阅读说明：本技术 使用光致发光无机纳米颗粒的毛细作用测试 (Capillary action test using photoluminescent inorganic nanoparticles ) 是由 帕斯卡尔·普瑞拉 马克西米利安·里奇伊 塞德里克·布兹格 安蒂戈尼·亚历桑德罗 蒂埃里·加库 于 2019-07-17 设计创作，主要内容包括：本发明涉及一种用于通过毛细作用测试来检测和/或定量液体样品中感兴趣的生物或化学物质的体外方法,所述毛细作用测试使用式A-(1-)-xLn-xVO-(4(1-y))(PO-4)-y(II)的光致发光无机纳米颗粒作为探针,其中Ln选自铕(Eu)、镝(Dy)、钐(Sm)、钕(Nd)、铒(Er)、镱(Yb)、铥(Tm)、镨(Pr)、钬(Ho)及其混合物；A选自钇(Y)、钆(Gd)、镧(La)、镥(Lu)及其混合物；0<x<1；且0≤y<1,所述方法进行通过在小于或等于320nm的波长下激发基质,在单光子吸收之后的所述纳米颗粒的发光检测,所述纳米颗粒具有的发射寿命短于100ms。本发明还涉及一种包括上述纳米颗粒作为探针的毛细作用测试以及这样的方法用于体外诊断目的的用途。(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 means of a capillary action test using formula A 1‑ x Ln x VO 4(1‑y) (PO 4 ) y (II) as a probe,wherein 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 y is not less than 0<The method performs luminescence detection of the nanoparticles after single photon absorption by excitation of a matrix at a wavelength less than or equal to 320nm, the nanoparticles having an emission lifetime shorter than 100 ms. The invention also relates to a capillary test comprising the above-described nanoparticles as probes and to the use of such a method for in vitro diagnostic purposes.)

1. 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 probe photoluminescent inorganic nanoparticles of formula (II):

A_1-xLn_xVO_4(1-y)(PO₄)_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), praseodymium (Pr), holmium (Ho) and mixtures thereof;

0< x <1, in particular 0.2. ltoreq. x.ltoreq.0.6, and more in particular x has a value of 0.4; and is

Y <1, in particular y has a value of 0;

the method employs luminescence detection of the nanoparticles after single photon absorption by excitation of the matrix at a wavelength less than or equal to 320nm, the nanoparticles having an emission lifetime shorter than 100 ms.

2. Method according to the preceding claim, wherein said luminescence detection is achieved by exciting the matrix at a wavelength comprised less than or equal to 300nm, in particular between 250nm and 300 nm.

3. The method according to any one of the preceding claims, wherein the liquid sample is a biological sample, in particular a sample taken from a human, and more particularly a sample selected from: blood, serum, plasma, saliva, urine, nasal smear, vaginal smear, expectorate, fecal waste and cerebrospinal fluid.

4. The method according to any one of the preceding claims, for detecting and/or quantifying molecules, proteins, nucleic acids, toxins, viruses, bacteria or parasites in a sample, in particular a biological sample.

5. The method according to any one of the preceding claims, wherein the photoluminescent nanoparticles have an average size greater than or equal to 5nm and strictly less than 1 μ ι η, in particular between 10nm and 500nm, and preferably between 20nm and 200 nm.

6. Method according to any one of the preceding claims, wherein Ln is selected from Eu, Dy, Sm, Yb, Er, Nd and mixtures thereof, in particular from Eu, Dy, Sm and mixtures thereof, and more in particular Eu.

7. The method according to any one of the preceding claims, wherein a is selected from Y, Gd, La and mixtures thereof, in particular a represents Y or Gd, in particular a represents Y.

8. The method according to any of the preceding claims, wherein the nanoparticles have on their surface an amount of tetraalkylammonium cations such that the nanoparticles have a zeta potential, referred to as zeta, in an aqueous medium at a pH ≥ 5, of less than or equal to-28 mV and wherein the ionic conductivity is strictly less than 100 μ S.cm^-1。

9. The method of any one of the preceding claims, wherein the nanoparticle is of formula a_1-xLn_xVO₄(III) wherein A, Ln and x are as defined in any one of claims 1, 6 and 7, in particular of formula Y_1-xEu_xVO₄(IV) wherein 0<x<1, in particular 0.2. ltoreq. x.ltoreq.0.6 and more in particular x has a value of 0.4.

10. The method according to any one of the preceding claims, which uses a capillary action test device, wherein the photoluminescent inorganic nanoparticles are conjugated with at least one reagent, in particular an antibody or antibody fragment, a peptide, a chemically modified nucleic acid or an aptamer, that specifically binds to a substance to be analyzed.

11. The method according to any one of the preceding claims, which uses a capillary action test device, wherein the photoluminescent inorganic nanoparticles are functionalized on the surface with one or more agents intended to promote their migration within the capillary action test device, in particular selected from stealth agents or passivating agents, and more in particular selected from silanized PEG chains, poloxamers and polylactic acids (PLA).

