ULTRA-SENSITIVE DETECTION METHOD USING PHOTOLUMINESCENT PARTICLES

Abstract

A process for ultrasensitive in vitro detection and/or quantification of a substance of interest in a sample is performed by detecting the luminescence emission by photoluminescent inorganic nanoparticles. The process includes (i) use of photoluminescent particles comprising a photoluminescent inorganic nanoparticle consisting of a crystalline matrix having at least 10.sup.3 rare-earth ions, and coupled to a targeting agent for the substance to be analyzed, under conditions conducive to their association with the sample substance to be analyzed; (ii) exciting the rare-earth ions of the particles by an illumination device having a power of at least 50 mW and an excitation intensity of at least 1 W/cm.sup.2; (iii) detecting the luminescence emission by the particles after single-photon absorption; and (iv) determining the presence and/or concentration of the substance by interpreting said luminescence measurement. This process can be used for in vitro diagnostic purposes and as an in vitro diagnostic kit.

Claims

1. A process for the ultrasensitive in vitro detection and/or quantification of a substance of biological or chemical interest in a sample, by detecting the luminescence emission by photoluminescent inorganic nanoparticles, comprising at least the following steps: (i) use of photoluminescent particles consisting, in whole or in part, of a photoluminescent inorganic nanoparticle consisting of a crystalline matrix having at least 10.sup.3 rare-earth ions, and coupled to at least one targeting agent for the substance to be analyzed, under conditions conducive to their association with the substance to be analyzed of the sample, said nanoparticles having an average size greater than or equal to 20 nm and strictly less than 1 m, and being capable of emitting luminescence after absorption of a photon; (ii) excitation of the rare-earth ions of the particles associated with the substance to be analyzed, by an illumination device, with a power of at least 50 mW, and an excitation intensity of at least 1 W/cm.sup.2; (iii) detection of luminescence emitted by particles after single-photon absorption, and (iv) determination of the presence and/or concentration of the substance by interpretation of said luminescence measurement, where appropriate by reference to a standard or calibration.

2. The process as claimed in claim 1, wherein the substance to be analyzed of said sample in step (i) is previously immobilized on the surface of a support, said surface being passivated so that said luminescent particles do not attach thereto in the absence of the substance to be analyzed.

3. The process as claimed in claim 1, for the detection and/or quantification of a substance of interest present in the sample in a content strictly below 10 pM.

4. The process as claimed in claim 1, wherein the sample is a biological sample.

5. The process as claimed in claim 1, for the detection and/or quantification of biomarkers, antibodies, DNA and/or RNA in a biological sample.

6. The process as claimed in claim 1, wherein said targeting agent is selected from a polyclonal or monoclonal antibody, an antibody fragment, a nanobody, an oligonucleide, a peptide, a hormone, a ligand, a cytokine, a peptidomimetic, a protein, a carbohydrate, a chemically modified protein, a chemically modified nucleic acid, a chemically modified carbohydrate which targets a known cell surface protein, an aptamer, a protein and DNA/RNA assembly or a chloroalkane used by HaloTag-type markers.

7. The process as claimed in claim 1, wherein the product between the doping rate of rare-earth ions and the quantum efficiency of the emission from the nanoparticle is maximized.

8. The process as claimed in claim 1, wherein the nanoparticles have an emission lifetime greater than or equal to 5 s.

9. The process as claimed in claim 1, wherein the nanoparticles have an average size of between 20 nm and 500 nm.

10. The process as claimed in claim 1, wherein the rare-earth ions are lanthanide ions selected from europium (Eu), dysprosium (Dy), samarium (Sm), praseodymium (Pr), neodymium (Nd), erbium (Er), ytterbium (Yb), cerium (Ce), holmium (Ho), terbium (Tb), thulium (Tm) and mixtures thereof.

11. The process as claimed in claim 1, wherein the crystalline matrix forming said photoluminescent inorganic nanoparticle is an oxide matrix; a halide matrix; or a chalcogenide matrix.

12. The process as claimed in claim 1, wherein the nanoparticles are of the following formula (I):
(A.sub.1-x Ln.sub.x).sub.a(M.sub.pO.sub.q)(I) wherein: M represents one or more elements capable of associating with oxygen (O) to form a crystalline compound; Ln corresponds to one or more luminescent lanthanide ion(s); A corresponds to one or more constituent ion(s) of the crystalline matrix whose electronic levels are not involved in the luminescence process; 0<x<1; and the values of p, q and a are such that the electroneutrality of (A.sub.1-xLn.sub.x).sub.a(M.sub.pO.sub.q) is respected.

13. The process as claimed in claim 1, the nanoparticles being of formula:
A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y(II) wherein: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu) and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), terbium (Tb) and mixtures thereof; 0<x<1; and 0y<1.

14. The process as claimed in claim 13, wherein said nanoparticles of formula (II) have on their surface tetraalkylammonium cations.

15. The process as claimed in claim 14, said nanoparticles being of formula (II)
A.sub.1-x Ln.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y.(NR.sub.4.sup.+).sub.z(II) wherein: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu) and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), terbium (Tb) and mixtures thereof; 0<x<1; 0y<1; R, which may be identical or different, represent a C.sub.1-C.sub.6-alkyl; and z represents the number of tetraalkylammonium cations NR.sub.4.sup.+ located on the surface of said nanoparticle.

16. The process as claimed in claim 1, wherein said nanoparticles are of formula Y.sub.1-xEu.sub.xVO.sub.4, wherein 0<x<1.

17. The process as claimed in claim 1, wherein the nanoparticles have, at the end of their synthesis, a zeta potential, denoted , less than or equal to 28 mV, in aqueous medium of pH5, and of ionic conductivity strictly less than 100 S.Math.cm.sup.1.

18. The process as claimed in claim 2, wherein step (i) comprises at least the following steps: (a) have a support whose surface is previously passivated and functionalized with a targeting agent for the substance to be detected/quantified; (b) contacting said sample to be analyzed with the support of step (a) under conditions conducive to the association of said substance with the targeting agent; and (c) contacting the photoluminescent particles coupled with at least one targeting agent with said support from step (b) to associate the particles with said substance immobilized on the surface of the support.

19. The process as claimed in claim 2, wherein said support is of the slide, multiwell plate, microplate, membrane gel, strip or microchannel type.

20. The process as claimed in claim 2, wherein the excitation in step (ii) is carried out with a laser excitation beam oriented so as to form an angle of incidence greater than or equal to 55 with the vertical of the support having at the surface said particles associated with the substance to be analyzed.

21. The process as claimed in claim 2, comprising the excitation of the particles by evanescent wave.

22. The process as claimed in claim 1, wherein the lifetime of emission by the nanoparticles is greater than or equal to 1 s, the detection of the light intensity in step (iii) comprising a time-resolved detection of the emission.

23. The process as claimed in claim 1, for the simultaneous detection and/or quantification of at least two different substances in a sample.

24. The process as claimed in claim 23, wherein the substances to be analyzed of said sample are immobilized in predefined and distinct areas on the surface of a support, said surface being passivated so that photoluminescent particles do not bind to it in the absence of the substances to be analyzed.

25. The process as claimed in claim 23, using at least two types of nanoparticles having distinct emission wavelengths and coupled to targeting agents for each of the substances to be analyzed.

26. The process as claimed in claim 1, wherein the laser illumination device in step (ii) comprises an optical arrangement, arranged in the path of the laser beam so as to control the size of the beam at the area of the support having the particles associated with the substance to be analyzed.

27. The process as claimed in claim 1, wherein the detection of the luminescence emission in step (iii) is performed by a light intensity detection device comprising a single detector.

28. The process as claimed in claim 27, wherein the detection device comprises an optical arrangement, for focusing the luminescence emission to the detector.

