam) to the bottom of a multi-well plate in 100 μL of PBS buffer (pH 7.2) and incubate overnight at 4°C with shaking. Wash the plate three times with 200 μL of modified 5X SSC (SSC: 750 mM NaCl, 75 mM sodium citrate, 0.05% SDS, 0.05% lauroyl sarcosine). Block the wells in 3% BSA PBS buffer, pH 7.2, with shaking at 4°C for 2 hours, and wash once with 200 μL of 5X SSC. Add 100 μL of 16.5 μM 5'-digoxigenin scavenging oligonucleotide and keep the plate at 4°C with shaking.

[0268] Experimental apparatus for luminescence measurement The detection device includes an illumination device and a luminescence detection device, as shown in Figure 2. This device was used for all the results presented below.

[0269] In the detection device shown in Figure 2, the illumination device consists of an electron light-emitting diode (1) and a 1 mm aperture located 3 mm from the diode (1). The light-emitting diode (Hex-S6060-DR250-W275-P100-V6.5, Laser Components) emits a wavelength of 275 nm in the UV-C range at an output of 100 mW. This makes it possible to illuminate a single well of a multi-well plate, with the bottom of each well located 20 mm from the diode. The detection device consists of a lens (20) for parallelizing the light emitted from nanoparticles in the sample and a focusing lens (26) for focusing the light emitted to a photomultiplier tube type detection module (28) (PMM02, Thorlabs) equipped with an interference filter that filters the emitted light at 617 nm. This photomultiplier tube may be configured not to detect irradiation in the UV range. This makes it possible to limit detection to the visible range and eliminate residual UV irradiation.

[0270] Alternative versions of the detection device are shown in Figures 3a and 3c. The detection device is described in detail below. The illumination device (Figure 3a) consists of a light-emitting diode (1) that emits UV-C range light at a wavelength of 275 nm and an output of 100 mW, and a system (2) for parallelizing the light and reducing the size of the laser beam. The parallelizing and beam size reduction system (2) shown consists of, starting from the diode and moving toward the sample, a parallelizing lens (3), a set of diaphragms (4) (two iris diaphragms, a "field" diaphragm, and a diaphragm that allows the beam to be completely stopped), a focusing lens (6), an iris diaphragm called an aperture diaphragm (8) at the focal point of the focusing lens, a parallelizing lens (10), a focusing lens (12), a dichroic mirror suitable for reflecting the illumination light and transmitting the light emitted by the sample (35), an objective lens (16) with a large numerical aperture, e.g., an numerical aperture equal to 0.79, and finally an illumination diaphragm (18). However, to avoid some wells of a multiwell plate being illuminated simultaneously, the illumination device may include only a diode and a suitable aperture to limit the "crosstalk" effect, as shown in Figure 2.

[0271] The detection device (Figure 3b) includes an objective lens (16), a focusing lens (22), a parallelizing lens, a 617 nm interference filter, and a focusing lens for focusing and parallelizing the luminescence emitted by nanoparticles in the sample following the light path from the sample, as well as a spectral filter (27) that removes 617 nm light, and a photomultiplier tube type detection module (28) (PMM02, Thorlabs). Figure 3c shows the entire detection apparatus including the illumination device and the detection device.

[0272] An analog-to-digital converter (NI9215, National Instruments) is used, and the signal is recorded using LabVIEW software.

[0273] All detection elements may or may not be located on the same axis (depending on the appropriate use of one or more mirrors). A slide holder transfer system (Z8253, KCH301, Thorlabs) was used to continuously observe several biological samples by scanning.

[0274] Time-resolved detection for measuring the amount of nanoparticles in the presence of unwanted signals. The light-emitting diode is driven by a voltage pulse via an NI-9215 input module (National Instruments), generating a UV excitation pulse to eliminate unwanted signals using time-resolved detection. Specifically, when a biological sample is illuminated by the diode, molecules other than the target nanoparticles emit fluorescence. This unwanted fluorescence can be eliminated by the UV excitation pulse and a signal detection frequency of 100 kHz using a photomultiplier tube (one signal acquisition every 10 μs). Because the emission duration of the particles of the present invention is long, time-resolved emission detection, particularly delayed emission detection, can be performed, as described in detail below.