12. The method according to any one of the preceding claims, using a capillary action test device comprising:

-a zone (1) for depositing the liquid sample and optionally a diluent;

-a zone (2), arranged downstream of said deposition zone, called "labeling zone", loaded with said photoluminescent inorganic nanoparticles coupled to at least one reagent that specifically binds said substance to be analyzed;

-a reaction zone (3), also called "detection zone", arranged downstream of said labeling zone (2), in which at least one capture reagent specific for said substance to be analyzed is immobilized;

-a control zone (4) located downstream of said detection zone, wherein at least one second capture reagent specific for a reagent specifically binding to said substance to be analyzed is immobilized; and

optionally, an absorbent pad (5) disposed downstream of the reaction zone and the control zone.

13. Method according to the preceding claim, comprising at least the following steps:

(i) applying a liquid sample to be analysed, and optionally a diluent, at the level of the deposition zone (1) of the capillary action test device;

(ii) incubating the device until luminescence generated by the photoluminescent nanoparticles is detected in the reaction zone (3) and/or until luminescence is detected in the migration control zone (4); and

(iii) reading and interpreting the results.

14. Method according to any one of the preceding claims, wherein reading of the result of the capillary test is achieved by detecting luminescence generated by probes immobilized at the level of the capillary test device at the end of the assay, in particular at the level of the detection zone (3) and optionally at the level of the control zone (4) of the device as defined in claim 12.

15. According to any one of the preceding claimsThe method of (a), wherein the nanoparticle is of formula Y_1-xEu_xVO₄(IV) by excitation of YVO at a wavelength between 230 and 320nm, in particular between 250 and 310nm and more particularly between 265 and 295nm₄The substrate is used for realizing luminescence detection.

16. Method according to any one of the preceding claims, wherein reading of the results of the capillary test is achieved by direct visual observation of the detection zone and optionally the control zone of the capillary test device, in particular as defined in claim 12, in particular using an emission filter.

17. The method according to any one of claims 1 to 15, wherein reading of the result of the capillary test is achieved using a detection device, in particular a CCD or CMOS camera, comprising an emission filter and a photon detector.

18. The method according to any one of claims 1 to 15 and 17, wherein the interpretation of the results, in particular for obtaining a quantitative characterization of the substance of interest, comprises determining the signal corresponding to the detection zone, the control zone and the background signal of the capillary action test device as defined in claim 12, subtracting the luminescence value of the background signal and then determining the ratio of the signal from the detection zone to the signal from the control zone.

19. A capillary action test device for detecting and/or quantifying a biological or chemical substance of interest in a liquid sample, the device comprising as a probe photoluminescent inorganic nanoparticles of the following formula (II):

A_1-xLn_xVO_4(1-y)(PO₄)_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), praseodymium (Pr), holmium (Ho) and mixtures thereof;

0< x <1, in particular 0.2. ltoreq. x.ltoreq.0.6, and more in particular x has a value of 0.4; and is

Y <1, in particular y has a value of 0;

the nanoparticles are capable of emitting luminescence after single photon absorption by exciting the matrix at a wavelength of less than or equal to 320nm, in particular less than or equal to 300nm, and more particularly between 250 and 300nm, the nanoparticles having an emission lifetime of less than 100 ms.

20. The device according to the preceding claim, wherein the nanoparticles are defined according to any one of claims 5 to 11.

21. The apparatus of claim 19 or 20, the apparatus being as defined in claim 12.

22. An in vitro diagnostic kit comprising at least:

-a capillary action test device according to any one of claims 19 to 21; and

-means for detecting the luminescence generated by the probes immobilized at the level of said device at the end of the assay, in particular at the level of said detection zone and/or control zone of the capillary action test device as defined in claim 21.

23. Use of the method according to any one of claims 1 to 18 or the capillary action test device according to any one of claims 19 to 21 for the purpose of in vitro diagnostics.

24. Use of the method according to any one of claims 1 to 18 or the capillary action test device according to any one of claims 19 to 21 for detecting and/or quantifying a substance of interest, in particular a pathogen, in an agricultural or food product or an environment.

25. Use of the method according to any one of claims 1 to 19 or the capillary action test device according to any one of claims 19 to 21 for detecting and/or quantifying illegal chemical substances, in particular drugs, or any other substance of interest to the police or to defense.

Drawings

FIG. 1:a schematic of a cross-sectional view of a strip for a lateral flow assay;

FIG. 2:schematic representation of a "sandwich" -type assay before (upper panel) and at the end of the assay (lower panel) application of a liquid sample to be analyzed comprising an analyte (11) at the level of the deposition zone (1);

FIG. 3:schematic representation of the procedure of a "competitive" assay according to two variants before (upper panel) and at the end of the assay (lower panel) the application of a liquid sample to be analyzed comprising an analyte (11) at the level of the deposition zone (1);

FIG. 4:images of the nanoparticles obtained according to example 1.1.a obtained by Transmission Electron Microscopy (TEM). (scale bars: 60nm (FIG. 4a) and 5nm (FIG. 4b), respectively);

FIG. 5:histogram of nanoparticle sizes determined from TEM images of a group of about 300 nanoparticles according to example 1.1. a.

FIG. 6:photographs of the strips after migration of the solutions containing 5, 0.5, 0.05ng/mL of h-FABP antigen, illuminated with UV lamps, according to the assay in example 3. 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 edge of the image. The luminescence signal shown in the photograph was analyzed by ImageJ. The results are shown in fig. 7.