29. (canceled)

30. An in vitro diagnostic kit comprising at least: photoluminescent particles consisting, in whole or in part, of a photoluminescent inorganic nanoparticle consisting of a crystalline matrix having at least 10.sup.3 rare-earth ions, and coupled to at least one targeting agent for the substance to be analyzed, under conditions conducive to their association with the substance to be analyzed of the sample, said nanoparticles having an average size greater than or equal to 20 nm and strictly less than 1 m, and being capable of emitting luminescence after absorption of a photon, said particles being surface functionalized with chemical groups, provided by molecules, and/or coupled to molecules, said chemical groups or molecules being capable of allowing coupling of said particles with a targeting agent for the substance to be analyzed; or said particles being already coupled to at least one targeting agent for the substance to be analyzed; and a detection and/or quantification system comprising at least: an illumination device, with a power of at least 50 mW or even at least 500 mW, and an optical arrangement for shaping the laser beam making it possible to obtain an excitation intensity at the level of the sample of at least 1 W/cm.sup.2, a device for detecting the light intensity emitted by the particles.

31. The diagnostic kit as claimed in claim 29, comprising at least one suitable support for immobilizing the substance to be analyzed of said sample.

32. An in vitro diagnostic method, said method implementing the ultrasensitive in vitro detection and/or quantification of a substance of biological or chemical interest in a sample, by detecting the luminescence emission by photoluminescent inorganic nanoparticles, comprising at least the following steps: (i) use of photoluminescent particles consisting, in whole or in part, of a photoluminescent inorganic nanoparticle consisting of a crystalline matrix having at least 10.sup.3 rare-earth ions, and coupled to at least one targeting agent for the substance to be analyzed, under conditions conducive to their association with the substance to be analyzed of the sample, said nanoparticles having an average size greater than or equal to 20 nm and strictly less than 1 m, and being capable of emitting luminescence after absorption of a photon; (ii) excitation of the rare-earth ions of the particles associated with the substance to be analyzed, by an illumination device, with a power of at least 50 mW, and an excitation intensity of at least 1 W/cm.sup.2; (iii) detection of luminescence emitted by particles after single-photon absorption, and (iv) determination of the presence and/or concentration of the substance by interpretation of said luminescence measurement, where appropriate by reference to a standard or calibration.

Description

FIGURES

[0356] FIG. 1: Schematic representation of the principle of detection of biomolecules: surface functionalized with a capture antibody (phase 1); contact of the sample to be analyzed (phase 2), washing (phase 3) and association of the photoluminescent particles coupled with a targeting agent (here an antibody) with the substance immobilized on the surface of the support followed by washing to remove the non-immobilized particles (phase 4);

[0357] FIG. 2: Schematic representation of a detection device according to the invention, composed notably of a laser source 1, a system for collimating and reducing the size of the laser beam 2, a mirror 3 for directing the laser beam, a support 6 for the samples, and a system for detecting the emitted photons comprising a lens with a large numerical aperture 10 and a photomultiplier 12;

[0358] FIG. 3: Transmission electron microscopy (TEM) images of the nanoparticles obtained in Example 1 (Scale bar: 60 nm (FIG. 3a) and 5 nm (FIG. 3b), respectively);

[0359] FIG. 4: Histogram of nanoparticle size determined from TEM images for a set of about 300 nanoparticles according to Example 1;

[0360] FIG. 5: X-ray diffractograms obtained for nanoparticles synthesized according to Example 1 (black line), and 3 (gray line);

[0361] FIG. 6: Schematic representation of the nanoparticles of the invention functionalized with citrate (FIG. 6a) or with polyacrylic acid (PAA) (FIG. 6b). For clarity, only one citrate or PAA molecule is shown. It is understood that each nanoparticle may have many citrate or PAA molecules on its surface.

[0362] FIG. 7: Schematic representation of the reactions for the coupling of a nanoparticle according to the invention with streptavidin, according to Example 1;

[0363] FIG. 8: Schematic representation of the coupling of streptavidin-coupled nanoparticles with a biotinylated antibody;

[0364] FIG. 9: Schematic representation of the use of a rotating mechanical chopper to obtain periodic illumination, and time profile of the excitation intensity obtained. In the case of the time profile shown, the resulting excitation intensity increases or decreases gradually due to the finite beam size and the time required for the mechanical chopper blade to expose or hide the entire excitation beam;

[0365] FIG. 10: Signals obtained in evanescent wave excitation mode (TIRF) after half a closing half-cycle of the chopper with pure water (a), nanoparticles (3 mM vanadate ion concentration drop) in water (b) and in serum (c): raw signal (FIG. 10-a) and luminescence signal in delayed detection (detection restricted to 150 s after the start of excitation beam blanking) (FIG. 10-b);

[0366] FIG. 11: Left: luminescence modelling with a two-level system. Right: averaged signal emitted by nanoparticles (Y.sub.0.6Eu.sub.0.4VO.sub.4) deposited on a slide, collected during 500 opening-closing cycles (circles) of the chopper, in comparison with its adjustment (solid line), determining the quantity of nanoparticles. Results obtained in HI-LO excitation mode (i.e. the excitation beam forming a wide angle with the vertical on the sample support);

[0367] FIG. 12: Schematic representation of a detection device according to the invention, with downward emission detection (FIG. 12-a) and upward emission detection (FIG. 12-b);

[0368] FIG. 13: Schematic representation of a detection device according to the invention, with laser beam chopper 4 and excitation in total internal reflection (using a Plexiglas 13 parallelepiped with a refractive index greater than or equal to that of the material serving as sample support);

[0369] FIG. 14: Calibration curve of the detection device according to variant 2, with chopper and with evanescent wave excitation, following the protocol described in Example 3.2.ii (FIG. 14-a); and according to variant 3, without chopper and without evanescent wave excitation, following the protocol described in Example 3.2.ii. (FIG. 14-b);

[0370] FIG. 15: Detection of recombinant insulin in solution by an ELISA kit (ABCAM item ab100578). The detection limit specified by the kit supplier is 9 pM (50 pg/mL);

[0371] FIG. 16: Detection of recombinant insulin in solution up to concentrations of 9 fM (0.05 pg/mL) with variant 1 of the detection device described in Example 3 (acquisition time: 30 s; signal readout every 100 ms). In the insert, the signal corresponding to a concentration equal to zero has been subtracted;

[0372] FIG. 17: Detection of recombinant insulin in solution up to concentrations of 9 fM (0.05 pg/mL) with variant 2 of the detection device described in Example 3, without chopper and without evanescent wave excitation (acquisition time: 1 s; signal readout every 10 s);

[0373] FIG. 18: Detection of recombinant insulin in solution up to concentrations of 9 fM (0.05 pg/mL) with variant 2 of the detection device described in Example 3, with chopper and with evanescent wave excitation (acquisition time: 1 s; signal readout every 10 s);

[0374] FIG. 19: Detection of insulin in different serum samples on a multi-well plate according to variant 1 of the detection device described in Example 3 (acquisition time: 30 s; signal readout every 100 ms);

[0375] FIG. 20: Detection of recombinant TSH in solution up to concentrations of 3.2 fM according to variant 1 of the detection device described in Example 3 with chopper (acquisition time: 1 s; signal readout every 10 s);

[0376] FIG. 21: Change in the detected luminescence signal according to variant 1 of the detection device as a function of the incubation time with antibody-nanoparticle conjugates according to Example 4;

[0377] FIG. 22: Schematic diagram of the principle of spatial multiplexing;

[0378] FIG. 23: Schematic representation of the two nanoparticle spots formed according to Example 5, and the luminescence signal detected for the different locations;

[0379] FIG. 24: Schematic representation of the detection and quantification of DNA/RNA using particles according to the invention coupled with single-stranded DNA. Single-stranded DNA partially complementary to the strand to be detected is fixed on a support. Then the sample containing the DNA or RNA to be detected is incubated with the functionalized support (left). After rinsing, the particles coupled with single-stranded DNA, partially complementary to the unpaired part of the DNA or RNA to be detected, are incubated with the support (middle). After rinsing, only the nanoparticles immobilized on the surface of the support after pairing with the DNA or RNA to be detected are present (right). They can be detected and quantified as described in the text.

