CAPILLARY ACTION TEST USING PHOTOLUMINESCENT INORGANIC NANOPARTICLES

Abstract

The present invention relates to an in vitro method for detecting and/or quantifying a biological or chemical substance of interest in a liquid sample, by a capillary action test using, as probes, photoluminescent inorganic nanoparticles, of formula A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y (II), in which Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho) and mixtures thereof; A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof; 0<x<1; and 0≤y<1, said method employing detection of the luminescence, with an emission lifetime shorter than 100 ms, of the nanoparticles, after one-photon absorption, by excitation of the matrix at a wavelength less than or equal to 320 nm.

It also relates to a capillary action test device comprising, as probes, the aforementioned nanoparticles, as well as the use of such a method for purposes of in vitro diagnostics.

Claims

1.-25. (canceled)

26. An in vitro method for detecting and/or quantifying a biological or chemical substance of interest in a liquid sample, by a capillary action test using, as probes, photoluminescent inorganic nanoparticles of the following formula (II):
Al.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y  (II) in which: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho) and mixtures thereof; 0<x<1; and 0≤y<1; said method employing detection of the luminescence, with an emission lifetime shorter than 100 ms, of the nanoparticles, after one-photon absorption, by excitation of the matrix at a wavelength less than or equal to 320 nm.

27. The method as claimed in claim 26, in which detection of the luminescence is effected by excitation of the matrix at a wavelength less than or equal to 300 nm.

28. The method as claimed in claim 26, in which the liquid sample is a biological sample.

29. The method as claimed in claim 26, for detecting and/or quantifying molecules, proteins, nucleic acids, toxins, viruses, bacteria or parasites in a sample.

30. The method as claimed in claim 26, in which said photoluminescent nanoparticles have an average size greater than or equal to 5 nm and strictly less than 1 μm.

31. The method as claimed in claim 26, in which Ln is selected from Eu, Dy, Sm, Yb, Er, Nd and mixtures thereof.

32. The method as claimed in claim 26, in which A is selected from Y, Gd, La and mixtures thereof.

33. The method as claimed in claim 26, in which said nanoparticles have tetraalkylammonium cations on their surface in an amount such that said nanoparticles have a zeta potential, designated ζ, less than or equal to −28 mV, in an aqueous medium of pH≥5, and with ionic conductivity strictly less than 100 μS.Math.cm.sup.−1.

34. The method as claimed in claim 26, in which said nanoparticles are of formula A.sub.1-xLn.sub.xVO.sub.4 (III), in which: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho) and mixtures thereof; and 0<x<1.

35. The method as claimed in claim 26, said method using a capillary action test device, in which said photoluminescent inorganic nanoparticles are coupled to at least one reagent specifically binding the substance to be analyzed.

36. The method as claimed in claim 26, said method using a capillary action test device, in which said photoluminescent inorganic nanoparticles are functionalized on the surface with one or more agents intended to facilitate their migration within the capillary action test device.

37. The method as claimed in claim 26, using a capillary action test device, comprising: a zone (1) for deposition of the liquid sample, and optionally of a diluent; a zone (2), arranged downstream of the deposition zone, called “labeling zone”, loaded with said photoluminescent inorganic nanoparticles coupled to at least one reagent specifically binding the substance to be analyzed; a reaction zone (3), also called “detection zone”, arranged downstream of the labeling zone (2), in which at least one capturing reagent specific to the substance to be analyzed is immobilized; a control zone (4), located downstream of the detection zone, in which at least one second capturing reagent specific to the reagent specifically binding the substance to be analyzed is immobilized; and optionally, an absorbent pad (5), arranged downstream of the reaction zone and of the control zone.

38. The method as claimed in claim 37, said method comprising at least the following steps: (i) applying the liquid sample to be analyzed, and optionally a diluent, at the level of the deposition zone (1) of the capillary action test device; (ii) incubating the device until the luminescence generated by the photoluminescent nanoparticles is detected in the reaction zone (3) and/or until the luminescence is detected in the migration control zone (4); and (iii) reading and interpreting the results.

39. The method as claimed in claim 26, in which reading of the results of the capillary action test is effected by detecting the luminescence generated by the probes immobilized, at the end of the assay, at the level of the capillary action test device.

40. The method as claimed in claim 26, in which said nanoparticles are of formula Y.sub.1-xEu.sub.xVO.sub.4, (IV), detection of the luminescence being effected by excitation of the YVO.sub.4 matrix at a wavelength between 230 and 320 nm.

41. The method as claimed in claim 26, in which reading of the results of the capillary action test is effected by direct, naked eye observation of the capillary action test device.

42. The method as claimed in claim 26, in which reading of the results of the capillary action test is effected using detection equipment comprising an emission filter and a photon detector.

43. The method as claimed in claim 37, in which interpretation of the results comprises determination of the signal corresponding to the detection zone, the control zone and the background signal of the capillary action test device, subtracting the value of luminescence of the background signal and then determining the ratio of the signal from the detection zone to the signal from the control zone.

44. A capillary action test device, useful for detecting and/or quantifying a biological or chemical substance of interest in a liquid sample, said device comprising, as probes, photoluminescent inorganic nanoparticles of the following formula (II):
A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y  (II) in which: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho) and mixtures thereof; 0<x<1; and 0≤y<1; said nanoparticles being able to emit luminescence, with an emission lifetime shorter than 100 ms, after one-photon absorption, by excitation of the matrix at a wavelength less than or equal to 320 nm.