FIG. 7:liquid samples containing 5, 0.5, 0.05 and 0ng/mL of h-FABP tested with test strips according to example 3 had the ratio R ═ S measured by ImageJ_D/S_D+S_CThe result of (1). These points represent the mean of R and the error bars represent the associated standard deviations of 3 and 2 bars, respectively;

FIG. 8:a schematic top view of a case comprising a strip for a lateral flow assay;

FIG. 9:a bar reader scheme using four groups of four UV LEDs ("LED # 1" to "LED # 4") to excite nanoparticles. The strip can be inserted into the reader at the level of the insertion rail (20). Reading is performed through an opening in the lid, in which a filter is positioned, which makes it possible to select the emission of the nanoparticles (centred on 617nm in the case of the present embodiment), and to exclude the excitation wavelength (centred on 280nm in the case of the present embodiment). It may be an interference filter or a high-pass filter. A camera, such as a CCD or CMOS camera of a cell phone, is positioned in front of the opening for recording images.

FIG. 10:description of the results of the bar was analyzed using a dedicated application operating under android (samsung). Left side: a black and white image with rectangular bars inside which the cumulative light emission levels of the detection, background signal and control zones were calculated from top to bottom. Right side: running a screenshot of the cell phone of the Android analysis application. We can see a black and white image with rectangular bars inside which the calculation of the cumulative level of luminescence is performed, as well as the "capture", "measure", "adjust" and "save" functions.

FIG. 11:(A) y synthesized according to the examples_0.6Eu_0.4VO₄Absorbance spectra of solutions of nanoparticles. (B) Emission spectra of nitrocellulose membranes, which were bonded to backing paper (backing card), as used in the lateral flow assay of the examples, were inserted into quartz absorption cells and excited at 280, 300 and 380nm (width of excitation slit: 5 nm). After excitation at 380nm, the emission is much more intense over the whole spectrum and more particularly at 617nm, where detection is from Y-based_0.6Eu_0.4VO₄The wavelength of the signal of the probe of the nanoparticle.

FIG. 12:with Y fixed at an emission wavelength of 617nm_0.6Eu_0.4VO₄The excitation spectrum of the nanoparticles (left part of the figure) and the emission spectrum with excitation wavelength fixed at 278nm (right part of the figure).

FIG. 13:YVO with emission wavelength fixed at 572nm₄Excitation spectrum of Dy 5% nanoparticles (FIG. 13-a) and emission spectrum with excitation wavelength fixed at 278nm (FIG. 13-b).

FIG. 14:YVO with emission wavelength fixed at 600nm₄Sm 3% nanoparticles (fig. 14-a) and emission spectra with excitation wavelength fixed at 278nm (fig. 14-b).

FIG. 15:Y_0.6Eu_0.4VO₄、Lu_0.6Eu_0.4VO₄、LuVO₄:Dy 10％、La_0.6Eu_0.4VO₄and GdVO₄Excitation spectrum of Dy 20% nanoparticles for Eu-containing³⁺Nanoparticles of ions, emission wavelength fixed at 617nm and for Dy containing³⁺Nanoparticles of ions, emission wavelength fixed at 573 nm.

FIG. 16:Lu_0.6Eu_0.4VO₄emission spectrum of nanoparticles at an excitation wavelength of 278nm (excitation LuVO)₄A substrate). The emission has a main peak at 617nm and two other peaks at 593 and 700 nm.

FIG. 17:LuVO₄emission spectrum of Dy 10% nanoparticles at an excitation wavelength of 278nm (excited LuVO)₄A substrate). The emission has two main peaks at 483 and 573 nm.

FIG. 18:La_0.6Eu_0.4VO₄emission spectrum of nanoparticles at an excitation wavelength of 278nm (excited LaVO)₄A substrate). The emission has a main peak at 617 and two other peaks at 593 and 700 nm.

FIG. 19:GdVO₄emission spectrum of Dy 20% nanoparticles at an excitation wavelength of 278nm (excited GdVO)₄A substrate). The emission has two main peaks at 483 and 573 nm.

FIG. 20:y (VO) where Y is 0, Y is 0.05, Y is 0.2, Y is 0.5, and Y is 1_4)1-y(PO₄)_yAbsorbance spectrum of Eu 20% nanoparticles. The initial concentration before dilution was approximately 50mM vanadate ion.

FIG. 21:y (VO) where Y is 0, Y is 0.05, Y is 0.2, Y is 0.5, and Y is 1_4)1-y(PO₄)_yEu 20% nanoparticles emission spectrum fixed at an excitation wavelength of 278 nm. The initial concentration before dilution was approximately 50mM vanadate ion.

FIG. 22:in the absence of antigen, Lu_0.6Eu_0.4VO₄-SA and Lu_0.9Dy_0.1VO₄Migration of SA nanoparticles on a "dipstick" strip containing BSA-biotin immobilised on a control line. UV at 312nmThe strip is observed under illumination by the lamp. By interference filter (for Eu)³⁺And Dy³⁺Emission of ions is Semrock FF01-620/14-25 and FF03-575/25) detection emission; images were taken with an Iphone 6 smartphone. Two clear bands were observed on the control line. The emission of nanoparticles that have migrated up to the absorbent pad can be seen on the right side of the image.