[0380] FIG. 25: Schematic representation of the detection and quantification of DNA/RNA using particles according to the invention coupled with streptavidin molecules.

[0381] (a) This DNA or RNA detection/quantification variant uses a single-stranded recognition DNA coupled to a streptavidin molecule at each end to be attached to the detection support, the single-stranded DNA or RNA to be detected and a single-stranded DNA partially complementary to the DNA coupled to a biotin molecule. The streptavidin-coupled DNA is, with its non-complementary part to the biotin-coupled DNA, at least partially complementary to the DNA to be detected.

[0382] (b) The surface of the support is passivated with PEG and functionalized with biotin. DNA coupled with streptavidin at each end attaches to the surface of the functionalized support. Then, following incubation with the DNA to be detected, it matches the DNA immobilized on the surface of the support. After rinsing, the biotins remaining on the surface are passivated with streptavidin. The detection DNA attached to a biotin is incubated with the surface and pairs with the complementary part of the DNA coupled to two molecules of streptavidin.

[0383] (c) After rinsing, the particles of the invention coupled to streptavidin molecules are incubated with the surface and bind to the single stranded detection DNA via interaction with biotin.

[0384] After rinsing, particles immobilized on the surface can be detected and quantified as described in the text. It is also possible to use recognition DNA coupled to a single streptavidin molecule at one end. In this case, the recognition DNA is attached to the surface of the support by only one of its two ends, as in variant 1.

[0385] FIG. 26: Excitation and emission spectra of the nanoparticle solution YVO.sub.4:Dy 3% prepared in Example 6: excitation spectrum in the region of direct absorption of Dy.sup.3+ ions for detection at =572 nm (FIG. 26-a) and emission spectrum for excitation at =278 nm corresponding to the excitation of the vanadate matrix (FIG. 26-b).

[0386] FIG. 27: Excitation and emission spectra of the nanoparticle solution YVO.sub.4:Sm 3% prepared in Example 7: excitation spectrum in the region of direct absorption of Sm.sup.3+ ions for detection at =600 nm (FIG. 27-a) and emission spectrum for excitation at =278 nm corresponding to the excitation of the vanadate matrix (FIG. 27-b).

EXAMPLES

Example 1

[0387] Preparation of Luminescent Particles

[0388] 1.1. Synthesis of Y.sub.0.6Eu.sub.0.4VO.sub.4 Nanoparticles

[0389] Ammonium metavanadate NH.sub.4VO.sub.3 is used as a source of metavanadate VO.sup.3 ions, orthovanadate VO.sub.4.sup.3 being obtained in situ by reaction with a base, in this case tetramethylammonium hydroxide, N(CH.sub.3).sub.4OH. Yttrium and europium nitrates were used as sources of Y.sup.3+ and Eu.sup.3+ ions.

[0390] A 10 mL aqueous solution of 0.1 M NH.sub.4VO.sub.3 and 0.2 M N(CH.sub.3).sub.4OH (solution 1) is freshly prepared.

[0391] A volume of 10 mL of another solution of Y(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 with 0.1 M ions (Y.sup.3++Eu.sup.3+) is added dropwise by means of a syringe plunger to solution 1 at a flow rate of 1 mL/min.

[0392] The molar concentration ratio between Y(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 is selected as a function of the desired ratio between Y.sup.3+ and Eu.sup.3+ ions in the nanoparticle, typically the molar ratio Y.sup.3+:Eu3.sup.+ is 0.6:0.4.

[0393] As soon as Y(NO.sub.3).sub.2/Eu(NO.sub.3).sub.3 solution is added, the solution becomes diffusive and appears milky/white without precipitate formation. Synthesis continues until Y(NO.sub.3).sub.2/Eu(NO.sub.3).sub.3 solution is fully added.

[0394] The final 20 mL solution must now be purified to remove excess counterions. To do this, centrifugations (typically three) at 11 000 g (Sigma 3K10, Bioblock Scientific) for 80 minutes each followed by redispersion by sonication (Bioblock Scientific, Ultrasonic Processor operating at 50% at 400 W power for 40 s) are used until a conductivity strictly below 100 S.Math.cm.sup.1 is reached.

[0395] Conductivity is measured using a chemical conductivity meter.

[0396] The synthesis of the nanoparticles Y.sub.0.6Eu.sub.0.4VO.sub.4 on the surface of which the tetramethylammonium cations are immobilized can be schematically summarized as follows:

NH.sub.4VO.sub.3+2(N(CH.sub.3).sub.4)OHVO.sub.4.sup.3+2N(CH.sub.3).sub.4.sup.++NH.sub.4.sup.+VO.sub.4.sup.+2N(CH.sub.3).sub.4.sup.++NH.sub.4.sup.++0.6Y(NO.sub.3).sub.3+0.4Eu(NO.sub.3).sub.3.fwdarw.Y.sub.0.6Eu.sub.0.4VO.sub.4+2N(CH.sub.3).sub.4.sup.++NH.sub.4.sup.++3NO.sub.3.sup.

[0397] Two synthesis tests (synthesis 1 and synthesis 2) are carried out.

[0398] Result

[0399] Visual observation of the nanoparticle solution according to the invention, after being left to rest for 16 hours in a vial, shows a uniformly diffusing solution.

[0400] The final solution remains very stable in water, even after several months in the final pH of the synthesis (about pH 5). The solution remains stable even in the synthesis medium (before removal of excess counterions), which has a high ionic strength (>0.1 M).

[0401] The zeta potential of the nanoparticles is determined with a DLS-Zeta Potential device (Zetasizer Nano ZS90, Malvern). The results of the measured zeta potentials for nanoparticles from both syntheses are summarized in Table 1 below.

TABLE-US-00001 TABLE 1 Synthesis 1 Synthesis 2 Conductivity (S/cm) 93 80 pH 4.81 4.96 Zeta potential 33.3 34.6

[0402] For transmission electron microscopy (TEM) observations, dilute solutions of nanoparticles are deposited on a carbon grid. The observations are made using a Philips CM30 microscope operating at 300 kV with a resolution of 0.235 nm.

[0403] The observation of nanoparticles by TEM (FIG. 3) shows that the nanoparticles have an elongated ellipsoid shape. Nanoparticle dimensions are determined from TEM images for a set of about 300 nanoparticles (FIG. 4). The nanoparticles of the invention have a major axis length, denoted a, of between 20 and 60 nm, with an average value of about 40 nm, and a minor axis length, denoted b, of between 10 and 30 nm, with an average value of about 20 nm.

[0404] The MET images (FIG. 3) do not distinguish between crystal planes, which is probably due to the fact that the nanoparticle is made up of several crystallites smaller than the size of the nanoparticle. The predominantly crystalline and polycrystalline nature of nanoparticles is confirmed by X-ray diffraction experiments.

[0405] The X-ray diffractogram obtained using a Philips X-pert diffractometer with the copper K.sub.1 line (=1.5418 ) is shown in FIG. 5. The diffracted intensity is recorded using an X'Celerator area detector (PANalytical).

[0406] The coherence length in a crystallographic direction and thus the average size of the crystallites constituting the nanoparticle in this crystallographic direction can be estimated from the width of the peaks in the RX diffractogram by applying the Scherrer formula. The coherence length values obtained for the different crystallographic directions are between 3 and 40 nm. Since the coherence length in at least one crystallographic direction is smaller than the size of the nanoparticle in that direction, it can be deduced that nanoparticles are imperfectly crystalline (polycrystallinity, defects or porosity). In the (200) direction (FIG. 3, peak at 225), the coherence length is 10.2 nm, slightly shorter than the coherence length for the nanoparticles in Example 3 (11.1 nm).

[0407] 1.2. Coupling of the Nanoparticles with Streptavidin Protein

[0408] i.a. Grafting of Citrate onto the Surface of Nanoparticles

[0409] After the synthesis of the nanoparticles according to Example 1.1. 250 L of Y.sub.0.6Eu.sub.0.4VO.sub.4 particles with a concentration of 5 mM vanadate ions are removed, the nanoparticle solution is centrifuged at 17 000 g for 30 minutes.