[0275] Time-modulated illumination makes it possible to limit the contribution of parasitic species to the luminescence signal from samples (serum, blood, etc.) or solid substrates that may be used (glass, plastic, etc.). Specifically, the nanoparticles used (e.g., YVO4:Eu or GdVO4:Eu) can be placed in an excited state with a long lifetime lasting hundreds of microseconds, compared to the nanosecond lifetime of conventional fluorescent dyes. This makes it possible to temporarily separate unwanted luminescence signals from the signals emitted by the nanoparticles.

[0276] The resulting modulation signal is the alternation between the decay phase (end of illumination) and the luminescence return phase (start of illumination) of all radiators present in the sample. The decay / return of the luminescence signal is determined by two different parameters: (i) the lifetime of the excited state of the radiator, and (ii) the dynamics of the on-time and off-time of the excitation beam.

[0277] Deformation of experimental apparatus Luminescent radiation from nanoparticles is As transmitted light (Figure 2), or By using a dichroic mirror (30) at 347 nm, the reflected light is obtained (Figures 3a to 3c). The light is focused.

[0278] To detect the concentration of each sample, several wells N, typically three wells, of a multiwell plate may be used. The measurement points for each concentration are presented as the mean and standard deviation of the N values ​​obtained for each well. The value measured for each well is the average of 60,000 values ​​recorded over 600 ms at an acquisition rate of 100 kHz (one voltage value is recorded every 10 μs).

[0279] Typically, the limit of detection (LOD) is determined by the concentration at which a signal greater than or equal to three times the sum of the signal obtained at zero concentration ("blank") and the standard deviation of the "blank" signal. The limit of quantification can be considered as the concentration at which a signal ten times greater than this standard deviation is produced. However, it is preferable to determine the limit of quantification experimentally.

[0280] Calibration of detection devices Prior to measurement, the detection device was calibrated using the substance to be detected at known concentrations.

[0281] Detection and quantification of substances in a sample The concentration of a substance in a sample is considered detectable if the resulting signal is at least equal to or greater than the sum of the signal from a sample of the same composition containing zero concentration of the substance, referred to as a "blank" sample, and three times the standard deviation of the signal from the "blank" sample.

[0282] To quantify the target substance (i.e., determine its concentration), the following protocol must be used. i) Perform a series of calibration measurements using the target substance at different known concentrations, for example, from commercially available or purified substances. Where possible, the calibration samples should be prepared with the same composition as the sample to be measured, or as close as possible. Adjust the obtained points (signal in mV and concentration of the target substance). ii) Perform measurements on the sample to be analyzed (generate signal values ​​in mV units). iii) Based on the measured signal (in mV) and the calibration curve and its adjustment obtained in step i), a concentration value for the substance is assigned to each measured sample.

[0283] To determine the concentration of a substance in a sample that can be quantified at a given coefficient of variation (CV), such as 25%, 20%, 15%, or 10%, the following protocol must be used. i) Perform a sufficient number of measurements on samples of known different increasing concentrations that exceed the limit of detection (LOD). ii) Determine the standard deviation and coefficient of variation related to the measurement variability for each of these concentrations and the concentrations 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, by normalizing it with respect to the concentration value. iv) If either of these two determined variation counts is greater than a predetermined variation count, repeat the measurement at a higher substance concentration.

[0284] The limit of quantification for a given CV corresponds to the concentration of the substance at which the coefficient of variation related to measurement variability and the coefficient of variation related to bias are equal to or less than a given CV.

[0285] Comparison of detection sensitivity between commercially available ELISA tests and the method of the present invention. For each analyte to be detected, the same 96-well plate, the same antibody, and substantially the same buffer as those used in commercially available ELISA tests are used. However, the enzyme HRP (horseradish peroxidase) is the one prepared in Example 1, surface-functionalized with streptavidin molecules and linked to the biotinylated antibody of each substance to be analyzed, obtained by the manufacturing method already described, using formula Y 0.6 EU 0.4 Replace with VO4 nanoparticles.