Examples

1. Preparation of photoluminescent Probe

1.1.Synthesis of photoluminescent inorganic nanoparticles

_{.6 0.4 4}1.1.a Y0 Synthesis of EuVO nanoparticles

Using ammonium metavanadate NH₄VO₃VO as metavanadate ion₃ ^-With a base (in this case, tetramethylammonium hydroxide N (CH)₃)₄OH) reaction and in-situ obtaining of original vanadate VO₄ ^3-. Yttrium nitrate and europium nitrate were used as Y³⁺And Eu³⁺A source of ions.

Fresh preparation of 0.1M NH₄VO₃And 0.2M of N (CH)₃)₄10mL of OH in aqueous solution (solution 1).

Ions (Y) were injected at a flow rate of 1mL/min by a syringe pump³⁺+Eu³⁺) Y (NO) at 0.1M₃)₃And Eu (NO)₃)₃A volume of 10mL of another solution (solution 2) was added dropwise to solution 1.

Selection of Y (NO)₃)₃With Eu (NO)₃)₃As Y in the nanoparticles³⁺And Eu³⁺Function of the desired ratio of ions, usually the molar ratio Y³⁺∶Eu³⁺The ratio of the components is 0.6: 0.4.

Once Y (NO) has been added₃)₃/Eu(NO₃)₃Solution, which became diffuse and appeared white/milky without formation of precipitate. The synthesis is continued until all Y (NO)₃)₃/Eu(NO₃)₃The solution was added until all was completed.

The final 20mL solution must now be purified to remove excess counterions. For this purpose, the following are used until the conductivity reaches strictly less than 100. mu.S.cm^-1: centrifugation (usually three times) for 80 minutes at 11000g (Sigma 3K10, Bioblock Scientific) was carried out, each time followed by redispersion by sonication (Bioblock Scientific, sonicator, maximum power 130W, 40s at 50% operation). The conductivity was measured using a chemical conductivity meter.

Y having tetramethylammonium cation fixed to its surface_0.6Eu_0.4VO₄The synthesis of nanoparticles can be described as follows:

VO₄ ^3-+2N(CH₃)₄ ⁺+NH₄ ⁺+0.6Y(No₃)₃+0.4Eu(NO₃)₃→Y_0.6Eu_0.4VO₄+2N(CH₃)₄ ⁺+NH₄ ⁺+3NO₃ ^-

results

The solution of nanoparticles after standing in the bottle for 16 hours was visually observed, showing a uniformly diffused solution.

Even after several months at the final pH of the synthesis (about pH 5), the final solution remained very stable in water. Despite having a high ionic strength (>0.1M), the solution comprised in the synthesis medium remains stable (before removal of excess counter ions).

After removal of the counter ion, the zeta potential of the nanoparticles, determined with a DLS-zeta potential instrument (Zetasizer Nano ZS90, Malvern), was-38.4 mV at pH 7.

Observation of the nanoparticles by TEM (fig. 4) shows that the nanoparticles have an elongated elliptical shape. The size of the nanoparticles was determined from TEM images of groups of about 300 nanoparticles (fig. 5). The nanoparticles of the invention have a major axis length, referred to as a, of between 20 and 60nm with an average value of about 40nm, and a minor axis length, referred to as b, of between 10 and 30nm with an average value of about 20 nm.

Y_0.6Eu_0.4VO₄The excitation and emission spectra of the nanoparticles are shown in fig. 12. The excitation spectrum has a peak at 278nm and the emission spectrum has a main peak at 617nm and two peaks at 593 and 700 nm.

YVO₄Eu in matrix³⁺The ions may be replaced by other luminescent lanthanide ions. In this case VO associated with the V-O charge transfer transition₄ ^3-The excitation and absorption spectra around the absorption peak of the vanadate ion remain unchanged. The emission spectrum is typical of the emission spectrum of each lanthanide ion.

_{0.95 0.05 4 4}Synthesis of b YDyVO (YVO: Dy 5%) nanoparticles

The synthesis was the same as in example 1.1.a, except that solution 2 was formed from ions (Y)³⁺+Dy³⁺) Y (NO) at 0.1M₃)₃And Dy (NO)₃)₃The composition is not the same. Solution 2 was added dropwise to solution 1 at a flow rate of 1mL/min by a syringe pump.

Selection of Y (NO)₃)₃With Dy (NO)₃)₃As Y in the nanoparticles³⁺And Dy³⁺Function of the desired ratio of ions, in this case the molar ratio Y³⁺:Dy³⁺0.95: 0.05.

The excitation and emission spectra of these nanoparticles are shown in fig. 13. Dy (Dy)³⁺The emission of the ion has two main peaks at 483 and 573 nm.

_{0.97 0.03 4 4}Synthesis of c YSmVO (YVO: Sm 3%) nanoparticles

The synthesis was the same as in example 1.1.a, except that solution 2 was formed from ions (Y)³⁺+Sm³⁺) Y (NO) at 0.1M₃)₃And Sm (NO)₃)₃The composition is not the same. Solution 2 was flowed at 1mL/min by syringe pumpThe amount was added dropwise to solution 1.