[0410] The pellet is removed and dispersed in 1 mL of a distilled water solution containing citrate ion (0.2 M concentration).

[0411] The solution is then sonicated for 5 minutes in an ice bath, centrifuged at 17 000 g for 30 minutes and the pellet is recovered and redispersed in distilled water containing citrate ion (0.2 M concentration). This step is repeated three times.

[0412] After this grafting, the particles are dispersed in distilled water, a solvent in which they are stable.

[0413] The functionalization of the nanoparticles by citrate can be replaced by functionalization by PAA (for example with a degree of polymerization between 3 and 10 000), using a PAA salt, such as a sodium or ammonium salt.

[0414] i.b. Grafting of PAAs onto the Surface of Nanoparticles

[0415] After the synthesis of the nanoparticles according to Example 1.1, 500 L of Y.sub.0.6Eu.sub.0.4VO.sub.4 particles with a concentration of 10 mM are removed and the nanoparticle solution is centrifuged at 17 000 g for 30 minutes.

[0416] The pellet is removed and dispersed in 1 mL of a distilled water solution containing 1800 Da molecular weight PAA (75 mM concentration).

[0417] The solution is then sonicated for 5 minutes, centrifuged at 17 000 g for 30 minutes and the pellet is recovered and redispersed in distilled water containing 1800 Da molecular weight PAA (75 mM concentration). This step is repeated three times.

[0418] After grafting, the particles are dispersed in distilled water, a solvent in which they are stable.

[0419] FIG. 6 schematically shows the nanoparticles of the invention functionalized with (a) citrate and (b) polyacrylic acid.

[0420] ii. Coupling Nanoparticles with Streptavidin

[0421] Nanoparticles (NPs) grafted with citrate ions or PAA are centrifuged at 16 000 g for 1 hour and the pellet is then recovered.

[0422] The coupling of the nanoparticles grafted on the surface by citrate with streptavidin is carried out according to the following protocol:

[0423] 1. Freshly prepare a mixed solution of EDC.sup.1/Sulfo-NHS.sup.2 (concentration 30 and 30 mg/mL, respectively) in MES buffer.sup.3 (pH 5-6).

[0424] 2. Disperse by sonication (ultrasonic bath) the pellet of NPs in 250 L of the solution prepared in step 1. Since centrifugation losses are low, the concentration of vanadate ions remains around 5 mM, giving a nanoparticle concentration of 48 nM. [0425] (The vanadate concentration of the nanoparticle solutions was determined by dissolving the particles in an acidic medium followed by a colorimetric determination of the vanadate ion concentration as described in reference [34] Abdesselem et al., ACS Nano 8, 11126-11137 (2014). The molar concentration of nanoparticles was determined from the concentration of vanadate ions, as described in reference [35].

[0426] 3. Prepare a 100 nM solution of streptavidin (SA) in pH 7.4 phosphate buffer with 10 mM NaCl. Dilute the streptavidin solution to a concentration determined by the number of grafted proteins per desired nanoparticle (for a streptavidin:NP ratio of 1:1, 5:1 and 10:1, choose concentrations of 4.8 nM, 24 nM and 48 nM, respectively). Preferably choose a ratio of at least 20:1, i.e. a SA concentration of 96 nM. Add 250 L of this solution to the nanoparticle solution.

[0427] 4. Allow to incubate for 2-4 hours at room temperature with stirring.

[0428] 5. Add 1 mL of PBST.sup.4 and vortex.

[0429] 6. Centrifuge at 17 000 g for 30 min and recover the pellet to remove non-NP-coupled proteins. Completely remove the supernatant. Redisperse the protein-coupled NPs in 1 mL of PBST and sonicate in an ultrasonic bath. Repeat this step twice.

[0430] 7. Recover protein-coupled NPs in 250 L of PBS.sup.5 with 1% BSA.sup.6.

[0431] 8. Store at 4 C. for immediate use or aliquot and store at 80 C.

[0432] Equipment used for functional testing: .sup.1 N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) (Sigma, cat # E1769)..sup.2 N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) (Sigma, cat #56485)..sup.3 2-(N-Morpholino)ethanesulfonic acid (MES) (10 mM, pH 5-6)..sup.4 Phosphate buffered saline pH 7.4 (10 mM NaCl)+0.05% Tween 20 (PBST)..sup.5 Bovine serum albumin (BSA) (Sigma, cat # A3059)..sup.6 Phosphate buffered saline (PBS) (pH 7.4, 10 mM NaCl).

[0433] The coupling of nanoparticles with streptavidin is shown schematically in FIG. 7.

[0434] The number of streptavidin (SA) molecules per nanoparticle (NP), denoted R, characterized at the end of the coupling protocol, is determined by the determination of streptavidin by the so-called BCA method, according to the protocol detailed above.

[0435] Protocol for the Characterization of the Streptavidin:Nanoparticle (SA:NP) Ratio

[0436] i. Determination of SA by the BCA Method

[0437] Principle

[0438] In an alkaline medium, proteins reduce Cu.sup.2+ to Cu. The salt of bicinchoninic acid (BCA) forms a colored complex with Cu.sup.2+ ions. This complex is quantifiable due to its absorption at 562 nm.

[0439] Procedure

[0440] BCA test kit from ThermoFisher (Pierce BCA Protein Assay Kit Cat. No. 23225) [0441] Preparation of the Cu.sup.2+/BCA test reagent according to the ThermoFisher kit protocol. [0442] Preparation of SA for the calibration curve. For the calibration curve, the test is performed three times.

TABLE-US-00002 TABLE 2 Streptavidin concentration of calibration solutions Tubes Final SA concentration (g/mL) A 2000 B 1500 C 1000 D 750 E 500 F 250 G 125 H 25 I 0 = blank

[0443] The procedure is as follows: [0444] Take a 96-well plate. Place 25 L of each tube of standard A-I (of known concentration of SA) or nanoparticle conjugated proteins to be assayed into the corresponding wells. [0445] Add 200 L of the Cu.sup.2+/BCA test reagent to each well. Homogenize, cover and incubate 30 min at 37 C. [0446] Read absorbances (A) at 562 nm as a function of the final concentration taking into account the dilution. Establish the calibration relationship A=f (SA concentration in g/mL) by linear regression.

[0447] The concentration of the proteins conjugated to the nanoparticles to be assayed is deduced from the linear regression equation obtained from the calibration curve.

[0448] ii. Characterization of the SA:NP Ratio

[0449] The mass concentration of streptavidin obtained with the BCA test is converted to a molar concentration using the following formula:

[00001]${\left[\mathrm{SA}\right]}_{\mathrm{molar}}.Math.\left(\mathrm{moles}.Math.\text{/}.Math.L\right)=\frac{\left[\mathrm{SA}\right].Math.\mathrm{mass}.Math..Math.\left(g.Math.\text{/}.Math.L\right)}{\mathrm{Molar}.Math..Math.\mathrm{mass}.Math..Math.\left(g.Math.\text{/}.Math.\mathrm{mole}\right)}$

[0450] To obtain the ratio (R) of the number of SA:NPs, we finally apply the following equation:

[00002]$R=\frac{\left[\mathrm{SA}\right].Math.\mathrm{molar}}{\left[\mathrm{NPs}\right].Math.\mathrm{molar}}$

[0451] Table 3 below summarizes the values obtained for the different concentration ratios between the streptavidin solution and the starting nanoparticle solution.

TABLE-US-00003 TABLE 3 Characterization of nanoparticle-streptavidin coupling for different concentration ratios between streptavidin solution and nanoparticle solution. Concentration ratios between streptavidin solution and nanoparticle solution 1:1 5:1 10:1 Determined number of streptavidin molecules 0.97 3.8 9.29 per nanoparticle after coupling

[0452] As can be seen from the results presented in Table 3, the number of streptavidin molecules per nanoparticle after coupling is of the same order as the concentration ratio in the initial solutions, thus indicating very good coupling efficiency. 1.3. Coupling Streptavidin-Coupled Nanoparticles (Nanoparticles-SA) with Biotinylated Antibodies

[0453] The streptavidin-coupled nanoparticles are then coupled to a biotinylated antibody, specific for the substance to be assayed (insulin or thyroid stimulating hormone (TSH)), as shown schematically in FIG. 8.