45. The device as claimed in claim 44, said device comprising: a zone (1) for deposition of the liquid sample, and optionally of a diluent; a zone (2), arranged downstream of the deposition zone, called “labeling zone”, loaded with said photoluminescent inorganic nanoparticles coupled to at least one reagent specifically binding the substance to be analyzed; a reaction zone (3), also called “detection zone”, arranged downstream of the labeling zone (2), in which at least one capturing reagent specific to the substance to be analyzed is immobilized; a control zone (4), located downstream of the detection zone, in which at least one second capturing reagent specific to the reagent specifically binding the substance to be analyzed is immobilized; and optionally, an absorbent pad (5), arranged downstream of the reaction zone and of the control zone.

46. An in vitro diagnostic kit, comprising at least: a capillary action test device useful for detecting and/or quantifying a biological or chemical substance of interest in a liquid sample, said device comprising, as probes, photoluminescent inorganic nanoparticles of the following formula (II):
A.sub.1-xLn.sub.xVO.sub.4(1-y)(PO.sub.4).sub.y  (II) in which: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof; Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho) and mixtures thereof; 0<x<1; and 0≤y<1; said nanoparticles being able to emit luminescence, with an emission lifetime shorter than 100 ms, after one-photon absorption, by excitation of the matrix at a wavelength less than or equal to 320 nm; and a device for detecting the luminescence generated by the probes immobilized at the level of the capillary action test device, at the end of the assay.

47. An in vitro diagnostic method, said method implementing an in vitro method for detecting and/or quantifying a biological or chemical substance of interest in a liquid sample as claimed in claim 1.

48. An in vitro diagnostic method, said method implementing an in vitro method for detecting and/or quantifying a biological or chemical substance of interest in a capillary action test device as claimed in claim 19.

49. A method for detecting and/or quantifying a substance of interest in an agricultural or food product or in the environment, said method implementing an in vitro method for detecting and/or quantifying a biological or chemical substance of interest in a liquid sample as claimed in claim 1.

50. A method for detecting and/or quantifying a substance of interest in an agricultural or food product or in the environment, said method implementing an in vitro method for detecting and/or quantifying a biological or chemical substance of interest in a capillary action test device as claimed in claim 19.

51. A method for detecting and/or quantifying an illegal chemical substance or any other substance of interest for the police or defense, said method implementing an in vitro method for detecting and/or quantifying a biological or chemical substance of interest in a liquid sample as claimed in claim 1.

52. A method for detecting and/or quantifying an illegal chemical substance or any other substance of interest for the police or defense, said method implementing an in vitro method for detecting and/or quantifying a biological or chemical substance of interest in a capillary action test device as claimed in claim 19.

Description

FIGURES

[0365] FIG. 1: Schematic representation, in cross-sectional view, of a strip for a lateral flow assay;

[0366] FIG. 2: Schematic representation of an assay of the “sandwich” type, before application of a liquid sample to be analyzed comprising the analyte (11) at the level of the deposition zone (1) (top figure) and at the end of the assay (bottom figure);

[0367] FIG. 3: Schematic representation of the procedure of a “competitive” assay according to two variants, before application of a liquid sample to be analyzed comprising the analyte (11) at the level of the deposition zone (1) (top figure) and at the end of the assay (bottom figure);

[0368] FIG. 4: Images obtained by transmission electron microscopy (TEM) of the nanoparticles obtained according to example 1.1.a. (Scale bar: 60 nm (FIG. 4a) and 5 nm (FIG. 4b), respectively);

[0369] FIG. 5: Histogram of nanoparticle size determined from TEM images for a set of about 300 nanoparticles according to example 1.1.a.

[0370] FIG. 6: Photographs of strips, according to the assay in example 3, after migration of a solution containing the h-FABP antigen at 5, 0.5, 0.05 ng/mL, illuminated by a UV lamp. The detection band can be seen on the left, and the control band on the right. The absorbent pad can be seen at the right-hand edge of the images. The luminescence signals shown in the photographs were analyzed by ImageJ. The results are shown in FIG. 7.

[0371] FIG. 7: Results for the ratio R=S.sub.D/S.sub.D+S.sub.C measured by ImageJ for liquid samples containing 5, 0.5, 0.05 and 0 ng/mL of h-FABP tested with the test strips according to example 3. The points represent the mean value of R and the error bars represent the associated standard deviation for the 3 and 2 strips, respectively;

[0372] FIG. 8: Schematic representation in top view of a case comprising a strip for a lateral flow assay;

[0373] FIG. 9: Scheme of the strip reader using four groups of four UV LEDs (“LEDs #1” to “LEDs #4”) for excitation of the nanoparticles. The strip may be inserted in the reader at the level of the insertion rail (20). Reading takes place through the opening in the cover, in which a filter is positioned which makes it possible to select the emission of the nanoparticles (centered at 617 nm in the case in the example) and to reject the excitation wavelength (centered at 280 nm in the case in the example). It may be an interference filter or a high-pass filter. A camera, for example the CCD or CMOS camera of a cellphone, is positioned in front of this opening for recording an image.

[0374] FIG. 10: Illustration of the analysis of the result for a strip using a dedicated application operating under Android (Samsung). Left: black and white image of the strip with the rectangles, inside which the cumulative levels of luminescence are calculated, from top to bottom, for the detection zone, the zone of the background signal and the control zone. Right: Screenshot of the cellphone on which the Android analysis application is running. We can see the black and white image of the strip with the rectangles, inside which calculation of the cumulative level of luminescence is performed, and the functions “Capture”, “Measure”, “Adjust” and “Save”.