[0286] Insulin detection The sample to be analyzed is recombinant insulin (ELISA kit, Abcam ab100578).

[0287] For detection based on nanoparticles, use the same multi-well plates, the same biotinylated detection antibody, insulin, and diluents A and B, as provided in the above Abcam kit coated with a commercially available capture antibody, as well as the washing buffer.

[0288] Prepare an insulin solution with diluent A to obtain a concentration of 10 ng / mL. Then, serially dilute the solution 10-fold (measure 3 times for each concentration, 50 "blank" samples) and incubate the microwell plate at 25°C for 2.5 hours while shaking at 300 rpm (Eppendorf Thermomixer C).

[0289] Simultaneously, the streptavidin-nanoparticle conjugate is incubated with 60 equivalents of biotinylation detection antibody in pH 7.4 phosphate-buffered saline (PBS) at 25°C for 1.5 hours. The solution is then centrifuged at 13,000 × g for 15 minutes. The pellet is resuspended in diluent B by pulse sonication on ice for 10 seconds on / 1 second off to obtain a final nanoparticle concentration of 4.5 nM.

[0290] Wash the microplate three times with washing buffer. Then, add 100 μL of nanoparticle-antibody conjugate solution and incubate the plate at 25°C for 1.5 hours while shaking at 300 rpm. Wash the plate three times with washing buffer and once with PBS pH 6.6. Then, add 200 μL of PBS pH 6.6 to each well and read the plate.

[0291] The standard deviation at zero concentration is 28 mV (see Figure 4). The measured signal value at a concentration of 10 fg / mL (i.e., 834 aM) was 409 mV, which is above the limit value of 289 mV, which is equal to the mean of the "blank" samples plus three times the standard deviation determined for the "blank" samples.

[0292] For comparison purposes, the minimum detection concentration indicated by the ELISA kit supplier is 50 pg / mL (i.e., 4.17 pM).

[0293] Therefore, the minimum concentration that can be detected using the ultra-high sensitivity detection method according to the present invention is as low as 1 / 5000th of the concentration that can be detected by ELISA using the same antibody as the ELISA kit.

[0294] Detection of interferon-gamma The sample to be analyzed is interferon-gamma (Thermofisher ELISA kit 88-7316-88).

[0295] For nanoparticle-based detection, 100 μL of capture antibody from a commercially available kit is diluted 250-fold with the kit's binding buffer and incubated in each well of a multi-well plate at 4°C for 16 hours with shaking at 300 rpm. The wells are then washed twice with the commercially available Elisa / Elispot diluent provided with the kit. Next, 200 μL of Elisa / Elispot diluent is added per well, and the mixture is blocked at 4°C for 2 hours with shaking at 300 rpm.

[0296] Prepare interferon-gamma at various concentrations in commercially available diluents (250 pg / mL to 25 fg / mL). Add 100 μL to each well in three measurements and incubate the plate at 4°C for 16 hours with shaking at 300 rpm. Then wash the wells three times with PBS. In parallel, mix YVO4 / Eu 20%:streptavidin 1 / 40 nanoparticles with 60 equivalents of detection antibody (4S.B3 Thermofischer 13-7319-85) in pH 7.4 PBS at room temperature for 1 hour with gentle shaking. Centrifuge the mixture at 13700×g for 15 minutes. Resuspend the pellet in PBS by pulse sonication on ice for 10 seconds on / 1 second off to obtain a nanoparticle concentration of 4.5 nM. Next, add 100 μL of the nanoparticle / 4S.B3 solution to each well and incubate the plate at 300 rpm at 25°C for 1.5 hours. The plate is washed three times with pH 7.4 PBS and read using the facility's reader (Figure 2).