[0454] The coupling protocol is as follows.

[0455] Between 10 and 50 g/mL of biotinylated antibodies is brought into contact with the excess nanoparticles-SA for 1 h using a rotating wheel to promote diffusion and interaction between biotin and streptavidin. The concentration of antibodies is selected so as to have 3 antibodies per NP. Thus, it is certain that all antibodies will bind to the nanoparticles-SA and there is no need for a step to remove unbound antibodies. The concentration of antibodies is therefore three times higher than the concentration of nanoparticles.

[0456] Alternatively, it is also possible to directly couple antibodies to nanoparticles grafted with citrate ions. In this case, the same protocol as above should be used by replacing the streptavidin solution with an antibody solution.

[0457] 1.4. Alternative Coupling Method by Direct Coupling of Nanoparticles with Antibodies

[0458] According to an alternative method, the coupling of the nanoparticles with the non-biotinylated antibodies can be carried out directly by coupling the antibodies to nanoparticles functionalized with APTES (transformation of the amino groups into carboxyl groups, activation of the carboxyl groups and direct reaction with the amino groups on the surface of the antibodies), according to the following detailed protocol.

[0459] 1.4.1. Coating Nanoparticles with a Silica Layer

[0460] After the synthesis of the nanoparticles in step 1.1, the nanoparticle solution is centrifuged at 17 000 g for 3 minutes to precipitate any nanoparticle aggregates and the supernatant is recovered. A size selection is made. To do this, several centrifugations at 1900 g for 3 min are carried out. Each centrifugation is followed by a redispersion of the nanoparticles in the sonicator, after which the size of the nanoparticles is determined using a DLS-Zeta Potential device (Zetasizer Nano ZS90, Malvern).

[0461] A volume of 25 mL of Y.sub.0.6Eu.sub.0.4VO.sub.4 particles with a concentration of 20 mM vanadate ions is prepared. A volume of 2.5 mL of another pure sodium silicate solution (Merck Millipore 1.05621.2500) is added dropwise with a pipette to coat the surface of the particles. We let this solution work under stirring for at least 5 hours.

[0462] The solution is then purified to remove excess silicate and sodium counterions. The solution is centrifuged at 11 000 g (Sigma 3K10, Bioblock Scientific) for 60 minutes and then redispersed by sonication (Bioblock Scientific, Ultrasonic Processor, operating at 50% at 400 W). This step is repeated until the conductivity of the solution is below 100 s/cm.

[0463] 1.4.2. Grafting of Amines onto the Surface of Nanoparticles

[0464] In a 500 mL triple-neck round-bottom flask, place 225 mL of absolute ethanol and add 265 L of 3-aminopropyltriethoxysilane (APTES) (Mw 221.37 g/mol Sigma Aldrich), which corresponds to a final concentration of 1.125 mM. This amount corresponds to 5 vanadate equivalents that are introduced. A refrigerant is then connected to the round-bottom flask. The assembly is placed on a round-bottom flask heater and placed under a hood. The mixture is refluxed at 90 C. A colloidal solution of nanoparticles (vanadate ion concentration [V]=3 mM) in 75 mL water at pH 9 is added dropwise to one of the inlets of the triple-neck flask by means of a peristaltic pump at a flow rate of 1 mL/min. The assembly is heated under stirring for 24 h.

[0465] After 48 hours, we use a rotary evaporator (rotavapor R-100, BUCHI) to partially concentrate the nanoparticles. The solution is rotated in a suitable round-bottom flask and heated in a bath at 50 C.

[0466] The recovered solution is purified by several centrifugations in an ethanol:water (3:1) solvent. After purification, a size sorting is carried out following the protocol described above.

[0467] 1.4.3 Grafting of Carboxyl to the Surface of Aminated Nanoparticles

[0468] Before starting the grafting, a solvent transfer is performed.

[0469] The grafting protocol is as follows.

[0470] Transfer the aminated NPs from EtOH:H.sub.2O buffer to DMF or DMSO by performing several centrifugations (13 000 g, 90 min). The pellet is redispersed by sonication between each centrifugation (20 s at 75%). Measure and determine the concentration of NPs.

[0471] Recover the NPs in 5 mL of DMF and then add 10% of the succinic acid anhydride to a glass beaker (i.e. 0.5 g in the 5 mL). Allow to react at least overnight in an inert atmosphere while stirring.

[0472] Wash the carboxylated NPs at least twice by centrifugation (13 000 g for 60 min, Legend Micro 17R, Thermo Scientific) to remove DMF and excess succinic acid anhydride.

[0473] Resuspend the carboxylated particles in water or pH 6 MES buffer by sonication (Bioblock Scientific, Ultrasonic processor).

[0474] 1.4.4 Direct Coupling of Nanoparticles with Antibodies

[0475] The coupling of the surface-grafted nanoparticles with COOH is carried out according to the protocol below: [0476] i) Freshly prepare a mixed solution of EDC/Sulfo-NHS (concentration 500 and 500 mg/mL, respectively) in MES buffer (pH 5-6). [0477] ii) In 3 mL of the previously prepared solution, add 90 nM NPs (in this case the nanoparticle concentration calculated from the vanadate ion concentration according to reference Casanova et al. [37]) and allow to react for 25 min at room temperature with stirring. [0478] iii) Quickly wash the NPs by at least 2 centrifugations (13 000 g for 60 min, Legend Micro 17R, Thermo Scientific) with Milli-Q water to remove excess reagents. [0479] iv) Recover the last pellet after sonication in sodium phosphate buffer at pH 7.3. Add the amount of antibody required according to the desired ratio (Protein:NPs), typically 2 M for a ratio of 20:1. [0480] v) Allow this solution to react for 2 to 4 hours at room temperature while stirring. [0481] vi) Add the blocking agent (1% glycine) to react with the free COOHs and block the residual reaction sites on the surface of the NPs. Allow to react for 30 min. [0482] vii) Wash the protein-coupled NPs several times by centrifugation using centrifugal filters (Amicon Ultra 0.5 mL, item UFC501096, Millipore) with pH 7.2 PBS. Transfer the NPs to their storage medium: phosphate buffer+Tween 20 (0.05%)+0.1% glycine+10% glycerol. Collect 100 L for BCA testing. The rest of the solution is aliquoted and frozen at 80 C.

[0483] The luminescent particles thus prepared according to either of the aforementioned methods, comprising photoluminescent nanoparticles Y.sub.0.6Eu.sub.0.4VO.sub.4 coupled to a targeting antibody for the substance to be analyzed (NPs-Ab), can be used to detect and/or quantify said substance in a biological sample.

Example 2

[0484] Preparation of the Support on the Surface of which is Immobilized the Substance to be Analyzed

[0485] i. Preparation of the Glass Slide Type Support

[0486] In a first step, a support is prepared consisting of glass slides whose surface is previously cleaned, passivated and functionalized with biotinylated antibodies (capture antibodies).

[0487] The protocol for cleaning, passivation and functionalization of the glass slides is as follows. The functionalization can be done either by printing or by depositing drops using a spotter.

[0488] Cleaning the Slides [0489] Wash the slides in deionized water with 4% Hellmanex II detergent (Hellma) in an ultrasonic tank heated to 40-50 C. for 15 minutes. [0490] Rinse the slides with deionized water in an ultrasonic tank heated to 40-50 C. for 15 minutes. [0491] Rinse with absolute ethanol in an ultrasonic tank heated to 40-50 C. for 15 minutes. [0492] Dry in a stream of N.sub.2 or under an extractor hood for 10-15 min. [0493] Just before the glass surface is functionalized, clean the slides one by one in a Plasma cleaner (Harrick Plasma, model PDC-002) for 2 minutes at medium intensity.