[0375] FIG. 11: (A) Absorbance spectrum of a solution of Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles synthesized according to the example. (B) Emission spectrum of a nitrocellulose membrane glued on a backing card, as used for the lateral flow assays of the example, inserted in a quartz cuvette, excited at 280, 300 and 380 nm (width of the excitation slit: 5 nm). The emission is far more intense after excitation at 380 nm over the whole spectrum and more particularly at 617 nm, the wavelength at which the signal from the probes based on Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles is detected.

[0376] FIG. 12: Excitation spectrum of the Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles (left-hand part of the figure) with the emission wavelength fixed at 617 nm and emission spectrum (right-hand part of the figure) with the excitation wavelength fixed at 278 nm.

[0377] FIG. 13: Excitation spectrum of the YVO.sub.4:Dy 5% nanoparticles (FIG. 13-a) with the emission wavelength fixed at 572 nm and emission spectrum (FIG. 13-b) with the excitation wavelength fixed at 278 nm.

[0378] FIG. 14: Excitation spectrum of the YVO.sub.4:Sm 3% nanoparticles (FIG. 14-a) with the emission wavelength fixed at 600 nm and emission spectrum (FIG. 14-b) with the excitation wavelength fixed at 278 nm.

[0379] FIG. 15: Excitation spectra of the Y.sub.0.6Eu.sub.0.4VO.sub.4, Lu.sub.0.6Eu.sub.0.4VO.sub.4, LuVO.sub.4:Dy 10%, La.sub.0.6Eu.sub.0.4VO.sub.4 and GdVO.sub.4:Dy 20% nanoparticles, for an emission wavelength fixed at 617 nm for the nanoparticles containing Eu.sup.3+ ions and at 573 nm for the nanoparticles containing Dy.sup.3+ ions.

[0380] FIG. 16: Emission spectrum of the Lu.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles for an excitation wavelength at 278 nm (excitation of the LuVO.sub.4 matrix). The emission has a main peak at 617 nm and two other peaks at 593 and 700 nm.

[0381] FIG. 17: Emission spectrum of the LuVO.sub.4:Dy 10% nanoparticles for an excitation wavelength at 278 nm (excitation of the LuVO.sub.4 matrix). The emission has two main peaks at 483 and 573 nm.

[0382] FIG. 18: Emission spectrum of the La.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles for an excitation wavelength at 278 nm (excitation of the LaVO.sub.4 matrix). The emission has a main peak at 617 and two other peaks at 593 and 700 nm.

[0383] FIG. 19: Emission spectrum of the GdVO.sub.4:Dy 20% nanoparticles for an excitation wavelength at 278 nm (excitation of the GdVO.sub.4 matrix). The emission has two main peaks at 483 and 573 nm.

[0384] FIG. 20: Absorbance spectra of the Y(VO.sub.4).sub.1-y(PO.sub.4).sub.y:Eu 20% nanoparticles for y=0, y=0.05, y=0.2, y=0.5 and y=1. The initial concentrations before dilution are of the order of 50 mM of vanadate ions.

[0385] FIG. 21: Emission spectra of the Y(VO.sub.4).sub.1-y(PO.sub.4).sub.y:Eu 20% nanoparticles for y=0, y=0.05, y=0.2, y=0.5 and y=1 for an excitation wavelength fixed at 278 nm. The initial concentrations before dilution are of the order of 50 mM of vanadate ions.

[0386] FIG. 22: Migration of the Lu.sub.0.6Eu.sub.0.4VO.sub.4—SA and Lu.sub.0.9Dy.sub.0.1VO.sub.4—SA nanoparticles on “dipstick” strips containing BSA-Biotin immobilized on the control line, in the absence of antigen. The strips are observed under illumination with a UV lamp at 312 nm. Emission detected through an interference filter (Semrock FF01-620/14-25 and FF03-575/25 for the emission of the Eu.sup.3+ and Dy.sup.3+ ions, respectively); image taken with an Iphone 6 smartphone. Two clear bands are observed on the control line. The emission of the nanoparticles that have migrated as far as the absorbent pad can be seen on the right-hand side of the image.

EXAMPLE

[0387] 1. Preparation of the Photoluminescence Probes

[0388] 1.1. Synthesis of Photoluminescent Inorganic Nanoparticles

[0389] 1.1.a Synthesis of Y.sub.0.6Eu.sub.0.4VO.sub.4 Nanoparticles

[0390] Ammonium metavanadate NH.sub.4VO.sub.3 is used as the source of metavanadate ions VO.sub.3.sup.−, the orthovanadate VO.sub.4.sup.3− being obtained in situ following reaction with a base, in this case tetramethylammonium hydroxide, N(CH.sub.3).sub.4OH. Yttrium nitrate and europium nitrate were used as sources of Y.sup.3+ and Eu.sup.3+ ions.

[0391] An aqueous solution of 10 mL of NH.sub.4VO.sub.3 at 0.1 M and 0.2 M of N(CH.sub.3).sub.4OH (solution 1) is freshly prepared.

[0392] A volume of 10 mL of another solution (solution 2) of Y(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 at 0.1 M of ions (Y.sup.3++Eu.sup.3+) is added dropwise by syringe pump to solution 1 at a flow of 1 mL/min.

[0393] The molar concentration ratio of Y(NO.sub.3).sub.3 to Eu(NO.sub.3).sub.3 is selected as a function of the desired ratio of the Y.sup.3+ and Eu.sup.3+ ions in the nanoparticle, typically the molar ratio Y.sup.3+:Eu.sup.3+ is 0.6:0.4.

[0394] Once the Y(NO.sub.3).sub.3/Eu(NO.sub.3).sub.3 solution has been added, the solution becomes diffusive and appears white/milky without formation of precipitate. The synthesis continues until all of the Y(NO.sub.3).sub.3/Eu(NO.sub.3).sub.3 solution has been added.

