Luminescent particles based on rare earth elements and use thereof as a diagnostic agent

Abstract

The present invention concerns a luminescent particle comprising a nanoparticle of formula
A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y  (I)
in which A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), and mixtures thereof; 0<x<1; and 0≤y<1; characterized in that the nanoparticle on its surface has tetraalkylammonium cations in an amount such that said nanoparticle has a zeta potential, ζ, of less than or equal to −28 mV in an aqueous medium with a pH≥5, more particularly with a pH≥5.5, and with an ionic conductivity >100 μS.Math.cm.sup.−1. It also concerns a method for preparing such luminescent particles, a colloidal suspension of these particles, and the use thereof as a diagnostic agent, and also a diagnostic kit comprising such luminescent particles.

Claims

1. A luminescent particle comprising a nanoparticle of formula:
A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y  (I) in which: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), and mixtures thereof; 0<x<1; and 0≤y<1; wherein the nanoparticle has, at the surface, tetraalkylammonium cations in an amount such that said nanoparticle has a zeta potential, ζ, of less than or equal to −28 mV in an aqueous medium with a pH≥5, and with an ionic conductivity of strictly less than 100 μS.Math.cm.sup.−1.

2. The particle as claimed in claim 1, wherein said tetraalkylammonium cations are associated with the nanoparticle via electrostatic interactions with the negatively charged O.sup.2− surface ions of the nanoparticle.

3. The particle as claimed in claim 1, wherein said tetraalkylammonium cations are cations of formula NR.sub.4.sup.+ where R, which are identical or different, represent a C.sub.1-C.sub.6-alkyl.

4. The particle as claimed in claim 1, wherein said tetraalkylammonium cations are selected from tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and mixtures thereof.

5. The particle as claimed in claim 1, wherein said nanoparticle has a zeta potential ζ of less than or equal to −30 mV in an aqueous medium with a pH≥6.5, and with an ionic conductivity of strictly less than 100 μS.Math.cm.sup.−1.

6. The particle as claimed in claim 1, said nanoparticle being of formula (I′)
A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y.Math.(NR.sub.4.sup.+).sub.z  (I′) in which: A is selected from Y, Gd, La, and mixtures thereof; Ln is selected from Eu, Dy, Sm, Nd, Er, Yb, and mixtures thereof; 0<x<1; 0≤y<1; R, which are 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.

7. The particle as claimed in claim 1, wherein y is 0.

8. The particle as claimed in claim 1, wherein A represents Y.

9. The particle as claimed in claim 1, wherein Ln represents Eu.

10. The particle as claimed in claim 1, wherein said nanoparticle is of formula Y.sub.1-xEu.sub.xVO.sub.4.Math.(NR.sub.4.sup.+).sub.z (III′), in which: 0<x<1; R, which are identical or different, represent a C.sub.1-C.sub.6-alkyl z represents the number of tetraalkylammonium cations NR.sub.4.sup.+ located on the surface of said nanoparticle.

11. The particle as claimed in claim 1, wherein the nanoparticle of formula (I) has an elongated ellipsoidal shape.

12. The particle as claimed in claim 1, wherein the product of the degree of doping, x, with Ln ions, and the quantum yield of the emission by the nanoparticle is maximized.

13. The particle as claimed in claim 1, wherein said nanoparticle is coupled to at least one targeting agent.

14. A method for preparing luminescent particles comprising a nanoparticle of formula:
A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y  (I) in which: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), and mixtures thereof; 0<x<1; and 0≤y<1; by coprecipitation reaction, in aqueous medium, from precursors of said elements A and Ln, and in the presence of orthovanadate (VO.sub.4.sup.3−) and optionally phosphate (PO.sub.4.sup.3−) ions; said reaction being performed in the presence of an effective amount of tetraalkylammonium cations such that said nanoparticle has a zeta potential, ζ, of less than or equal to −28 mV in an aqueous medium with a pH≥5, and with an ionic conductivity of strictly less than 100 μS.Math.cm.sup.−1.