[0494] Passivation of the Slides

[0495] The passivation molecule used is a silane-polyethylene glycol (PEG) with 10 kDa PEG, marketed by Laysanbio under the item number M-SIL-5K.

[0496] Passivation only involves one stage of silanization of the glass. The longer the PEG, the better the resulting passivation will be.

[0497] The passivation protocol is as follows: [0498] Dissolve PEG at 10 mg/mL in absolute ethanol (EtOHAbs, molar concentration 2 mM or 1 mM depending on the size of the PEG respectively 5 kDa or 10 kDa). To dissolve the longer PEGs at 10 mg/mL, it may be necessary to heat them for a few minutes at 40 C. [0499] A few seconds before use, add in this solution a little H.sub.2O and acetic acid (AcAc) to have the proportions v/v/v: EtOHAbs/H.sub.2O/AcAc: 95/5/0.2. [0500] At the bottom of an airtight dish place a square of parafilm, then place a 30 L drop of the solution prepared above on top, then the glass slide (from the plasma cleaner) on top to take the drop as a sandwich. Put a small reservoir of ethanol in the hermetically sealed dish to saturate the air with ethanol and so that the 30 L does not dry out. Allow to incubate overnight. [0501] Rinse with deionized water, then dry in a stream of nitrogen. Finally, place the strips on a hot plate at 110 C. for 5 min.

[0502] Functionalization of the Slides by Overnight Incubation

[0503] The protocol for functionalizing the slides previously passivated with biotinylated antibodies is as follows. This is the protocol used for the results shown in the figures.

[0504] Incubation of the slide in a solution containing the capture antibodies overnight at 4 C. The antibody solution contains 10-25 g/mL antibodies in a solution of PBS at pH 7.4 or carbonate buffer at pH 9 according to the supplier's instructions.

[0505] Alternatively, the following two protocols can be used:

[0506] Functionalization by Streptavidin Printing and Biotinylated Antibodies [0507] Cut a patch of PDMS (polydimethylsiloxane) with dimensions corresponding to the surface that is to be functionalized. [0508] Place the patch on a flat surface. Incubate on the patch 15-30 L of streptavidin at 20 g/mL in phosphate-buffered saline (PBS) for 1 min. [0509] Rinse immediately with H.sub.2O (using a wash bottle) and pass under a stream of N.sub.2 (10 sec). [0510] Turn the patch over and place its inked surface on the slide for 1 min. [0511] Remove the patch, being careful not to drag it across the surface. [0512] After functionalization of the glass slide with streptavidin, incubate the necessary amount (1-10 g/mL in bicarbonate buffer pH 7.4) of biotinylated antibodies for 2 h at room temperature or overnight at 4 C. [0513] Remove the antibody incubation solution and wash with 100 L of PBS buffer 3 times. [0514] Add 100 L of BSA 5% w/v (5 g in 100 mL) PBS and incubate for 1 h.

[0515] Functionality by Spotter

[0516] Spotter functionalization is performed with the SPRi-Arrayer device from Horiba according to the supplier's protocol using a 500 m diameter needle to deposit drops containing the antibodies (1-10 g/mL in bicarbonate buffer pH 7.4).

[0517] ii. Immobilization of the Substance to be Analyzed of the Samples on the Surface of the Support

[0518] The protocol is as follows. [0519] Add the samples to be assayed (insulin solution, insulin in serum or TSH solution) to each well and incubate for 2 hours. This time is the same as that indicated for the corresponding conventional ELISAs. [0520] After 2 hours incubation, remove the solution and wash with 100 L wash buffer (0.2% PBS 2Tween 20) 3 times.

Example 3

[0521] Ultrasensitive Detection and Quantification

[0522] 3.1. Association of Luminescent Particles with the Substance to be Analyzed

[0523] The protocol is as follows: [0524] Add the nanoparticles coupled to the biotinylated antibodies prepared as described in Example 1 and incubate for 1 hour. The incubation time required to maximize the signal during luminescence measurement can be determined beforehand, as described in Example 4. [0525] After 1 hour incubation, remove the solution and wash with 100 L wash Buffer (0.2% PBS 2Tween 20) 4 times. [0526] Add 100 L of PBS and perform the luminescence measurement as described below.

[0527] 3. 2 Luminescence Measurement

[0528] i. Experimental Arrangement for Measuring Luminescence

[0529] The detection arrangement is as follows. It is shown schematically in FIG. 2 and FIGS. 12 and 13.

[0530] The detection device consists of a 1 W laser source (1) with a wavelength of 465 nm (ML-6500-465, Modulight or F465-HS-1W, Laser2000), a collimation and laser beam size reduction system (2) consisting of two lenses (two bi-convex lenses of f=100 mm and f=30 mm (Thorlabs)), optionally a mechanical chopper (4) (MC2000B-EC, Thorlabs), a slide support (6) for the samples, and a detection system for the emitted photons.

[0531] To collect the luminescence emission by the nanoparticles in the sample, a high numerical aperture lens (10) (bi-convex lens, =50.8 mm, f=100 mm (Thorlabs)) and two interference filters 62014-25 (FF01-620/14-25, Semrock) to spectrally eliminate interfering signals and a photomultiplier (12) (PMM02, Thorlabs) are used.

[0532] An analog-to-digital converter (NI9215, National Instrument) allows the signal to be recorded using LabVIEW software.

[0533] All detection elements are located on the same axis. Thus, the arrangement is both more ergonomic and easier to adjust. A translation system of the slide support (Z8253, KCH301 Thorlabs) was implemented in order to be able to observe several biological samples successively by scanning.

[0534] Time-Resolved Detection for Measuring the Number of Nanoparticles in the Presence of Interfering Signals

[0535] A mechanical chopper can be placed in the path of the laser beam to create laser excitation slots to eliminate interfering signals. Indeed, during the illumination of the biological sample by the laser beam, it is possible that molecules other than the nanoparticles of interest emit fluorescence. The use of a mechanical chopper and a signal detection frequency of 100 kHz by the photomultiplier (signal acquisition every 10 s) makes it possible to avoid this interfering fluorescence.

[0536] It is thus possible due to the long emission time by the particles of the invention, to make a time-resolved detection of the emission, in particular a delayed detection of the emission, as described in detail below.

[0537] A time-modulated illumination allows to limit the contribution to the luminescence signal of interfering species present in the sample (serum, blood, etc.) or in the solid substrates used (glass, plastic, etc.). Indeed, the nanoparticles used (YVO.sub.4:Eu or GdVO.sub.4:Eu for example) can be placed (by illumination at 465 nm) in an excited state with a long lifetime, of the order of a few hundred s, in comparison with the lifetime of usual fluorophores, which are in the nanosecond range. This allows the temporal separation of the interfering luminescence signals from the signal emitted by the nanoparticles.

[0538] The modulation can be implemented by the use of a rotating mechanical chopper, in order to obtain periodic illumination at frequencies between 100 and 1000 Hz. FIG. 9 schematically shows the illumination time profile obtained by mechanical chopping of the excitation laser.

[0539] The modulated signal obtained is the alternation of a decay phase (chopper closing) and a luminescence return phase (chopper opening) of all the emitters present in the sample. The decay/return of the luminescence signal is determined by two separate parameters: (i) the lifetimes of the excited states of the emitters, and (ii) the dynamics of occultation/uncovering of the exciter beam by the mechanical chopper. FIG. 10-a shows examples of luminescence decline after initiation of chopper closure (t=0). Digital post-processing analysis of the signal allows the signal due to nanoparticles to be isolated, for example by the following two methods.

[0540] Method 1: Delayed Detection.

[0541] In the phase of luminescence decay, the contribution of the interfering emitters is concentrated at short times, when the excitation beam is not yet fully occluded (FIG. 10-a). By removing this part (FIG. 10-b, detection restricted to 170 s after the beginning of the occultation of the excitation beam by the chopper blade), a signal due exclusively to nanoparticles is obtained (superposition of curves (b) and (c), the contribution of the interfering fluorescence of the slide, serum and water is eliminated), which thus allows an estimation of their number despite the presence of other emitters.