Selection of Y (NO)₃)₃With Sm (NO)₃)₃As Y in the nanoparticles³⁺And Sm³⁺Function of the desired ratio of ions, in this case the molar ratio Y³⁺:Sm³⁺The ratio of the components is 0.97: 0.03.

The excitation and emission spectra of these nanoparticles are shown in fig. 14.

Furthermore, YVO₄Y of the substrate³⁺The ions may be substituted by other ions, such as Gd³⁺、Lu³⁺And La³⁺Replacement (see examples below). GdVO for all these substrates₄、LuVO₄And LaVO₄And V and⁵⁺-O₂ ^-VO related to charge transfer transition₄ ^3-Excitation and absorption spectra around the absorption peak of vanadate ion versus YVO₄The matrix remains unchanged. Then, GdVO is present in these substrates₄、LuVO₄And LaVO₄Middle, Eu³⁺The ions may be replaced by other luminescent lanthanide ions. The emission spectrum is typical of the emission spectrum of each lanthanide ion. Various representative combinations of host and luminescent lanthanide ions are presented below.

_{0.6 0.4 4}Synthesis of d LuEuVO nanoparticles

The synthesis was the same as in example 1.1.a, except that solution 2 was made of ions (Lu)³⁺+Eu³⁺) Lu (NO) at 0.1M₃)₃And Eu (NO)₃)₃The composition is not the same. Solution 2 was added dropwise to solution 1 at a flow rate of 1mL/min by a syringe pump.

Selection of Lu (NO)₃)₃With Eu (NO)₃)₃As Lu in the nanoparticles³⁺And Eu³⁺Function of the desired ratio of ions, in this case the molar ratio Lu³⁺:Eu³⁺0.6: 0.4.

Lu_0.6Eu_0.4VO₄The excitation spectrum of the nanoparticles is shown in fig. 15, and the emission spectrum is shown in the figure16 are shown. LuVO₄Eu in matrix³⁺Emission spectrum of ion relative to YVO₄The emission spectrum in the matrix was virtually unchanged (FIG. 12) and had a main peak at 617nm and two peaks at 593 and 700 nm.

₄Synthesis of e LuVO 10% Dy nanoparticles

The synthesis was the same as in example 1.1.a, except that solution 2 was made of ions (Lu)³⁺+Dy³⁺) Lu (NO) at 0.1M₃)₃And Dy (NO)₃)₃The composition is not the same. Solution 2 was added dropwise to solution 1 at a flow rate of 1mL/min by a syringe pump.

Selection of Lu (NO)₃)₃With Dy (NO)₃)₃As Lu in the nanoparticles³⁺And Dy³⁺Function of the desired ratio of ions, in this case the molar ratio Lu³⁺:Dy³⁺0.9: 0.1.

LuVO₄Excitation spectrum of Dy 10% nanoparticles is shown in fig. 16, and emission spectrum is presented in fig. 17. LuVO₄Dy in matrix³⁺Emission spectrum of ion relative to YVO₄The emission spectrum in the matrix was virtually unchanged (fig. 13) and had two emission peaks at 483 and 573 nm.

_{0.6 0.4 4}Synthesis of fLaEuVO nanoparticles

The synthesis was the same as in example 1.1.a, except that solution 2 was made of ions (La)³⁺+Eu³⁺) La (NO) of 0.1M₃)₃And Eu (NO)₃)₃The composition is not the same. Solution 2 was added dropwise to solution 1 at a flow rate of 1mL/min by a syringe pump.

Selecting La (NO)₃)₃With Eu (NO)₃)₃As the molar concentration ratio of La in the nanoparticles³⁺And Eu³⁺Function of the desired ratio of ions, in this case the molar ratio La³⁺:Eu³⁺0.6: 0.4.

La_0.6Eu_0.4VO₄Nano meterThe excitation spectrum of the particles is shown in fig. 15, and the emission spectrum is presented in fig. 18. LaVO₄Eu in matrix³⁺Emission spectrum of ion relative to YVO₄The emission spectrum in the matrix was virtually unchanged (FIG. 12) and had a main peak at 617nm and two peaks at 593 and 700 nm.

₄Synthesis of nanoparticles of GdVO 20% Dy 1.1

The synthesis of these nanoparticles proceeds as follows starting from orthovanadate precursors. Freshly prepared 0.1M 10mL of NaVO₄The aqueous solution (solution 1) was adjusted to a pH between 12.6 and 13 with 1M NaOH solution.

Ions (Gd) were injected at a flow rate of 1mL/min by a syringe pump under stirring³⁺+Dy³⁺) Gd (NO) at 0.1M₃)₃And Dy (NO)₃)₃A volume of 10mL of another solution (solution 2) was added dropwise to solution 1.

Selecting Gd (NO)₃)₃With Dy (NO)₃)₃As Gd in the nanoparticles³⁺And Dy³⁺Function of the desired ratio of ions, in this case the molar ratio Gd³⁺:Dy³⁺0.8: 0.2.

Upon addition of Gd (NO)₃)₃/Dy(NO₃)₃A milky white precipitate formed. The synthesis is continued until all Y (NO)₃)₃/Eu(NO₃)₃The solution was added until all was completed. The solution was stirred for 30min until the pH stabilized at 8-9.