[0395] The final solution of 20 mL must now be purified to remove the excess counterions. For this purpose, centrifugations (typically three) at 11000 g (Sigma 3K10, Bioblock Scientific) for 80 minutes, each followed by redispersion by sonication (Bioblock Scientific, Ultrasonic Processor, maximum power of 130 W operating at 50% for 40 s) are used until a conductivity strictly below 100 μS.Math.cm.sup.−1 is reached. The conductivity is measured using a chemical conductivity meter.

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

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

[0397] Visual observation of the solution of nanoparticles, after being left to stand for 16 hours in a bottle, shows a uniformly diffusing solution.

[0398] The final solution remains very stable in water, even after several months at the final pH of the synthesis (about pH 5). The solution remains stable including in the synthesis medium (before removing the excess counterions), although of high ionic strength (>0.1 M).

[0399] After removal of the counterions, the zeta potential of the nanoparticles, determined with a DLS-Zeta Potential apparatus (Zetasizer Nano ZS90, Malvern), is −38.4 mV at pH 7.

[0400] Observation of the nanoparticles by TEM (FIG. 4) shows that the nanoparticles are of elongated ellipsoidal shape. The dimensions of the nanoparticles are determined from TEM images for a set of about 300 nanoparticles (FIG. 5). The nanoparticles of the invention have a length of the major axis, designated a, between 20 and 60 nm, with an average value of about 40 nm, and a length of the minor axis, designated b, between 10 and 30 nm, with an average value of about 20 nm.

[0401] The excitation and emission spectrum of the Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles is shown in FIG. 12. The excitation spectrum has a peak at 278 nm and the emission spectrum has a main peak at 617 nm and two peaks at 593 and 700 nm.

[0402] The Eu.sup.3+ ions in the YVO.sub.4 matrix can be replaced with other luminescent lanthanide ions. In this case, the excitation and absorption spectrum around the absorption peak of the VO.sub.4.sup.3− vanadate ions associated with a V-O charge transfer transition remains unchanged.

[0403] The emission spectrum is typical of the emission spectrum of each lanthanide ion.

[0404] 1.1.b Synthesis of Y.sub.0.95Dy.sub.0.05 VO.sub.4 (YVO.sub.4:Dy 5%) Nanoparticles

[0405] The synthesis is identical to that in example 1.1.a., apart from solution 2, which consists of Y(NO.sub.3).sub.3 and Dy(NO.sub.3).sub.3 at 0.1 M of ions (Y.sup.3++Dy.sup.3+). Solution 2 is added dropwise by syringe pump to solution 1 at a flow of 1 m/min.

[0406] The molar concentration ratio of Y(NO.sub.3).sub.3 to Dy(NO.sub.3).sub.3 is selected as a function of the desired ratio of the Y.sup.3+ and Dy.sup.3+ ions in the nanoparticle, in this case the molar ratio Y.sup.3+:Dy.sup.3+ is 0.95:0.05.

[0407] The excitation and emission spectra of these nanoparticles are shown in FIG. 13. The emission of the Dy.sup.3+ ions has two main peaks at 483 and 573 nm.

[0408] 1.1.c Synthesis of Y.sub.0.97Sm.sub.0.03VO.sub.4 (YVO.sub.4:Sm 3%) Nanoparticles

[0409] The synthesis is identical to that in example 1.1.a, apart from solution 2, which consists of Y(NO.sub.3).sub.3 and Sm(NO.sub.3).sub.3 at 0.1 M of ions (Y.sup.3++Sm.sup.3+). Solution 2 is added dropwise by syringe pump to solution 1 at a flow of 1 m/min.

[0410] The molar concentration ratio of Y(NO.sub.3).sub.3 to Sm(NO.sub.3).sub.3 is selected as a function of the desired ratio of the Y.sup.3+ and Sm.sup.3+ ions in the nanoparticle, in this case the molar ratio Y.sup.3+:Sm.sup.3+ is 0.97:0.03.

[0411] The excitation and emission spectra of these nanoparticles are shown in FIG. 14.

[0412] Moreover, the Y.sup.3+ ions of the YVO.sub.4 matrix can be replaced with other ions such as Gd.sup.3+, Lu.sup.3+ and La.sup.3+ (see next examples). For all these matrixes GdVO.sub.4, LuVO.sub.4 and LaVO.sub.4, the excitation and absorption spectrum around the absorption peak of the VO.sub.4.sup.3− vanadate ions associated with a V.sup.5+—O.sub.2.sup.− charge transfer transition remains unchanged relative to the YVO.sub.4 matrix. Moreover, in these matrixes GdVO.sub.4, LuVO.sub.4 and LaVO.sub.4, the Eu.sup.3+ ions can be replaced with other luminescent lanthanide ions. The emission spectrum is typical of the emission spectrum of each lanthanide ion. Different representative combinations of the matrixes and luminescent lanthanide ions are presented hereunder.

[0413] 1.1.d Synthesis of Lu.sub.0.6Eu.sub.0.4VO.sub.4 Nanoparticles

[0414] The synthesis is identical to that in example 1.1.a, apart from solution 2, which consists of Lu(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 at 0.1 M of ions (Lu.sup.3++Eu.sup.3+). Solution 2 is added dropwise by syringe pump to solution 1 at a flow of 1 m/min.

[0415] The molar concentration ratio of Lu(NO.sub.3).sub.3 to Eu(NO.sub.3).sub.3 is selected as a function of the desired ratio of the Lu.sup.3+ and Eu.sup.3+ ions in the nanoparticle, in this case the molar ratio Lu.sup.3+:Eu.sup.3+ is 0.6:0.4.