[0542] Method 2: Time-Resolved Detection

[0543] The luminescence of nanoparticles and interfering fluorophores can be modelled by a two-level system (FIG. 11). In this case, the shape of the luminescence signal PL(t) can be determined from the characteristics of the transmitters:

PL(t)=kN*=G[I(t),k,N,]

where I(t) is the excitation time profile; N is the total number of emitters of a type and N* is the number of emitters on the excited state; k its de-excitation rate; its quantum efficiency; by numerically solving the system of differential equations resulting from the model.

[0544] The total signal can then be written,

PL(t)=G[I(t),k.sub.NP,N.sub.NP,.sub.NP]+F[I(t),k.sub.F,N.sub.F,.sub.F]+BG

where NP and F denote nanoparticles and interfering fluorophores, respectively, and BG denotes the background signal.

[0545] Adjustment of the signal recorded by this function then makes it possible to determine the various parameters, notably N.sub.NP and k.sub.NP (FIG. 11). It is robust, as k.sub.NP<<k.sub.F. The determination of the number of nanoparticles adhering to the N.sub.NP surface is then carried out unambiguously, despite the presence of interfering emitters in the sample contributing to the raw signal. In practice, the mechanical masking kinetics of illumination at the frequencies used (100-1000 Hz) is significantly slower than the fluorescence decay of interfering emitters k.sub.F10.sup.9 s.sup.1, so that F [I(t), k.sub.F, N.sub.F, .sub.F]K.Math.I(t).

[0546] The measurement of N.sub.NP is then carried out by the following method: (i) experimental determination of the illumination time profile I(t)/I(0) by measuring the autofluorescence of a calibration sample without nanoparticles; (ii) measurement of the luminescence signal of the target sample; and (iii) non-linear adjustment (least squares method) of this signal by a function

[00003]$\mathrm{PL}\left(t\right)=M.\frac{I\left(t\right)}{I\left(0\right)}+G\left[I\left(t\right),k_{\mathrm{NP}},N_{\mathrm{NP}},{}_{\mathrm{NP}}\right]+\mathrm{BG}.$

This adjustment allows the identification of the parameters M, k.sub.NP and .sub.NPN.sub.NP to within an instrumental multiplicative factor: this last parameter then indicates, for fixed detection conditions, the number of nanoparticles deposited on the surface.

[0547] Variants of the Experimental Arrangement

[0548] The nanoparticles are excited: [0549] either by using a high angle of incidence (angle between the direction of propagation of the laser beam and the vertical to the sample support) between 60 and 63 (as shown schematically in FIG. 12), [0550] or by using a total internal reflection fluorescence (TIRF) type illumination arrangement, in particular using a Plexiglas 13 parallelepiped with a refractive index greater than or equal to that of the glass slide serving as sample support (as shown schematically in FIG. 13). This configuration provides an angle of incidence at the glass/water or glass/serum interface greater than 61.04, resulting in total reflection of the beam at this interface and evanescent wave excitation of the nanoparticles anchored to the substance to be analyzed.

[0551] Three variants of the experimental arrangement were used.

[0552] Variant 1: Reflected (downward) detection without chopper (except where specified) and without evanescent wave excitation (FIG. 12-a). In the majority of cases, the acquisition time is fixed at 30 s with a voltage value recorded every 100 ms. The diameter of the collection lens is 30 mm instead of 50.8 mm.

[0553] Variant 2: Transmission detection (upwards) (FIG. 12-b). Measurements can be made with or without a chopper and with or without evanescent wave excitation. The acquisition time is fixed at 1 s with a voltage value recorded every 10 s. This variant includes the blade support translation system (Z8253, KCH301 Thorlabs).

[0554] Variant 3: Transportable mounting with transmission detection (downwards). Measurements can be made with or without a chopper and with or without evanescent wave excitation. The acquisition time is fixed at 1 s with a voltage value recorded every 10 s.

[0555] For each sample concentration to be detected, several measurements (N measurements, N greater than or equal to 5) at different positions of the slide are performed. Each measurement is the average of 100 000 values recorded for 1 s with an acquisition rate of 100 kHz (1 voltage value recorded every 10 s) except in the case of variant 1 of the experimental arrangement where the acquisition time was 30 s with a voltage value recorded every 100 ms.

[0556] The signal value indicated on the graphs and its error bar correspond, respectively, to the mean of the N measurements and their standard deviation. In most cases, the signal value for a concentration of the molecule to be detected equal to zero was subtracted from all the measured values for the different concentrations. Thus the signal value for a concentration equal to zero, appears at zero. However, the standard deviation indicated is a measure of the ability to detect a given concentration. Typically, the detection limit is considered to be determined by the concentration generating a signal equal to 3 times the standard deviation of the signal obtained at zero concentration (blank). The limit of quantification is determined by the concentration generating a signal 10 times greater than this standard deviation at zero concentration.

[0557] For experiments performed with evanescent wave excitation, a collection of the luminescence from above is preferable. Indeed, the luminescence emission by the sample downwards is refracted towards wide angles by the Plexiglas parallelepiped and, as a result, a smaller fraction is collected by the collection lens in the presence of the parallelepiped allowing the evanescent wave excitation to take place.

[0558] ii. Calibration of the Detection Device

[0559] Prior to the measurements, the detection device was calibrated with nanoparticles in solution, according to variants 2 (upward detection with chopper and with evanescent wave excitation) and 3 (downward detection, without chopper and without evanescent wave excitation).

[0560] The calibration protocol is as follows:

[0561] The glass slides are activated beforehand with the plasma cleaner. [0562] deposition of a solution of nanoparticles diluted in PBS of known concentration on the glass slide; [0563] incubation for 2 hours; [0564] rinse at least three times with ultrapure water.

[0565] The acquisition time is fixed at 1 s with a voltage value recorded every 10 s.

[0566] The calibration curves of the detection device obtained from the two variants are shown in FIGS. 14-a and 14-b.

[0567] iii. Results of Detection/Quantification Tests

[0568] Detection and Quantification of a Substance in a Sample

[0569] The concentration of a substance in a sample is detectable when the signal obtained is at least three times greater than the standard deviation of the signal for a sample of the same composition containing zero concentration of the substance.

[0570] In order to carry out the quantification of the substance to be analyzed (i.e. to determine its concentration), the following protocol must be implemented:

[0571] i) perform a series of calibration measurements with the substance to be analyzed at different known concentrations, for example from commercially available substances or from purification. If possible, calibration samples should be prepared with the same or as close as possible to the composition of the samples to be measured. Make an adjustment of the points obtained (signal in mV versus concentration of the substance to be analyzed);

[0572] ii) carry out measurements of the samples to be analyzed (obtaining the signal value in mV);

[0573] iii) assign to each measured sample a concentration value of the substance from the measured signal (in mV) and from the calibration curve carried out in step i) and its fit.

[0574] The concentration of a substance in a sample is quantifiable when the signal obtained is at least 10 times greater than the standard deviation of the signal for a sample of the same composition containing zero concentration of the substance.

[0575] Detection of Insulin

[0576] The samples analyzed are either solutions of recombinant insulin in PBS+5% BSA or insulin in serum (samples provided by Cerba Specimen Services with insulin concentrations previously determined by reference techniques).

[0577] For recombinant insulin samples in solution in PBS+5% BSA, the recombinant insulin provided by the ELISA kit for the associated calibration experiments was used. The various samples were prepared by successive dilutions according to the protocol indicated by the kit supplier.

[0578] For serum insulin samples, the solutions were used as is for the highest concentrations. As very low concentration samples were not available, they were prepared by diluting the samples containing the lowest available concentrations in PBS+5% BSA.