The final 20mL of solution had to be purified, as in example 1.1.a, to remove excess counter ions. For this purpose, the following are used until the conductivity reaches strictly less than 100. mu.S.cm^-1: centrifugation (usually three times) for 15 minutes at 11000g (Sigma 3K10, Bioblock Scientific) was followed each time by redispersion by sonication (Bioblock Scientific, sonicator with maximum power of 130W, operating at 50% for 40 s).

GdVO₄Excitation spectrum of Dy 20% nanoparticles is shown in fig. 15, and light emission is performedThe spectra are presented in fig. 19. GdVO₄Dy in matrix³⁺Emission spectrum of ion relative to YVO₄The emission spectrum in the matrix was virtually unchanged (fig. 13) and had two emission peaks at 483 and 573 nm.

_{4)1-y 4 y}Synthesis of 1.1.h Y (VO (PO): Eu 20% nanoparticles

Also synthesizes VO contained in different types₄ ^3-:PO₄ ^3-VO in matrix of ratio₄ ^3-And PO₄ ^3-Nanoparticles of a mixture of ions.

The synthesis was identical to that in example 1.1.a, except that solution 1 was made up of ions (VO) at a total concentration of 0.1M₃ ^-+PO₄ ^3-) 0.1. y M of Na₃PO₄0.1. cndot. (1-y) M NH₄VO₃And 0.2. sup. (1-y) M of N (CH)₃)₄OH composition. 10mL of the aqueous solution (solution 1) having the above concentration was freshly prepared. NPs were prepared where y ═ 0, y ═ 0.05, y ═ 0.2, y ═ 0.5, and y ═ 1.

PO₄ ^3-The ions showed no absorption at 278 nm. Thus, 100% PO is contained₄ ^3-The ionic nanoparticles did not have an absorption peak at 278nm (see fig. 20). The emission spectra of these nanoparticles are presented in fig. 21 and are the same for all values of y different from 1 (for y ═ 1, no emission is observed). They have a main peak at 617 and two additional peaks at 593 and 700 nm. These emission spectra are virtually identical.

1.2.Covalent coupling of nanoparticles to proteins (anti-h-FABP antibodies)

According to the following protocol, Y obtained as described in point 1.1.a_0.6Eu_0.4VO₄The nanoparticles are conjugated to antibodies.

1.2.1. Coating nanoparticles with a silica layer

At the end of the synthesis of the nanoparticles, the solution of nanoparticles was centrifuged at 17000g for 3 minutes to precipitate any aggregates of nanoparticles and the supernatant was recovered. The selection is made by size. For this purpose, several centrifugations at 1900g were carried out for 3 min. After each centrifugation, the nanoparticles were redispersed with a sonicator and then sized using a DLS-zeta potential instrument (Zetasizer Nano ZS90, Malvern).

Preparation of 25mL volume of Y with 20mM concentration of vanadate ion_0.6Eu_0.4VO₄And (3) granules. Another pure sodium silicate solution (Merck Millipore 1.05621.2500) was added dropwise in a volume of 2.5mL by pipette to coat the surface of the particles. The solution was allowed to act for at least five hours with stirring.

The solution is then purified to remove excess silicate and sodium counter ions. The solution was centrifuged at 11000g (Sigma 3K10, Bioblock Scientific) for 60 minutes and then redispersed by sonication (Bioblock Scientific, sonicator, operating at 50% power at 400W). This step was repeated until the conductivity of the solution was less than 100. mu.S/cm.

1.2.2. Grafting of amines onto the surface of nanoparticles

225mL of absolute ethanol was placed in a 500-mL three-necked flask and 265. mu.L of APTES (3-aminopropyltriethoxysilane) (Mw 221.37g/mol Sigma Aldrich) was added, corresponding to a final concentration of 1.125 mM. This amount corresponds to 5 equivalents of vanadate introduced. The condenser was then attached to the flask. The whole was placed on a flask heater and under a fume hood. The mixture was heated at 90 ℃ under reflux. At one inlet of the three-necked flask, a colloidal solution of nanoparticles at pH 9 in 75mL of water (vanadate ion concentration [ V ] ═ 3mM) was added dropwise at a flow rate of 1mL/min using a peristaltic pump. The whole was heated with stirring for 24 h.

After 48 hours, the nanoparticles were partially concentrated using a rotary evaporator (rotavapor R-100, BUCHI). The solution was spun in a suitable flask and heated in a bath at 50 ℃.

The recovered solution was purified by several centrifugations in ethanol water (3:1) solvent. After purification, sorting by size was performed following the protocol described above.

1.2.3. Grafting of carboxyl groups onto aminated nanoparticle surfaces

Solvent transfer was performed before grafting was initiated.

The grafting protocol is as follows.

Reaction of aminated NPs from EtOH: H₂The O buffer was transferred to DMF or DMSO and centrifuged several times (13000g,90 min). The pellets were redispersed by sonication between each centrifugation (20 s at 75%). The concentration of NP is measured and determined.

NPS was recovered in 5mL of DMF, and then 10% succinic anhydride was added to a glass beaker (i.e. 0.5g in 5 mL). The reaction was allowed to proceed under stirring under an inert atmosphere at least overnight.