15. The method as claimed in claim 14, said method comprising at least the steps of: (i) providing an aqueous solution, called solution (1), comprising orthovanadate ions (VO.sub.4.sup.3−), and optionally phosphate ions (PO.sub.4.sup.3−), and tetraalkylammonium cations; (ii) admixing the aqueous solution (1) with an aqueous solution, called solution (2), comprising said precursors of the elements A and Ln, under conditions conducive to the formation by coprecipitation of the nanoparticles of formula (I); and (iii) recovering the nanoparticles of formula (I) with tetraalkylammonium cations located on their surface that are formed at the end of step (ii).

16. The method as claimed in claim 14, wherein the tetraalkylammonium cations are selected from tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and mixtures thereof.

17. The method as claimed in claim 14 for preparing particles of formula (I′)
A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y.Math.(NR.sub.4.sup.+).sub.z  (I′) in which: A is selected from Y, Gd, La, and mixtures thereof; Ln is selected from Eu, Dy, Sm, Nd, Er, Yb, and mixtures thereof; 0<x<1; 0≤y<1; R, which are 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.

18. The method as claimed in claim 14, wherein the orthovanadate ions (VO.sub.4.sup.3−) are generated in situ by reacting a metavanadate salt, with a base.

19. The method as claimed in claim 14, said method further comprising one or more steps of coupling the particles with one or more targeting agents.

20. A colloidal aqueous suspension comprising particles as defined in claim 1.

21. A method of diagnostics, comprising: coupling the luminescent particles as defined in claim 1 to one or more targeting agents of a substance of biological or chemical interest to obtain a diagnostic probe; implementing said diagnostic probe in an in vitro, ex vivo, or in vivo diagnostic technique; and detecting and/or quantifying said substance of biological or chemical interest.

22. A diagnostic kit comprising at least particles as defined in claim 1, or a colloidal aqueous suspension of these particles.

23. The particle as claimed in claim 6, wherein z is between 100 and 10,000.

24. A colloidal aqueous suspension comprising particles obtained by the method defined in claim 14.

25. A method of diagnostics, comprising: coupling the luminescent particles obtained by the method of claim 14 to one or more targeting agents of a substance of biological or chemical interest to obtain a diagnostic probe; implementing said diagnostic probe in an in vitro, ex vivo, or in vivo diagnostic technique; and detecting and/or quantifying said substance of biological or chemical interest.

26. A diagnostic kit comprising at least particles obtained by the method defined in claim 14, or a colloidal aqueous suspension of these particles.

Description

FIGURES

(1) FIG. 1: Micrographs obtained by transmission electron microscopy (TEM) of the nanoparticles obtained according to example 1 (Scale bar: 60 nm (FIG. 1a) and 5 nm (FIG. 1b), respectively);

(2) FIG. 2: Size histogram of the nanoparticles, determined from TEM micrographs for a collective of approximately 300 nanoparticles according to example 1;

(3) FIG. 3: X-ray diffractograms obtained for the nanoparticles synthesized according to example 1 (black line), and 3 (gray line);

(4) FIG. 4: Schematic representation of the nanoparticles of the invention functionalized with citrate (FIG. 4a) or with polyacrylic acid (PAA) (FIG. 4b). For clarity, a single citrate or PAA molecule is represented. It will be appreciated that each nanoparticle may contain numerous PAA or citrate molecules on its surface.

(5) FIG. 5: Schematic representation of the reactions for coupling a nanoparticle according to the invention with streptavidin, according to example 4;

(6) FIG. 6: Schematic representation of the coupling of the nanoparticles coupled with streptavidin with a biotinylated antibody.

(7) It will be appreciated that FIGS. 4 to 6 are diagrams of principle, especially as regards the structure of the surface molecules, which may have a number of configurations other than that shown in these figures.