[0297] The results are shown in Figure 5. In buffer solution (Figure 5a), the signal value measured at a concentration of 160 fg / mL was 160.8 mV, exceeding the detection limit of 149.1 mV, which is equal to the mean of the "blank" samples (133.8 mV) plus three times the standard deviation determined for the "blank" samples. In serum (Figure 5b), the signal value measured at a concentration of 800 fg / mL was 149.7 mV, exceeding the detection limit of 148.3 mV, which is equal to the mean of the "blank" samples (138.5 mV) plus three times the standard deviation determined for the "blank" samples.

[0298] For comparative purposes, interferon-gamma was detected under the conditions specified in the ELISA kit. The experimental conditions followed were those indicated by the ELISA kit supplier. The results for measurements in buffer are shown in Figure 6a, and for measurements in serum are shown in Figure 6b. The minimum concentrations measured were 5 pg / mL in buffer and 62.5 pg / mL in serum.

[0299] Therefore, the minimum concentration that can be detected using the ultra-high sensitivity detection method according to the present invention is low, being 1 / 31st of the concentration detectable by ELISA when the measurement is performed in a buffer solution, and 1 / 78th of the concentration detectable by ELISA when the measurement is performed in serum.

[0300] Detection of HIV-p24 The sample to be analyzed is HIV-p24 (QC221 Quantikine Immunoassay Control Set 896 HIV-1 Gag p24, Biotechne).

[0301] For nanoparticle-based detection, 100 μL of Biotechne's capture antibody MAB73602 (10 μg / μL) is diluted in PBS and incubated in each well of a multi-well plate at 4°C for 16 hours with shaking at 300 rpm. Simultaneously, streptavidin-nanoparticle conjugates are incubated with 30 equivalents of biotinylated detection antibody (Biotechne, NBP3-06466-100 μL) in pH 7.4 PBS at 25°C for 1.5 hours. The solution is then centrifuged at 13,000 × g for 15 minutes. The pellet is resuspended in PBS by pulsed sonication (10 seconds on / 1 second off) on ice to obtain a final nanoparticle concentration of 4.5 nM.

[0302] Wash the microplate three times with pH 7.4 PBS wash buffer containing 0.01% Tween 20 as recommended by the antibody supplier. Then, add 100 μL of nanoparticle-antibody conjugate solution and incubate the plate at 25°C for 1.5 hours with shaking at 300 rpm. Wash the plate three times with wash buffer and once with pH 6.6 PBS. Then, add 200 μL of pH 6.6 PBS to each well and read the plate with the reader shown in Figure 2.

[0303] The standard deviation relative to zero concentration is 5.4 mV. The measured signal value for a concentration of 500 fg / mL (i.e., 20.8 fM) was 251 mV, which exceeds the limit of 246 mV, which is equal to the mean of the "blank" samples plus twice the standard deviation determined for the "blank" samples, consistent with the definition of commercially available tests (see Figure 7).

[0304] For comparison purposes, the minimum detectable concentration indicated by the antibody supplier (Biotechne HIV-1 Gag P-24 DuoSet ELISA kit) is 15.6 pg / mL (i.e., 650 fM). This detection limit was defined as the concentration that produces a signal equal to or greater than the mean signal of the "blank" sample plus twice the standard deviation of the "blank" sample.

[0305] According to the ultra-high sensitivity detection method of the present invention, the minimum measurable concentration using the device in Figure 2 is 500 fg / mL (i.e., 20.8 fM), and therefore the minimum detectable concentration is small, about 1 / 32nd of the concentration detectable by ELISA using the same antibody.