Carboxylated NPs were washed at least twice by centrifugation (13000g for 60min, Legend Micro17r, Thermo Scientific) to remove DMF and excess succinic anhydride.

The carboxylated particles were resuspended in water or MES buffer at pH 6 by sonication (Bioblock Scientific, sonicator).

1.2.4. Coupling nanoparticles with anti-h-FABP antibodies

The coupling of the nanoparticles surface-grafted with COOH is carried out according to the following scheme:

1.a mixed solution of EDC/sulfo-NHS (at 500 and 500mg/mL, respectively) in MES buffer (pH 5-6) was freshly prepared.

2. 90nM NP (in this case, this is the nanoparticle concentration calculated from the vanadate ion concentration according to Casanova et al [37 ]) was added to a previously prepared 3mL solution and reacted at room temperature for 25min with stirring.

3. Excess reagent was removed by rapidly washing the NPs with MilliQ water at least 2 times (13000g for 60min, Legend Micro17R, Thermo Scientific).

4. After sonication in sodium phosphate buffer at pH 7.3, the final pellet was recovered. The required amount of protein (anti-h-FABP antibody, Ref 4F29,10E1, Hytest) was added as a function of the required ratio (protein: Np), typically 2. mu.M, at a ratio of 20:1, and 5mg/mL mPEG-silane (MW:10kD, Laysan Bio 256-.

5. The solution was allowed to react for between 2 and 4h at room temperature with stirring.

6. A blocker (1% glycine) was added so that it reacted with free COOH and blocked residual reaction sites on the NP surface. The reaction was carried out for 30 min.

7. The protein-coupled NPs were washed several times by centrifugation with PBS pH 7.2 using a centrifugal filter (Amicon Ultra 0.5mL, Ref UFC501096, Millipore). Transfer of NPs to their storage media: phosphate buffer + Tween 20 (0.05%) + 0.1% p-hydroxyphenylglycine + 10% glycerol. 100 μ L was taken for BCA assay. The remaining solution was divided into aliquots and frozen at-80 ℃.

And, with Y_0.6Eu_0.4VO₄In the same way as for the nanoparticles, all nanoparticles synthesized according to examples 1.1.b to 1.1.h can be coupled to antibodies.

1.3.Passive coupling of nanoparticles to proteins (anti-h-FABP antibodies)

Instead of covalent coupling as in example 1.2, passive coupling of nanoparticles to antibodies can also be performed as follows.

1mL of the nanoparticle solution (5 mM vanadate ion) was centrifuged at 15000g for 15 min.

The pellets were dissolved in 800 μ L MilliQ water and then redispersed by sonication (Bioblock Scientific, sonicator, maximum power 130W, 40s at 50% run).

100. mu.L of a 250. mu.g/mL antibody solution was added to 2mM potassium phosphate buffer, pH 7.4.

Incubation for 1 hour while spinning.

Addition of 100. mu.L of 20mM potassium phosphate buffer pH 7.4/1% BSA.

Centrifugation at 15000g for 15min and removal of the supernatant.

The pellets were dissolved in 1mL of 2mM potassium phosphate buffer pH 7.4/0.1% BSA. Redispersion was carried out by sonication (Bioblock Scientific, sonicator, maximum power 130W, 40s at 50% run).

Centrifugation at 15000g for 15min and removal of the supernatant.

The pellets were dissolved in 250. mu.L of 2mM potassium phosphate buffer pH 7.4/0.1% BSA. Redispersion was carried out by sonication (Bioblock Scientific, sonicator, maximum power 130W, 40s at 50% run).

2. Test prepared on strips of the "Sandwich" type

In order to develop a rapid test for qualitatively or quantitatively determining the presence of a protein, it is necessary to optimize various parameters and to find a compromise between reaction time and test sensitivity.

As shown in fig. 1, the production of the assay strip was performed by combining four basic parts:

use of substantially inert glass fibers as the marking zone (2) (in english terminology "conjugate pad") (GFDX 103000, Millipore).

Use of surface-modified polyester as deposition area (in english terminology "sample pad") (1) (Ref CFSP173000, Millipore). They have the advantage that they have a weak non-specific interaction with proteins, excellent traction and good handling properties.

Nitrocellulose membrane (NC) (HF180MC100, Millipore) was used as the device (10) for capillary action. It has optimal properties for fluid migration and protein immobilization. The NC film glue is glued to a support (6) of non-porous adhesive plastic ("backing paper").

Cellulose (CFSP173000, Millipore) was used as absorbent pad (5) (due to its high absorbent capacity).

To detect h-FABP (human fatty acid binding protein) as cardiac biomarker:

it is necessary to deposit the antibody on the NC film before assembling the individual components.

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

2. The antibody solution was deposited on the NC membrane using a "dispenser" (Claremont Bio Automated Lateral Flow Reagent Dispenser (ALFRD)). Using a syringe pump, each strip deposited a volume of 0.7. mu.L/2 mm all the way along the NC film (about 30cm long, several strips will be made from it). Drying was carried out at 37 ℃ for 1 h.