EXAMPLES

Example 1

Synthesis of Y.SUB.0.6.Eu.SUB.0.4.VO.SUB.4 .Nanoparticles According to the Invention

(8) The source of metavanadate VO.sub.3.sup.− ions used is ammonium metavanadate NH.sub.4VO.sub.3, the orthovanadate VO.sub.4.sup.3− being obtained in situ following reaction with a base, presently 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.

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

(10) A volume of 10 mL of another solution of Y(NO.sub.3).sub.3 and of Eu(NO.sub.3).sub.3 with an ion (Y.sup.3++Eu.sup.3+) concentration of 0.1 M is added dropwise to solution 1 using a syringe driver at a rate of 1 mL/min.

(11) The molar concentration ratio between Y(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 is selected according to the desired ratio between ions Y.sup.3+ and Eu.sup.3− in the nanoparticle; typically, the molar ratio Y.sup.3+:Eu.sup.3+ is 0.6:0.4.

(12) When the Y(NO.sub.3).sub.2/Eu(NO.sub.3).sub.3 solution is added, the solution becomes diffuse and appears white/milky without forming precipitate. The synthesis continues until all of the Y(NO.sub.3).sub.2/Eu(NO.sub.3).sub.3 solution has been added.

(13) The eventual 20 mL solution must now be purified to remove the excess of counterions. To do this, centrifugations (typically three) at 11 000 g (Sigma 3K10, Bioblock Scientific) for 80 minutes, followed in each case by redispersion by sonication (Branson Sonifier 450 operating at 50% with a power of 400 W) are used until the resulting conductivity is strictly less than 100 μS.Math.cm.sup.−1.

(14) The conductivity is measured using a chemical conductimeter.

(15) The synthesis of Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles with tetramethylammonium cations immobilized on their surface may be represented schematically 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.3−+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.0.5Eu.sub.0.4VO.sub.4+2N(CH.sub.3).sub.4.sup.++NH.sub.4.sup.−+3NO.sub.3

(16) Two trial syntheses (“synthesis 1” and “synthesis 2”) are performed.

(17) Result

(18) Visual observation of the solution of nanoparticles according to the invention, after having been left at rest for 16 hours in a flask, shows a uniformly diffuse solution.

(19) The eventual solution remains very stable in water, even after several months in the final pH of the synthesis (approximately pH 7-9). The solution remains stable, including in the synthesis medium (before the removal of the excess of counterions), but with a high ionic strength (>0.1 M).

(20) The zeta potential of the nanoparticles is determined using a DLS-Zeta Potential instrument (Zetasizer Nano ZS90, Malvern). The results of the zeta potentials measured for the nanoparticles from the two syntheses are collated in table 1 below.

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

(22) The observation of the nanoparticles by TEM (FIG. 1) shows the nanoparticles to have an elongated ellipsoidal shape. The dimensions of the nanoparticles are determined from TEM micrographs for a collective of approximately 300 nanoparticles (FIG. 2). The nanoparticles of the invention have a major axis length, a, of between 20 and 60 nm, with an average value of approximately 40 nm, and a minor axis length, b, of between 10 and 30 nm, with an average value of approximately 20 nm.

(23) The TEM micrographs (FIG. 1) do not allow the crystalline planes to be made out, a fact probably attributable to the nanoparticle being composed of a number of crystallites whose size is smaller than the size of the nanoparticle. The primarily crystalline and polycrystalline nature of the nanoparticles is confirmed by X-ray diffraction experiments.

(24) The X-ray diffractogram obtained by means of a Philips X-pert diffractometer using the K.sub.α1 ray of copper (λ=1.5418 Å) is represented in FIG. 3. The diffraction intensity is recorded using an X'Celerator area detector (PANalytical).