[0579] Detection by ELISA

[0580] For comparison, the detection of recombinant insulin was carried out in solution by an ELISA kit in a 96-well plate. The experimental conditions followed are those indicated by the ELISA kit supplier. The absorbance of the well that was not incubated with insulin (zero concentration) was subtracted from the absorbance values measured for the wells that were incubated with the different insulin concentrations. Two wells were used for each concentration value and the standard deviation for these two measurements is shown as the error bar in FIG. 15. The lowest concentration measured is 187 pg/mL (or 33 pM). The standard deviation for zero concentration is 0.0035. The measured absorbance value for the concentration 187 pg/mL (or 33 pM) is 0.0085, just below the limit value of 0.0105 equal to 3 times the standard deviation determined for zero concentration. According to the supplier's specifications (ABCAM item ab100578), detection by a conventional ELISA kit will not detect concentrations below 50 pg/mL (or 9 pM) (FIG. 15).

[0581] Ultrasensitive Detection According to the Invention

[0582] FIG. 16 shows the signal obtained for samples of recombinant insulin in solution for different concentrations, with the lowest concentration being 0.05 pg/mL (or 9 fM), with variant 1 of the detection device. The acquisition time is 30 seconds, with a voltage value recorded every 100 ms.

[0583] In the inset in FIG. 16, the signal without insulin has been subtracted.

[0584] The minimum detectable concentration is thus 1000 times lower than the concentration detectable by ELISA (50 pg/mL or 9 pM) using the same antibodies as the ELISA kit.

[0585] FIG. 17 shows the signal obtained for samples of recombinant insulin in solution for different concentrations, with the lowest concentration being 0.05 pg/mL (or 9 fM), with variant 2 of the detection device (top detection), without chopper and without evanescent wave excitation. The acquisition time is 1 second, with a voltage value recorded every 10 s.

[0586] The minimum concentration detected is thus 1000 times lower than the concentration detectable by ELISA (50 pg/mL or 9 pM) using the same antibodies as the ELISA kit.

[0587] FIG. 18 shows the signal obtained for samples of recombinant insulin in solution for different concentrations, with the lowest concentration being 0.05 pg/mL (or 9 fM), with variant 2 of the detection device (top detection), with chopper and with evanescent wave excitation. The acquisition time is 1 second, with a voltage value recorded every 10 s.

[0588] The minimum concentration detected is thus 1000 times lower than the concentration detectable by ELISA (50 pg/mL or 9 pM) using the same antibodies as the ELISA kit.

[0589] FIG. 19 shows the signal obtained for serum samples containing insulin (samples provided by CERBA Specimen Services and diluted in PBS+5% BSA for the lowest concentrations. To obtain the lowest concentration samples, the sample containing insulin at approximately 8 pM was diluted on an IBIDI multiwell plate with variant 1 of the detection device.

[0590] The acquisition time is 30 seconds, with a voltage value recorded every 100 ms.

[0591] The minimum concentration detected is 9 fM (or 0.05 pg/mL), 1000 times lower than the concentration detectable by ELISA (50 pg/mL or 9 pM) using the same antibodies as the ELISA kit.

[0592] TSH Detection

[0593] The samples analyzed are solutions of recombinant TSH with a molecular weight of 15 639 Da in PBS+5% BSA. The antibodies used are those of the ABCAM kit item ab 100660.

[0594] Ultrasensitive Detection According to the Invention

[0595] FIG. 20 shows the signal obtained for recombinant TSH samples in solution for different concentrations, with the lowest concentration being 3.2 fM (1.4 fg/mL), using variant 1 of the detection device with chopper. The acquisition time is 1 second, with a voltage value recorded every 10 s.

[0596] In comparison, the minimum concentration that can be detected by the ELISA kit is typically less than 4 pg/mL according to the supplier. Thus, the minimum concentration detected according to the invention is more than 1000 times lower than the concentration detectable by the ELISA kit.

Example 4

[0597] Incubation Time Adjustment

[0598] The incubation time required to maximize the signal in the case of the detection of recombinant insulin in solution (50 pg/mL insulin) can be determined beforehand by luminescence measurements obtained for different incubation times between sample supports with the substance to be analyzed (here recombinant insulin) immobilized on their surfaces (immobilization carried out according to the protocol described in Example 2ii) and antibody-nanoparticle conjugates (obtained according to the protocol described in Example 1 with 10 g/mL antibody), step carried out according to the protocol described in Example 3.i.

[0599] The measurements were made with variant 1 of the detection device described in Example 3. The acquisition time is 30 seconds, with a voltage value recorded every 100 ms.

[0600] FIG. 21 shows the change in the detected luminescence signal as a function of the incubation time.

[0601] It appears necessary to incubate the antibody-coupled nanoparticles with the surface on which the substance to be analyzed (in this case recombinant insulin) is immobilized for at least 45 minutes to maximize the detected luminescence signal. This time required may vary depending on the substance to be analyzed and the antibody used.

Example 5

[0602] Spatial Multiplexing Experiment

[0603] To illustrate the possibility of performing multiplexed detection according to the process of the invention (schematic diagram of spatial multiplexing shown in FIG. 22), the following experiment was performed.

[0604] Two areas or spots containing nanoparticles were created by depositing two drops of two solutions of nanoparticles as obtained according to the protocol described in Example 1 (part 1.1, nanoparticles as obtained after synthesis) of two different concentrations (4 mM and 8 mM) on glass slides cleaned according to the protocol described in Example 2. Here the drops containing the nanoparticles were allowed to dry before the luminescence measurements began, without a rinsing step.

[0605] The diagram in FIG. 23 shows the diameter of the laser beam at the sample level according to the direction of travel (3 mm), the diameter (2 mm) and the distance between the two nanoparticle spots (1 mm).

[0606] The measurements are carried out with variant 2 of the experimental arrangement described in Example 3 (upward detection, with chopper and without evanescent wave excitation). Thanks to the motorized displacement system of the sample support, the signal emitted by the nanoparticles was detected at several locations: before the first spot (position 1), in the first spot (position 4), between the two spots (position 6), in the second spot (position 7) and finally after the second spot (position 9). For each position of the excitation laser beam, the acquisition time is 1 second, with a voltage value recorded every 10 s.

[0607] FIG. 23 shows the luminescence signal detected for the different locations mentioned above. The two peaks with different locations and amplitudes show the possibility of differentiating spatially organized deposits, as is necessary for a multiplexed measurement.

Example 6

[0608] Synthesis of 3% YVO.sub.4:Dy Nanoparticles.

[0609] The protocol used is even the same as that for the synthesis of the Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles indicated in point 1.1. of Example 1 above, with the only difference being that instead of the precursor Eu(NO.sub.3).sub.3, we use the precursor Dy(NO.sub.3).sub.3 at a concentration of 0.03 M.

[0610] Result

[0611] FIG. 26 shows the luminescence excitation and emission spectra of a solution of these nanoparticles after their synthesis. The excitation spectrum shows peaks of direct excitation of Dy.sup.3+ ions. The following steps of functionalization and coupling to streptavidin (Example 1, point 1.2.) and then to a biotinylated targeting agent (Example 1, point 1.3.), or direct coupling to a targeting agent (Example 1, point 1.4.), can be reproduced identically with these nanoparticles, thus producing probes emitting at a different emission wavelength.

Example 7

[0612] Synthesis of 3% YVO.sub.4:Sm Nanoparticles.

[0613] The protocol used is even the same as that for the synthesis of the Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles indicated in point 1.1. of Example 1 above, with the only difference being that instead of the precursor Eu(NO.sub.3).sub.3, we use the precursor Sm(NO.sub.3).sub.3 at a concentration of 0.03 M.

[0614] Result

[0615] FIG. 27 shows the luminescence excitation and emission spectra of a solution of these nanoparticles after their synthesis. The excitation spectrum shows peaks of direct excitation of Sm.sup.3+ ions. The following steps of functionalization and coupling to streptavidin (Example 1, point 1.2.) and then to a biotinylated targeting agent (Example 1, point 1.3.), or direct coupling to a targeting agent (Example 1, point 1.4.), can be reproduced identically with these nanoparticles, thus producing probes emitting at a different emission wavelength.

REFERENCES

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