3. After antibody deposition, NC membranes were incubated with 1% BSA diluted in PBS at 0.04% (pH 7.4) + Tween 20 at 37 ℃ for 30min to inactivate the immobilization sites not occupied by the antibody.

4. Abs coupled to NPs were deposited on the label zone ("conjugate pad") using a dispenser. 3 μ L volume/4 mm was applied all the way along the glass fiber membrane. Dried at room temperature for 1h and then blocked with 1% BSA diluted in PBS (pH 7.4). Drying at room temperature.

Assembling strip

1. Various structures (cellulose used as an absorbent pad, and a deposition region, a labeling region (on which Ab + NP is deposited)) were assembled on the adhesive portion of NC to which Ab had been fixed. The components are fixed on a plastic carrier of the NC in the following order: the label zone ("conjugate pad"), the deposition zone ("sample pad"), and finally the absorbent pad. In order to better perform fluid transfer by capillary action, the respective components are installed to overlap each other as shown in the description (fig. 1).

2. The assembled film was cut into individual pieces 4mm wide using a paper cutter.

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

3. Strip test procedure

Using Y conjugated to antibody prepared in example 1.2_0.6Eu_0.4VO₄The nanoparticles were prepared into strips.

Several concentrations of h-FABP from 5ng/mL to 0.05ng/mL were measured (Ref.8F65, Hytest). The recombinant h-FABP is diluted to the desired concentration with buffer or with serum.

1. Before testing, the strips were prepared and analyzed as described above at point 2.

2. A400 μ L sample was deposited in a vertically placed vial.

3. The strip was dipped into the bottle, with the deposition area ("sample pad") oriented downward. The bottom bar was tapped to start migration. The strips were kept standing vertically in the tube for 10 min.

4. The results of the strips were read using UV lamps (Vilber Lourmat, VL-8.MC, 8W, at 312nm, and 8W, at 254 nm) (digital photographs were taken and analyzed by ImageJ, see fig. 6 and 7) or using the reader presented in fig. 8 and 9. Fig. 10 illustrates the analysis of the results in bands using a dedicated application on a cell phone operating under Android. The reader uses 4 sets of 4 LEDs at 278nm and an interference filter (620/15, Semrock) for detection. High pass filters such as the RG605 filter (Schott) may also be used for detection.

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

Qualitative interpretation of the results

The qualitative interpretation of the results is as follows:

if there are two bands: test positive

If a single control band is present: test is negative

If there is a single test strip, the test is invalid

If no band is present: the test is invalid.

Quantitative interpretation of the results

Fig. 10 shows an example of quantitative analysis starting from a digital photograph taken with a mobile phone using a dedicated application. When left on the "capture" on the phone screen, the recording of a black and white image will be triggered. Then, after staying "measure", the application asks the user to point with a finger on the phone screen to the detection zone and then the control zone, so that the application programCalculating the cumulative light level L inside a rectangle containing the detection region_DAnd the cumulative light emission level L inside a rectangle of the same size containing the control zone_C. Staying on "the adjustment" triggers an optimization of the two rectangular positions corresponding to the detection and control zones in order to maximize the measurement 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 is used to determine the background signal L_BAnd for calculating the signal S_D/C＝L_D/C-L_B. Application calculates and then displays the ratio R ═ S_D/S_COr R ═ S_D/(S_D+S_C). The R value can be compared to a calibration table, and concentration values in ng/mL are also provided. The result may be saved with a "save" function for later comparison with the next result.

Three strips were prepared for strip testing of each of the samples comprising h-FABP antigen. For the case where the sample does not contain antigen, only two strips are prepared.

The graph in fig. 7 shows the ratio R ═ S measured by ImageJ for different liquid samples containing 5, 0.5, 0.05 and 0ng/mL of h-FABP_D/(S_D+S_C) The results obtained. The points in FIG. 7 represent the mean values of R and the error bars represent the associated standard deviations of the different bars tested (three bars in the case of samples containing h-FABP; two bars in the case of samples not containing h-FABP).

The method according to the invention therefore advantageously allows the detection of h-FABP in a sample in an amount of less than or equal to 5ng/mL, in particular less than or equal to 0.5ng/mL, or even up to values as low as 0.05 ng/mL. In other words, h-FABP may be detected at a level of less than or equal to 330pM, in particular less than or equal to 33pM, or even up to a level as low as 3.3 pM.

_{0.6 0.4 4 0.9 0.1 4}Migration of LuEuVO-SA and LuDyVO-SA nanoparticles on a "Sandwich" type "dipstick" strip

According to example 1.3 (passive coupling), respectivelyLu synthesized according to examples 1.1.d and 1.1.e_0.6Eu_0.4VO₄And Lu_0.9Dy_0.1VO₄The nanoparticles were coupled to Streptavidin (SA). A "dipstick" strip was prepared according to example 2 by immobilizing BSA-biotin on nitrocellulose membrane to recognise NP coupled to streptavidin. In the absence of antigen, Lu was used according to example 3_0.6Eu_0.4VO₄-SA and Lu_0.9Dy_0.1VO₄SA nanoparticle manufacturing test strips. The strips were visualized under UV lamp excitation (312 nm). Two clear strong bands were formed at the level of the control line (fig. 22).

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