(25) The coherence length in one crystallographic direction, and therefore the average size of the crystallites making up the nanoparticle in this crystallographic direction, can be estimated from the width of the peaks in the XR diffractogram, by application of the formula of Scherrer. The coherence length values obtained for the various crystallographic directions are between 3 and 40 nm. The coherence length in at least one crystallographic direction is smaller than the size of the nanoparticle in that direction, and from this it may be deduced that the crystallinity of the nanoparticles is imperfect (polycrystallinity, defects or porosity). In the direction (200) (FIG. 3, peak at) 2θ≅25°), the coherence length is 10.2 nm, which is slightly weaker than the coherence length for the nanoparticles of example 3 (11.1 nm).

Example 2 (Not Inventive)

Synthesis of Y.SUB.0.6.Eu.SUB.0.4.VO.SUB.4 .Nanoparticles from Sodium Metavanadate, without Bulky Counterions

(26) For the synthesis by coprecipitation of metal salts, yttrium and europium nitrates were used as sources of Y.sup.3+ and Eu.sup.3+ ions. The source of metavanadate VO.sub.3.sup.− ions used is sodium metavanadate NaVO.sub.3, the orthovanadate VO.sub.4.sup.3− being obtained in situ following reaction with a base, presently sodium hydroxide, NaOH.

(27) An aqueous solution of 10 mL of 0.1 M NaVO.sub.3 and 0.2 M NaOH (solution 1) is freshly prepared.

(28) A volume of 10 mL of another solution of Y(NO.sub.3).sub.3 and of Eu(NO.sub.3).sub.3 with an ion (Y.sup.3++Eu.sup.3+) concentration of 0.1 M is added dropwise to solution 1 using a syringe driver at a rate of 1 mL/min.

(29) The molar concentration ratio between Y(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 is selected according to the desired ratio between ions Y.sup.3+ and Eu.sup.3− in the nanoparticle; typically, the molar ratio Y.sup.3+:Eu.sup.3+ is 0.6:0.4.

(30) When the Y(NO.sub.3).sub.2/Eu(NO.sub.3).sub.3 solution is added, a milky precipitate appears. The synthesis continues until all of the Y(NO.sub.3).sub.2/Eu(NO.sub.3).sub.3 solution has been added.

(31) The eventual 20 mL solution must now be purified to remove the excess of counterions. To do this, centrifugations (typically three) at 11 000 g (Sigma 3K10, Bioblock Scientific) for 80 minutes, followed in each case by redispersion by sonication (Branson Sonifier 450 operating at 50% with a power of 400 W) are used until the resulting conductivity is strictly less than 100 μS.Math.cm.sup.−1.

(32) Result

(33) A comparison of the resulting solution with the solution of nanoparticles according to the invention as obtained in example 1 (for the same final concentration of vanadate ions), after 16 hours at rest, shows that, in contrast to the solution from example 1, the solution is less diffuse (less white); a slight deposit has formed on the base of the flask.

(34) The zeta potential of the nanoparticles, determined as described in example 1, is presented in table 1 below.

Example 3 (Not Inventive)

Synthesis of Y.SUB.0.6.Eu.SUB.0.4.VO.SUB.4 .Nanoparticles from Sodium Orthovanadate, without Bulky Counterions

(35) For the synthesis by coprecipitation of metal salts, yttrium and europium nitrates were used as sources of Y.sup.3+ and Eu.sup.3+ ions. As a source of orthovanadate ions, VO.sub.4.sup.3−, sodium orthovanadate Na.sub.3VO.sub.4 is used.

(36) An aqueous solution of 10 mL of 0.1 M Na.sub.3VO.sub.4 (solution 1) is freshly prepared. The pH is measured and adjusted if necessary to give a value of between 12.6 and 13.

(37) A volume of 10 mL of another solution of Y(NO.sub.3).sub.3 and of Eu(NO.sub.3).sub.3 with an ion (Y.sup.3++Eu.sup.3+) concentration of 0.1 M is added dropwise to solution 1 using a syringe driver at a rate of 1 mL/min.