[0306] Detection of the SARS-CoV-2 n1 gene The optimal scheme used for detection is shown in Figure 8. The sample to be analyzed is the PCR product of the SARS-CoV-2 n1 gene. Anti-digoxigenin antibody (Ab64509, Abcam) is added to the bottom of a multi-well plate at a concentration of 10 μg / mL in 100 μL of pH 7.2 PBS buffer and incubated overnight at 4°C with shaking. The plate is washed three times with 200 μL of modified 5X SSC (750 mM NaCl, 75 mM sodium citrate, 0.05% SDS, 0.05% lauroyl sarcosine). The wells are blocked at 4°C for 2 hours with shaking in 3% BSA pH 7.2 PBS buffer and washed once with 200 μL of modified 5X SSC. Add 100 μL of 16.5 μM digoxigenin-5' capture oligo (5'-GACCCCAAAATCAGCGAAAT) and maintain the plate at 4°C with shaking. Simultaneously, denaturate 60 μL of 43 nM target DNA in solution [0.1 M NaOH, 0.01% Tween 20] at room temperature for 15 minutes for each concentration, and add 540 μL of 40 nM 3'-biotin detection oligo (5'-CAGATTCAACTGGCAGTAACCAGA) in modified 5X SSC. After washing the wells with 200 μL of modified 5X SSC, add 100 μL of this target DNA detection oligo conjugate solution to the plate wells. Maintain the plate at 4°C for 30 minutes with shaking. 4.5 nM YVO 4: Add 100 μL of Eu 20% nanoparticle-streptavidin conjugate and incubate at 4°C for 1 hour with shaking. Then wash the wells five times with modified 5X SSC and twice with PBS, and read them with the specially designed UV reader shown in Figure 2.

[0307] The standard deviation of the "blank" sample is 22.8 mV. The signal value measured at a concentration of 50 fM was 348 mV, which is just above the limit of 335 mV, which is equal to the mean of the "blank" sample plus three times the standard deviation determined for the "blank" sample. That is, the detection limit for the SARS-CoV-2 double-stranded n1 gene is 50 fM (Figure 9a). If the target substance is a synthesized SARS-CoV-2 double-stranded n1 gene (Eurogentec), the minimum detectable concentration is 5 fM (Figure 9b). If the target substance is a synthesized SARS-CoV-2 single-stranded n1 gene (Eurogentec), the minimum detectable concentration is 0.5 fM (Figure 9c). This detection limit is comparable to the results obtained in literature

[14] that does not include enzymes or nucleic acid amplification.

[0308] Detection of the SARS-CoV-2 n1 gene using nanoparticles annealed at 1000°C. Equation Y obtained by the method already described 0.95 EU 0.5 Nanoparticles containing VO4 were functionalized and ligated to streptavidin as previously described, and then ligated to biotinylated oligonucleotides as previously described. Next, the same protocol as in previous examples for the detection of the SARS-CoV-2 n1 gene was used. The signal value measured at a concentration of 10 aM was 524.6 mV, well above the limit value of 508 mV, which is equal to the mean of the "blank" sample plus three times the standard deviation determined for the "blank" sample. In this case, a sensitivity limit of less than 10 aM was obtained because the number of photons emitted per unit time from these nanoparticles increased in relation to the number of ions, i.e., the larger size of the nanoparticles, and the higher quantum yield. This is comparable to the detection limit of conventional PCR (approximately 1 aM, corresponding to 30,000 nucleic acid molecules / mL). That is, this result was obtained without amplification and without the use of enzymes.

[0309] Detection of phagemids carrying the SARS-CoV-2 n1 gene The optimal scheme used for detection is shown in Figure 15. The sample to be analyzed is a phagemide carrying the SARS-CoV-2 n1 gene produced by the XL1-Blue MRF' strain according to the protocol of reference

[37] . This was constructed on a pTA131 plasmid using the SLIC protocol of reference