[0416] The excitation spectrum of the Lu.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles is shown in FIG. 15 and the emission spectrum is presented in FIG. 16. The emission spectrum of the Eu.sup.3+ ions in the LuVO.sub.4 matrix is practically unchanged relative to that in the YVO.sub.4 matrix (FIG. 12) and has a main peak at 617 nm and two peaks at 593 and 700 nm.

[0417] 1.1.e Synthesis of LuVO.sub.4:Dy 10% Nanoparticles

[0418] The synthesis is identical to that in example 1.1.a, apart from solution 2, which consists of Lu(N03).sub.3 and Dy(N03).sub.3 at 0.1 M of ions (Lu.sup.3++Dy.sup.3+). Solution 2 is added dropwise by syringe pump to solution 1 at a flow of 1 m/min.

[0419] The molar concentration ratio of Lu(NO.sub.3).sub.3 to Dy(NO.sub.3).sub.3 is selected as a function of the desired ratio of the Lu.sup.3+ and Dy.sup.3+ ions in the nanoparticle, in this case the molar ratio Lu.sup.3+ Dy.sup.3+ is 0.9:0.1.

[0420] The excitation spectrum of the LuVO.sub.4:Dy 10% nanoparticles is shown in FIG. 16 and the emission spectrum is presented in FIG. 17. The emission spectrum of the Dy.sup.3+ ions in the LuVO.sub.4 matrix is practically unchanged relative to that in the YVO.sub.4 matrix (FIG. 13) and has two emission peaks at 483 and 573 nm.

[0421] 1.1.f Synthesis of La.sub.0.6Eu.sub.0.4VO.sub.4 Nanoparticles

[0422] The synthesis is identical to that in example 1.1.a, apart from solution 2, which consists of La(NO.sub.3).sub.3 and Eu(NO.sub.3).sub.3 at 0.1 M of ions (La.sup.3++Eu.sup.3+). Solution 2 is added dropwise by syringe pump to solution 1 at a flow of 1 m/min.

[0423] The molar concentration ratio of La(NO.sub.3).sub.3 to Eu(NO.sub.3).sub.3 is selected as a function of the desired ratio of the La.sup.3+ and Eu.sup.3+ ions in the nanoparticle, in this case the molar ratio La.sup.3+:Eu.sup.3+ is 0.6:0.4.

[0424] The excitation spectrum of the La.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles is shown in FIG. 15 and the emission spectrum is presented in FIG. 18. The emission spectrum of the Eu.sup.3+ ions in the LaVO.sub.4 matrix is practically unchanged relative to that in the YVO.sub.4 matrix (FIG. 12) and has a main peak at 617 nm and two peaks at 593 and 700 nm.

[0425] 1.1.g Synthesis of GdVO.sub.4:Dy 20% Nanoparticles

[0426] The synthesis of these nanoparticles is carried out starting from an orthovanadate precursor as follows. An aqueous solution of 10 mL of NaVO.sub.4 at 0.1 M (solution 1) is freshly prepared and its pH is adjusted to between 12.6 and 13 with 1 M NaOH solution.

[0427] A volume of 10 mL of another solution (solution 2) of Gd(NO.sub.3).sub.3 and of Dy(NO.sub.3).sub.3 at 0.1 M of ions (Gd.sup.3++Dy.sup.3+) is added dropwise by syringe pump to solution 1 with stirring, at a flow of 1 mL/min.

[0428] The molar concentration ratio of Gd(NO.sub.3).sub.3 to Dy(NO.sub.3).sub.3 is selected as a function of the desired ratio of the Gd.sup.3+ and Dy.sup.3+ ions in the nanoparticle, in this case the molar ratio Gd.sup.3+:Dy.sup.3+ is 0.8:0.2.

[0429] Once the Gd(NO.sub.3).sub.3/Dy(NO.sub.3).sub.3 solution is added, a milky precipitate forms. The synthesis continues until all of the Y(NO.sub.3).sub.3/Eu(NO.sub.3).sub.3 solution has been added. The solution is stirred for 30 min until the pH stabilizes at 8-9.

[0430] The final solution of 20 mL must be purified as in example 1.1.a to remove the excess counterions. For this purpose, centrifugations (typically three) at 11000 g (Sigma 3K10, Bioblock Scientific) for 15 minutes, each followed by redispersion by sonication (Bioblock Scientific, Ultrasonic Processor with a maximum power of 130 W operating at 50% for 40 s) are used until a conductivity strictly below 100 μS.Math.cm.sup.−1 is reached.

[0431] The excitation spectrum of the GdVO.sub.4:Dy 20% nanoparticles is shown in FIG. 15 and the emission spectrum is presented in FIG. 19. The emission spectrum of the Dy.sup.3+ ions in the GdVO.sub.4 matrix is practically unchanged relative to that in the YVO.sub.4 matrix (FIG. 13) and has two emission peaks at 483 and 573 nm.

[0432] 1.1.h Synthesis of Y(VO.sub.4).sub.1-v(PO.sub.4).sub.v:Eu 20% Nanoparticles

[0433] Nanoparticles containing a mixture of VO.sub.4.sup.3− and PO.sub.4.sup.3− ions in the matrix at different VO.sub.4.sup.3−:PO.sub.4.sup.3− ratios were also synthesized.