(38) The molar concentration ratio between Y(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 is selected according to the desired ratio between ions Y.sup.3+ and Eu.sup.3− in the nanoparticle; typically, the molar ratio Y.sup.3+:Eu.sup.3+ is 0.6:0.4.

(39) When the Y(NO.sub.3).sub.2/Eu(NO.sub.3).sub.3 solution is added, a milky precipitate appears. The synthesis continues until all of the Y(NO.sub.3).sub.2/Eu(NO.sub.3).sub.3 solution has been added.

(40) The eventual 20 mL solution must now be purified to remove the excess of counterions. To do this, centrifugations (typically three) at 11 000 g (Sigma 3K10, Bioblock Scientific) for 80 minutes, followed in each case by redispersion by sonication (Branson Sonifier 450 operating at 50% with a power of 400 W) are used until the resulting conductivity is strictly less than 100 μS.Math.cm.sup.−1.

(41) Results

(42) A comparison of the resulting solution with the solution of nanoparticles according to the invention as obtained in example 1 (for the same final concentration of vanadate ions), after 16 hours at rest, shows that, in contrast to the solution from example 1, the majority of the nanoparticles have deposited on the base of the flask; the upper part of the solution is almost transparent, indicating an absence of diffusion and hence of nanoparticles in solution.

(43) The zeta potential of the nanoparticles, determined as described in example 1, is presented in table 1 below.

(44) The X-ray diffractogram obtained using a Philips X-pert diffractometer is represented in FIG. 3.

(45) As for example 1, the average size of the crystallites making up the nanoparticle in the crystallographic direction (200) may be estimated by application of the formula of Scherrer, and gives a value of between 3 and 40 nm. The size of the crystallites is smaller overall than the dimensions of the nanoparticle, and it can be deduced from this that the nanoparticles synthesized according to example 3 are also polycrystalline.

(46) TABLE-US-00001 TABLE 1 comparison of the zeta potential ζ of nanoparticles obtained according to example 1 in accordance with the invention (synthesis based on ammonium metavanadate with bulky counterions), and according to examples 2 and 3 not in accordance with the invention (synthesis based on sodium metavanadate without bulky counterions, and synthesis based on orthovanadate without bulky counterions). Example 1 (inventive) Example Example Synthesis Synthesis 2 (not 3 (not Examples 1 2 inventive) inventive) Conductivity 93 80 53 66 (μS/cm) pH 4.8 5.0 5.4 9.6 Zeta potential ζ* −33.3 −34.6 −2.30 −18.00 * zeta potential measurements made after purification to give a conductivity <100 μS .Math. cm.sup.−1 and after dilution in acidic Milli-Q ® water.

(47) The comparison of the zeta potentials of the nanoparticles obtained according to examples 1 to 3 confirm that the nanoparticles according to the invention (example 1) exhibit an improved colloidal stability with |ζ|>30 mV by comparison with the nanoparticles obtained without tetraalkylammonium cations (examples 2 and 3).

(48) An alternative synthesis of the nanoparticles Y.sub.0.6Eu.sub.0.4VO.sub.4 according to the invention was carried out according to the same protocol as in the synthesis of example 1, with the only difference being that the solution of 10 mL of Y(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 with an ion (Y.sup.3−+Eu.sup.3+) concentration of 0.1 M was added to solution 1 not dropwise, but in a single go.

(49) At the end of the synthesis, after purification, the zeta potential is −35 mV at a pH of 7.8.

Example 4

Coupling of the Nanoparticles with the Protein Streptavidin

(50) i. Grafting of Citrate to the Surface of the Nanoparticles

(51) At the outcome of the synthesis of the nanoparticles according to example 1, the nanoparticle solution is centrifuged at 17 000 g for 3 minutes, to precipitate any nanoparticle aggregates, and the supernatant is recovered.