[38] with EcoR1 and Not1 restriction enzymes. Anti-digoxigenin antibody (Ab64509, Abcam) was added to the bottom of a multiwell plate at 10 μg / mL in 100 μL of pH 7.2 PBS buffer and incubated overnight at 4°C with shaking. The plate was washed three times with 200 μL of modified 5X SSC (750 mM NaCl, 75 mM sodium citrate, 0.05% SDS, 0.05% lauroyl sarcosine). Block the wells in 3% BSA pH 7.2 PBS buffer with salmon sperm DNA added while shaking, at 4°C for 2 hours, and wash once with 200 μL of modified 5X SSC. Add 100 μL of 16.5 μM digoxigenin-5' capture oligo (5'-GACCCCAAAATCAGCGAAAT) and maintain the plate at 4°C while shaking. Simultaneously, denaturate 16.5 μL of 18 nM target phagemid in a solution of [0.1 M NaOH, 0.01% Tween 20] at room temperature for 15 minutes, add 43.55 μL of modified 5X SSC, and then add 540 μL of 40 nM 3'-biotin detection oligo (5'-CAGATTCAACTGGCAGTAACCAGA) in modified 5X SSC. A 10-fold dilution series was prepared for six different concentrations of phagemids ranging from 500 pM to 5 fM in modified 5X SSC containing 40 nM 3'-biotin detection oligo. After washing the wells with 200 μL of modified 5X SSC, 100 μL of each concentration of phagemid DNA-detection oligo conjugate was added to the plate wells. The plate was incubated at 4°C for 30 minutes with shaking. 100 μL of 9 nM YVO4:Eu 20% nanoparticle-streptavidin conjugate was added, and the samples were incubated at 4°C for 1 hour with shaking. The wells were then washed twice with 5X modified SSC and PBS, and read using the specially designed UV reader shown in Figure 2.

[0310] The standard deviation of the "blank" sample is 17.3 mV. The signal value measured at a concentration of 50 fM is 268 mV, which is greater than the limit value of 261 mV, which is equal to the mean of the "blank" sample plus three times the standard deviation determined for the "blank" sample. That is, the detection limit for phagemids containing the SARS-CoV-2 n1 gene is 50 fM, as shown in Figure 15a.

[0311] Detection of phagemids carrying the SARS-CoV-2 n1 gene using nanoparticles annealed at 1000°C. Equation Y obtained by the method already described 0.95 EU 0.5 Nanoparticles containing VO4 were functionalized and ligated to streptavidin as previously described, and then ligated to biotinylated oligonucleotides as previously described. Next, the same protocol as in previous examples for the detection of the SARS-CoV-2 n1 gene was used, except that the detection range was set to 50 pM to 5 aM. The signal value measured at a concentration of 5 fM was 348.4 mV, which is just above the limit value of 338.8 mV, which is equal to the mean of the "blank" sample plus three times the standard deviation determined for the "blank" sample. In this case, the sensitivity limit increased as shown in Figure 15b because the number of photons emitted per unit time from these nanoparticles increased in relation to the number of ions, i.e., the larger size of the nanoparticles, and the higher quantum yield.

[0312] These two examples demonstrate the ability of the present invention to detect long single-stranded nucleic acids with increased detection sensitivity for phage genomes containing 3657 base pairs. [Explanation of symbols]

[0313] 1. Phase 1 (Surface Functionalization) 2. Phase 2 (Contact with the sample to be analyzed) 3. Phase 3 (Washing / Nucleic Acid Detection: Combination of Oligonucleotide Conjugate and Functionalized Surface) 4. Phase 4 (Combination of photoluminescent particles linked to a molecule that enables binding of a washing / targeting agent to a detection oligonucleotide in solution, and a nucleic acid detection oligonucleotide conjugate) 5. Phase 5 (Detection of luminescence emission from immobilized nanoparticles after excitation) 35 samples 38 Nanoparticles / Photoluminescent Particles 40. Target substance / sample / target antibody / biological or chemical target substance 42 Targeting agents / Single-stranded DNA / Detectable oligonucleotides / Biotin / Linking agents / Drugs that target target substances 44. Streptavidin / Conjugate / A molecule that enables the binding of drugs targeting a specific substance. 46-carbon chain 48 Detected DNA 52 Target substances 55 Capture antibodies / Single-stranded DNA / Capture oligonucleotides / Capture agents

[0314] (References)

[0315] [Table 1] [Table 2] [Table 3]