[0434] The synthesis is identical to that in example 1.1.a apart from solution 1, which consists of 0.1.Math.y M of Na.sub.3PO.sub.4, 0.1.Math.(1−y) M NH.sub.4VO.sub.3 at a total concentration of 0.1 M of ions (VO.sub.3.sup.−+PO.sub.4.sup.3−) and 0.2.Math.(1−y) M of N(CH.sub.3).sub.4OH. An aqueous solution of 10 mL with the above concentrations (solution 1) is freshly prepared. NPs with y=0, y=0.05, y=0.2, y=0.5 and y=1 were prepared.

[0435] The PO.sub.4.sup.3− ions do not display absorption at 278 nm. Thus, the nanoparticles containing 100% of PO.sub.4.sup.3− ions do not have an absorption peak at 278 nm (see FIG. 20). The emission spectra of these nanoparticles are presented in FIG. 21 and are identical for all the values of y different from 1 (no emission observed for y=1). They have a main peak at 617 and two additional peaks at 593 and 700 nm. These emission spectra are practically identical.

[0436] 1.2. Covalent Coupling of the Nanoparticles to Proteins (Anti-h-FABP Antibodies)

[0437] The Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles, obtained as described at point 1.1.a., are coupled to antibodies according to the following protocol.

[0438] 1.2.1. Coating the Nanoparticles with a Layer of Silica

[0439] At the end of synthesis of the nanoparticles, the solution of nanoparticles is centrifuged at 17 000 g for 3 minutes, to precipitate any aggregates of nanoparticles, and the supernatant is recovered. Selection by size is carried out. For this purpose, several centrifugations are carried out at 1900 g for 3 min. Each centrifugation is followed by redispersion of the nanoparticles with the sonicator, and then the size of the nanoparticles is determined using DLS-Zeta Potential apparatus (Zetasizer Nano ZS90, Malvern).

[0440] A volume of 25 mL of Y.sub.0.6Eu.sub.0.4VO.sub.4 particles with a concentration of 20 mM of vanadate ions is prepared. A volume of 2.5 mL of another solution of pure sodium silicate (Merck Millipore 1.05621.2500) is added dropwise by pipette in order to coat the surface of the particles. This solution is left to act with stirring for at least five hours.

[0441] The solution is then purified in order to remove the excess silicate and the sodium counterions. The solution is centrifuged at 11000 g (Sigma 3K10, Bioblock Scientific) for 60 minutes and then redispersed by sonication (Bioblock Scientific, Ultrasonic Processor, operating at 50% at a power of 400 W). This step is repeated until the conductivity of the solution is below 100 μS/cm.

[0442] 1.2.2. Grafting of Amines on the Surface of the Nanoparticles

[0443] Put 225 mL of absolute ethanol in a 500-mL flask of the three-necked type, and add 265 μL of APTES (3-aminopropyltriethoxysilane) (Mw 221.37 g/mol Sigma Aldrich), which corresponds to a final concentration of 1.125 mM. This quantity corresponds to 5 equivalents of vanadate that are introduced. A condenser is then attached to the flask. The whole is placed on a flask heater and put under a hood. The mixture is heated under reflux at 90° C. On one of the inlets of the three-necked flask, a colloidal solution of nanoparticles (concentration of vanadate ions [V]=3 mM) in 75 mL of water at pH 9 is added dropwise using a peristaltic pump with a flow rate of 1 mL/min. The whole is heated with stirring for 24 h.

[0444] After 48 hours, a rotary evaporator (rotavapor R-100, BUCHI) is used for partially concentrating the nanoparticles. The solution is rotated in a suitable flask, and heated in a bath at 50° C.

[0445] The solution recovered is purified by several centrifugations in ethanol:water (3:1) solvent. After purification, sorting by size is carried out following the protocol described above.

[0446] 1.2.3. Grafting of Carboxyls on the Surface of the Aminated Nanoparticles

[0447] Solvent transfer is carried out before beginning the grafting.

[0448] The grafting protocol is as follows.

[0449] Transfer the aminated NPs from the EtOH:H.sub.2O buffer to DMF or DMSO, performing several centrifugations (13000 g, 90 min). The pellet is redispersed by sonication between each centrifugation (20 s at 75%). Measure and determine the concentration of the NPs.

[0450] Recover the NPS in 5 mL of DMF and then add 10% of succinic acid anhydride to a glass beaker (i.e. 0.5 g in 5 mL). Leave to react at least overnight under an inert atmosphere, with stirring.

[0451] Wash the carboxylated NPs at least twice by centrifugations (13000 g for 60 min, Legend Micro 17r, Thermo Scientific) in order to remove the DMF and the excess succinic acid anhydride.

[0452] Resuspend the carboxylated particles in water or MES buffer at pH6 by sonication (Bioblock Scientific, Ultrasonic processor).

[0453] 1.2.4. Coupling the Nanoparticles to the Anti-h-FABP Antibodies

[0454] Coupling of the nanoparticles surface-grafted with COOH is carried out according to the following protocol: [0455] 1. Freshly prepare a mixed solution of EDC/Sulfo-NHS (concentration 500 and 500 mg/mL, respectively) in MES buffer (pH 5-6). [0456] 2. Add 90 nM of NPs (in this case it is the concentration of nanoparticles calculated from the concentration of vanadate ions according to the reference Casanova et al. [37]) to 3 mL of solution prepared beforehand, and leave to react for 25 min at room temperature, with stirring. [0457] 3. Wash the NPs quickly by at least 2 centrifugations (13000 g for 60 min, Legend Micro 17R, Thermo Scientific) with MilliQ water to remove the excess reagents. [0458] 4. Recover the last pellet after sonication in sodium phosphate buffer at pH 7.3. Add the required amount of protein (anti h-FABP Antibody, Ref 4F29, 10 E1, Hytest) as a function of the required ratio (Protein:Nps), typically 2 μM for a ratio of 20:1, and 5 mg/mL of mPEG-silane (MW: 10 kD, Laysan Bio 256-586-9004). [0459] 5. Leave this solution to react for between 2 and 4 h at room temperature, with stirring. [0460] 6. Add the blocking agent (glycine at 1%) so that it reacts with the free COOH and blocks the residual reaction sites on the surface of the NPs. Leave to react for 30 min. [0461] 7. Wash the NPs coupled to the proteins several times by centrifugation using centrifuging filters (Amicon Ultra 0.5 mL, Ref UFC501096, Millipore) with PBS pH 7.2. Transfer the NPs to their storage medium: phosphate buffer+Tween 20 (0.05%)+0.1% glycin+10% glycerol. Take 100 μL for the BCA assays. The rest of the solution is divided into aliquots and frozen at −80° C.