(52) Approximately 250 μL of Y.sub.0.6Eu.sub.0.4VO.sub.4 particles with a vanadate ion concentration of 5 mM are removed and are dispersed in 1 mL of a distilled water solution containing the citrate ion (concentration 0.2 M).

(53) The solution is then sonicated for 5 minutes and subsequently centrifuged at 17 000 g for 3 minutes. This step is repeated 3 times.

(54) At the end of this grafting, the particles are dispersed in the distilled water, a solvent in which they are stable.

(55) The functionalization of the nanoparticles with citrate may be replaced by functionalization with PAA (for example, with a degree of polymerization of between 3 and 10 000) by employing a salt of PAA, as for example a sodium or ammonium salt.

(56) FIG. 4 shows schematically the nanoparticles of the invention functionalized with (a) citrate and (b) polyacrylic acid.

(57) ii. Coupling of the Nanoparticles with Streptavidin

(58) The nanoparticles (NPs) grafted with the citrate ions are centrifuged at 16 000 g for 1 hour, and the pellet is then recovered.

(59) The coupling of the nanoparticles, grafted on their surface with citrate, with streptavidin is carried out according to the following protocol: 1. Freshly prepare a mixed solution of EDC.sup.1/Sulfo-NHS.sup.2 (concentrations 30 and 30 mg/mL, respectively) in MES buffer.sup.3 (pH 5-6). 2. By sonication (ultrasound bath) disperse the pellet of the NPs in 250 μL of the solution prepared in step 1. Since the losses during the centrifugations are low, the concentration of vanadate ions remains around 5 mM, giving a nanoparticle concentration of 48 nm. (The vanadate concentration of the nanoparticle solutions was determined by dissolving the particles in an acidic medium, followed by colorimetric determination of the concentration of vanadate ions, as described in the reference Abdesselem et al., ACS Nano 8, 11126-11137 (2014). The molar concentration of the nanoparticles was determined from the concentration of vanadate ions as described in the reference Casanova et al., Appl. Phys. Lett 89, 253103 (2006).) 3. Prepare a solution of streptavidin (SA) at 100 nm in phosphate buffer pH 7.4 with NaCl at 10 mM. Dilute the streptavidin solution to a concentration determined by the desired number of proteins grafted per nanoparticle (for a streptavidin:NPs ratio of 1:1, 5:1 and 10:1, select, respectively, concentrations of 4.8 nm, 24 nm and 48 nm). Add 250 μL of this solution to the nanoparticle solution. 4. Incubate for between 2 and 4 h at ambient temperature. 5. Add 1 mL of PBST.sup.4 and vortex. 6. Carry out centrifugation at 6 500 g for 30 min and recover the pellet to remove the proteins not coupled to the NPs. Discard all of the supernatant. Redisperse the NPs coupled to the proteins in 1 mL of PBST and sonicate in an ultrasound bath. Repeat this step twice. 7. Recover the NPs coupled to the proteins in 250 μL of PBS.sup.5 with 1% of BSA.sup.6. 8. Keep at 4° C. for immediate use or aliquot and keep at −80° C.

(60) The coupling of the nanoparticles with streptavidin is represented schematically in FIG. 5.

(61) Materials used for the functionalization trial: .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).

(62) The number of molecules of streptavidin (SA) per nanoparticle (NP), denoted R, characterized at the end of the coupling protocol, is determined by assaying the streptavidin by the BCA method, according to the protocol detailed above.

(63) Protocol for Characterizing the Streptavidin:Nanoparticles (SA:NPs) Ratio

(64) i. Assay of SA by the BCA method

(65) Principle 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 can be quantified by its absorption at 562 nm.

(66) Procedure BCA assay kit from ThermoFisher (Pierce™ BCA Protein Assay Kit Cat. Num. 23225) Preparation of the Cu.sup.2+/BCA test reagent according to the protocol in the ThermoFisher kit. Preparation of the SA for the standard curve. For the calibration curve, the assay is carried out three times.