Claims

1. A method for ultra-high-sensitivity in vitro detection and / or quantification of a biological or chemical target substance (40) in a sample (35), particularly a biological sample, by detecting luminescence radiation emitted by photoluminescent inorganic nanoparticles, wherein at least the following: (i) Equation (I): A １－ｘ Ln ｘ VO ４（１－ｙ） (PO ４ ) ｙ (I) A step of preparing photoluminescent particles (38) formed entirely or partially from photoluminescent inorganic nanoparticles containing vanadate or a vanadate / phosphate matrix, A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), and mixtures thereof, and 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 when Ln represents Eu. 0 < x < 1, especially 0.02 ≤ x ≤ 0.5, especially 0.05 ≤ x ≤ 0.4, more specifically x is 0.4, 0.2, 0.1, or 0.05, 0 ≤ y < 1, in particular y is equal to 0, The photoluminescent inorganic nanoparticles are linked to an agent for direct (42) or indirect (44) linkage to a target substance, in the process, (ii) A step of bringing the photoluminescent particles (38) into contact with the target substance (40) under conditions that link the target substance (40) and the binders (42, 44), (iii) A step of exciting a matrix of photoluminescent inorganic nanoparticles of formula (I) by irradiation having a wavelength of 240 nm to 330 nm, wherein the irradiation is emitted by an illumination device having an output of 50 mW to 500 mW. (iv) A step of detecting the luminescence emission from the photoluminescent inorganic nanoparticles, particularly by time-resolved detection, and (v) A step of determining the presence and / or concentration of the target substance by analyzing the measurement results of luminescence radiation from the particles. Includes, A method having sensitivity for detecting biological or chemical target substances in a sample at concentrations of 10 pM or less.

2. The method according to claim 1, having a sensitivity of less than 1 pM, less than 0.1 pM, less than 0.01 pM, or 1 fM or less, or more preferably 0.1 fM (i.e., 100 aM) or less, or more preferably 0.01 fM (i.e., 10 aM) or less, for detecting a biological or chemical target substance in a sample, in particular without amplification and without the use of enzymes.

3. The method according to claim 1 or 2, wherein the binder is a targeting agent (42) that is directly bound to the target substance in step (ii), or a molecule (44) that enables the binding of a targeting agent that is bound to the target substance before the photoluminescent particles come into contact with the target substance.

4. The method according to any one of claims 1 to 3, comprising the step of linking nanoparticles to a binder, wherein at least one nanoparticle is linked to a plurality of binders, and the nanoparticles are dissolved in a proportion of binders greater than the proportion of nanoparticles, particularly at least two binders per nanoparticle, or at least ten binders per nanoparticle, or 10 to 80 binders per nanoparticle.

5. The method according to claim 4, wherein each of the photoluminescent particles comprises a plurality of nanoparticles that are bound together by the binder to form a nanoparticle aggregate, and preferably the photoluminescent particles are colloidally dispersed in a solution in step (ii) of contacting the target substance.

6. The method according to any one of claims 1 to 5, using at least two different types of photoluminescent particles, each comprising nanoparticles of formula (I), which are linked to different binders suitable for binding to different sites of the same analyte, and in particular to different oligonucleotides that recognize different regions of nucleic acid-type analytes.

7. The method according to any one of claims 1 to 6, wherein the nanoparticles are each linked to a plurality of binders, in particular a plurality of oligonucleotides selected to bind to different zones of nucleic acids in several zones of the analyte.

8. In step (i), the target substance of the sample is first fixed to the surface of the support, the surface is passivated to prevent the luminescent particles from binding to the surface when the target substance is not present, and step (i) is, in particular, at least the following: (a) A step of providing a support whose surface is pre-passivated and functionalized with a drug for capturing a substance to be detected / quantified, such as a monoclonal antibody known as a capture antibody, (b) A step of bringing the sample to be analyzed into contact with the support from step (a) under conditions that promote the combination of the substance and the capture agent, and (c) A step of bringing the photoluminescent particles, linked to at least one binder, into contact with the support from step (b), thereby directly or indirectly combining the particles with the material fixed on the surface of the support. The method according to any one of claims 1 to 7, including the method described in any one of claims 1 to 7.