[0462] Moreover, all of the nanoparticles synthesized according to examples 1.1.b to 1.1.h can be coupled to antibodies, in the same way as for the Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles.

[0463] 1.3. Passive Coupling of the Nanoparticles to Proteins (Anti-h-FABP Antibodies)

[0464] Passive coupling of the nanoparticles to antibodies, instead of the covalent coupling in example 1.2, can also be carried out as follows. [0465] Centrifuge a solution of 1 mL of nanoparticles (concentration of 5 mM of vanadate ions) for 15 min at 15 000 g. [0466] Take up the pellet in 800 μL of MilliQ water and then redisperse by sonication (Bioblock Scientific, Ultrasonic Processor, maximum power of 130 W operating at 50% for 40 s). [0467] Add 100 μL of a solution of antibodies at 250 μg/mL in potassium phosphate buffer 2 mM pH 7.4. [0468] Incubate while rotating for 1 hour. [0469] Add 100 μL of potassium phosphate buffer 20 mM pH 7.4/1% BSA. [0470] Centrifuge for 15 min at 15 000 g and remove the supernatant. [0471] Take up the pellet in 1 mL of potassium phosphate buffer 2 mM pH 7.4/0.1% BSA. Redisperse by sonication (Bioblock Scientific, Ultrasonic Processor, maximum power of 130 W operating at 50% for 40 s). [0472] Centrifuge for 15 min at 15 000 g and remove the supernatant. [0473] Take up the pellet in 250 μL of potassium phosphate buffer 2 mM pH 7.4/0.1% BSA. Redisperse by sonication (Bioblock Scientific, Ultrasonic Processor, maximum power of 130 W operating at 50% for 40 s).

[0474] 2. Preparing the Test on Strips of the “Sandwich” Type

[0475] To develop rapid tests for determining the presence of a protein qualitatively or quantitatively, it is necessary to optimize the various parameters and find compromises between the reaction time and the sensitivity of the test.

[0476] Manufacture of the assay strip, as shown in FIG. 1, is carried out by combining four essential parts: [0477] Essentially inert glass fiber is used as the labeling zone (2) (“Conjugate Pad” in English-language terminology) (GFDX 103000, Millipore). [0478] As deposition zone (“Sample Pad” in English-language terminology) (1) (Ref CFSP173000, Millipore), surface-modified polyesters are used. They have the advantage that they have weak nonspecific interactions with proteins, an excellent traction force as well as good handling properties. [0479] The nitrocellulose membrane (NC) (HF180MC100, Millipore) is used as the means for capillary action (10). It possesses optimal properties for migration of fluids and for immobilization of proteins. The NC membrane is glued on a support (6) of nonporous adhesive plastic (“backing card”). [0480] Cellulose (CFSP173000, Millipore) is used as absorbent pad (5) for its high absorbent capacity.

[0481] For detecting h-FABP (human fatty-acid binding protein), which is a cardiac biomarker:

[0482] Before assembling the various components, it is necessary to deposit the antibodies on the NC membrane.

[0483] 1. A solution of mouse monoclonal antibodies directed against h-FABP (ref. 4F29, 9F3, Hytest) is diluted in PBS (pH 7.4) at a concentration of 1 mg/mL. This solution will be used for the test band (3). Another solution of goat polyclonal IgG antibodies (Ref ab6708, Abcam) directed against the mouse antibodies is diluted in PBS (pH7.4) at a concentration of 1 mg/mL. The latter is used for the control band (4).

[0484] 2. The antibody solutions are deposited on the NC membrane using a “dispenser” (Claremont Bio Automated Lateral Flow Reagent Dispenser (ALFRD)). Using a syringe pump, a volume of 0.7 μL/2 mm is deposited for each band all the way along the NC membrane (about 30 cm long, from which several strips will be made). Leave to dry for 1 h at 37° C.

[0485] 3. After depositing the antibodies, incubate the NC membrane with 1% BSA diluted in PBS (pH 7.4)+Tween 20 at 0.04% for 30 min at 37° C. to passivate the fixation sites not occupied by antibodies.

[0486] 4. Deposit the Abs coupled to the NPs, on the labeling zone (“conjugate pad”), using the dispenser. A volume of 3 μL/4 mm is applied all the way along the glass fiber membrane. Leave to dry for 1 h at room temperature before blocking with BSA 1% diluted in PBS (pH7.4). Leave to dry at room temperature.

[0487] Assembling the Strip

[0488] 1. Assemble the various structures (cellulose which serves as absorbent pad and deposition zone, the labeling zone on which Ab+NPs is deposited) on the adhesive parts of the NC on which the Abs are already immobilized. The components are fixed on the plastic support of the NC in the following order: labeling zone (“conjugate pad”), deposition zone (“sample pad”) and finally absorbent pad. For better migration of the fluid by capillarity, the various components are mounted so as to overlap one another as shown in the illustration (FIG. 1).