(67) TABLE-US-00002 TABLE 2 streptavidin concentration of the standard solutions Tubes Final SA concentration (μg/mL) A 2000 B 1500 C 1000 D 750 E 500 F 250 G 125 H 25 I 0 = white

(68) The procedure is as follows: Take a 96-well plate. Place 25 μL of each standard tube A to I (with a known concentration of SA) or of nanoparticle-conjugated proteins for assay in the corresponding wells. Add 200 μL of the Cu.sup.2+/BCA test reagent to each well. Homogenize, cover, and incubate at 37° C. for 30 min. Read off the absorbances (A) at 562 nm as a function of the final concentration, taking account of the dilution. Establish the standardization relationship A=f (concentration of SA in μg/mL) by linear regression. The concentration of the nanoparticle-conjugated proteins for assay is deduced from the equation of the linear regression obtained with the standard curve.

(69) ii. Characterization of the SA:NPs Ratio

(70) The mass concentration of streptavidin as obtained with the BCA assay is converted to molar concentration via the following formula:

(71) ${\left[\mathrm{SA}\right]}_{\mathrm{molar}}\left(\mathrm{mol}\text{/}L\right)=\frac{\left[\mathrm{SA}\right]\mathrm{mass}\left(g\text{/}L\right)}{\mathrm{Molar}\mathrm{mass}\left(g\text{/}\mathrm{mol}\right)}$

(72) To give the ratio (R) of the number of SA:NPs, we apply, finally, the following

(73) $R=\frac{\left[\mathrm{SA}\right]\mathrm{molar}}{\left[\mathrm{NPs}\right]\mathrm{molar}}$
equation:

(74) Table 3 below collates the values obtained for the various concentration ratios of the streptavidin solution to the starting nanoparticle solution.

(75) TABLE-US-00003 TABLE 3 Characterization of nanoparticle-streptavidin coupling for various concentration ratios of the streptavidin solution to the nanoparticle solution. Concentration ratios of the streptavidin 1:1 5:1 10:1 solution to the nanoparticle solution Number of streptavidin molecules determined 0.97 3.8 9.29 per nanoparticle at the end of coupling

(76) As is apparent from the results presented in table 3, the number of molecules of streptavidin per nanoparticle after coupling is of the same order as the concentration ratio in the initial solutions, and thus indicates highly effective coupling.

(77) The nanoparticles coupled in this way to the streptavidin may be used advantageously in an in vitro diagnostic technique, by coupling the nanoparticles to a biotinylated antibody, as illustrated schematically in FIG. 6.

(78) Alternatively, it is also possible for antibodies to be coupled directly to the nanoparticles grafted with citrate ions. In that case, the same protocol as above should be used, with the streptavidin solution replaced with a solution of antibody.

Example 5 (Not Inventive)

Synthesis of Y.SUB.0.6.Eu.SUB.0.4.VO.SUB.4 .Nanoparticles from Sodium Metavanadate, with Addition of Tetraalkylammonium Ions at the End of Synthesis

(79) A colloidal suspension of Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles is prepared from sodium salts, in accordance with the synthesis of example 2.

(80) Purification is carried out by dialysis.

(81) One equivalent (relative to the vanadate ions) of tetramethylammonium hydroxide N(CH.sub.3).sub.4OH is added, the suspension is left for 20 minutes, and it is then dialyzed again to remove the excess counterions until the resulting conductivity is strictly less than 100 μS.Math.cm.sup.−1.

(82) The zeta potential, determined as described in example 1, at the resulting suspension pH of 8.9 is −2.20 mV.

(83) It is not possible to obtain the same result as with the method of the invention using a standard method of nanoparticle synthesis and then, at the end of the synthesis, adding bulky tetraalkylammonium ions.

REFERENCES

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