9. The method according to any one of claims 1 to 8, using at least two nanoparticles doped with different rare-earth metal ions having different emission wavelengths, each linked to a different direct or indirect binder that links to one of two different target substances.

10. A lighting device having a wavelength of 240 nm to 330 nm, more preferably 260 to 330 nm, more preferably 260 nm to 310 nm, more preferably 270 to 290 nm, preferably a light-emitting diode (LED) type (1), and preferably an output of 50 to 500 mW, more preferably 50 to 150 mW, and In step (iii), a device for detecting the intensity of light emitted by the nanoparticles, particularly a single detector of the type of photomultiplier tube, photodiode, or avalanche photodiode that detects only visible light, or a photosensitive device array type detector consisting of a two-dimensional surface of detection pixels such as a CCD or EM-CCD camera or a CMOS camera. The method according to any one of claims 1 to 9, carried out using an apparatus including the following:

11. The method according to any one of claims 1 to 10, wherein the detection is time-resolved, and the time-resolved detection is obtained by electronic or mechanical chopping of an incident UV-B and / or UV-C beam.

12. The aforementioned nanoparticles are, in particular, at least, obtained by colloidal transformation of rare earth metal hydroxycarbonate particles. (a) Metavanadate salts, especially ammonium metavanadate (NH ４ VO ３ ), and optionally a step of preparing an aqueous solution (1) by mixing a phosphate in an aqueous medium, (b) under conditions that promote the formation of said hydroxycarbonate nanoparticles by coprecipitation, preparing hydroxycarbonate nanoparticles of formula A １－ｘ Lnx ３＋ CO ３ OH from precursors of elements A and Ln, particularly in the form of salts, especially nitrates, and a source of excess bicarbonate ions, especially urea, (b') A step of adding an aqueous solution (1) to hydroxycarbonate nanoparticles in a colloidal suspension under conditions that promote the formation of nanoparticles of formula I by coprecipitation, and (c) Steps to recover nanoparticles of formula I The method according to any one of claims 1 to 11, prepared by a process comprising the steps described above.

13. The method according to any one of claims 1 to 12, wherein in step (i), preferably the nanoparticles are mixed with a protective agent before linking the nanoparticles with the binder, post-synthesis annealing is performed at a temperature of 500°C to 1500°C, more specifically 800°C to 1300°C, and the protective agent is then removed by a method suitable for the protective agent, in particular by acid dissolution.

14. Use of the method according to any one of claims 1 to 13 for in vitro diagnostic purposes.

15. In particular, at least equation (I): A １－ｘ Ln ｘ VO ４（１－ｙ） (PO ４ ) ｙ (I) Photoluminescent particles formed whole or partially from photoluminescent inorganic nanoparticles comprising vanadate or a vanadate / phosphate matrix, wherein A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), and mixtures thereof, and 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 when Ln represents Eu. 0 < x < 1, especially 0.02 ≤ x ≤ 0.5, especially 0.05 ≤ x ≤ 0.4, more specifically x is 0.4, 0.2, 0.1, or 0.05, 0 ≤ y < 1, in particular when y is 0, The particles are surface-functionalized with chemical groups provided by molecules such as citric acid or polyacrylic acid, such as carboxyl, amino, thiol, aldehyde, or epoxy groups, and / or linked to molecules such as streptavidin, and the chemical groups or molecules are suitable for enabling the linkage of the particles to an agent for targeting the target substance, or Photoluminescent particles, wherein the particles are already linked to at least one agent for targeting the target substance, and at least, A lighting device, preferably of the light-emitting diode (LED) type (1), having an output power of preferably 50 to 500 mW, and more preferably 50 to 150 mW, with a wavelength of 240 nm to 330 nm, more preferably 260 to 330 nm, more preferably 260 nm to 310 nm, and even more preferably 270 to 290 nm, and A device for detecting the intensity of light emitted by the aforementioned particles. Detection and / or quantitative systems including, Optionally, a suitable support for immobilizing the target substance of the sample. An in vitro diagnostic kit for carrying out the method according to any one of claims 1 to 13, including the method described in any one of claims 1 to 13.

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