[0489] 2. Cut the assembled membrane using a paper cutter into separate pieces 4 mm wide.

[0490] 3. The strips are then stored in aluminum bags in the presence of a moisture absorber (desiccant) in an atmosphere with a humidity below 30%.

[0491] 3. Strip Test Procedure

[0492] The strip is prepared using Y.sub.0.6Eu.sub.0.4VO.sub.4 nanoparticles coupled to the antibodies prepared in example 1.2.

[0493] Several concentrations of h-FABP (Ref. 8F65, Hytest) were measured from 5 ng/mL to 0.05 ng/mL. The recombinant h-FABP is diluted to the desired concentrations with buffer or with serum. [0494] 1. Bring the strip prepared as described in point 2 above, and the samples to be assayed, back to room temperature before carrying out the test. [0495] 2. Deposit 400 μL of the sample in a bottle in the vertical position. [0496] 3. Immerse the strip in the bottle, orienting the deposition zone (“sample pad”) downwards. Tap the strip on the bottom to start migration. Keep the strip in the vertical position in the tube for 10 min. [0497] 4. Read the results of the strips using a UV lamp (Vilber Lourmat, VL-8.MC 8W at 312 nm and 8 W at 254 nm) (taking digital photographs and analysis by ImageJ, see FIGS. 6 and 7) or using the reader presented in FIGS. 8 and 9. FIG. 10 illustrates analysis of the result for a strip using a dedicated application on a cellphone operating under Android. The reader uses 4 groups of 4 LEDs at 278 nm and an interference filter (620/15, Semrock) for detection. A high-pass filter such as an RG605 filter (Schott) may also be used for detection.

[0498] The absorption spectrum of the nanoparticles is presented in FIG. 11 (A). The absorption peak is located at 280 nm with a full width at half maximum of about 50 nm. The emission spectrum of the UV lamp is centered at 310 nm with a full width at half maximum of 40 nm. The emission spectrum of the UV LED is centered at 278 nm with a full width at half maximum of 10 nm.

[0499] Qualitative Interpretation of the Results

[0500] Qualitative interpretation of the results is as follows: [0501] if two bands are present: the test is positive [0502] if the single control band is present: the test is negative [0503] if the single test band is present: the test is invalid [0504] if no band is present: the test is invalid.

[0505] Quantitative Interpretation of the Results

[0506] FIG. 10 shows an example of quantitative analysis starting from a digital photograph taken with a cellphone, using a dedicated application. On resting on “Capture” on the phone's screen, recording of a black and white image is triggered. Then, after resting on “Measure”, the application asks the user to point with a finger on the phone's screen to the detection zone and then the control zone so that the application calculates the cumulative luminescence level inside a rectangle containing the detection zone, L.sub.D, and the cumulative luminescence level inside a rectangle of the same size containing the control zone, L.sub.C. Resting on “Adjust” triggers optimization of the position of the two rectangles corresponding to the detection zone and the control zone so as to maximize the measured signal. The cumulative emission level inside a rectangle of the same size located in the middle of the space between the detection zone and the control zone serves for determining the background signal, L.sub.B, and for calculating the signals S.sub.D/C=L.sub.D/C−L.sub.B. The application calculates and then displays the ratio R=S.sub.D/S.sub.C or R=S.sub.D/(S.sub.D+S.sub.C). The value of R can be compared against a calibration table for also supplying a concentration value in ng/mL. The result can be saved with the “Save” function for later comparison with the next results.

[0507] Three strips were prepared for the strip test of each of the samples comprising the h-FABP antigen. For the case of the sample not containing the antigen, only two strips were prepared.

[0508] The graph in FIG. 7 shows the results obtained for the ratio R=S.sub.D/(S.sub.D+S.sub.C), measured by ImageJ for the different liquid samples containing 5, 0.5, 0.05 and 0 ng/mL of h-FABP. The points in FIG. 7 represent the mean values of R, and the error bars represent the associated standard deviation for the different strips tested (three strips in the case of the samples containing h-FABP; two strips in the case of the sample not containing it).

[0509] Thus, the method according to the invention advantageously allows h-FABP to be detected in a sample, at a content less than or equal to 5 ng/mL, in particular less than or equal to 0.5 ng/mL, or even down to a value as low as 0.05 ng/mL. In other words, h-FABP can be detected at a content less than or equal to 330 pM, in particular less than or equal to 33 pM, or even down to a content as low as 3.3 pM.

[0510] 4. Migration of the Lu.sub.0.6Eu.sub.0.4VO.sub.4-SA and Lu.sub.0.9Dy.sub.0.1VO.sub.4-SA Nanoparticles on “Dipstick” Strips of the “Sandwich” Type

[0511] Lu.sub.0.6Eu.sub.0.4VO.sub.4 and Lu.sub.0.9Dy.sub.0.1VO.sub.4 nanoparticles synthesized according to examples 1.1.d and 1.1.e, respectively, were coupled to streptavidin (SA) according to example 1.3 (passive coupling). “Dipstick” strips were prepared according to example 2 by immobilizing BSA-Biotin on the nitrocellulose membrane for recognizing the NPs coupled to streptavidin. Test strips were made with the Lu.sub.0.6Eu.sub.0.4VO.sub.4—SA and Lu.sub.0.9Dy.sub.0.1VO.sub.4—SA nanoparticles according to example 3, in the absence of antigen. The strips were visualized under UV lamp excitation (312 nm). Two clear, intense bands formed at the level of the control line (FIG. 22).

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