METHOD FOR DETECTING OXIDISING SPECIES

Abstract

The invention relates to a method which can be used to analyse oxidising species in a sample, comprising the steps which consist of: placing (12) the sample in contact with a first photoluminescent agent, and a second optically active agent, at least the first photoluminescent agent including nanoparticles doped with rare earth elements and being oxidisable, the luminescence of the first photoluminescent agent varying, at least at one first wavelength, with the amount of oxidising species, and the signal emitted by the second optically active agent being constant or varying, at least at one second wavelength other than the first wavelength, with the amount of oxidising species in a direction opposite to the luminescence of the first photoluminescent agent; energising (16) the first and second agents in the sample; measuring (18) the light intensity of the sample at least at said first and second wavelengths; and assessing the presence and/or the amount of oxidising species by interpreting (20) said measured light intensities.

Claims

1. A method that can be used for analyzing oxidizing species in a sample, comprising the steps consisting of: i) contacting the sample with a photoluminescent first agent and an optically active second agent, the photoluminescent first agent at least comprising nanoparticles doped with rare earths and oxidizable, with the luminescence of the photoluminescent first agent varying, at at least one first wavelength, with the quantity of oxidizing species, and the signal emitted by the optically active second agent being, at at least one second wavelength different from the first wavelength, constant with the quantity of oxidizing species, or varying with the quantity of oxidizing species in a direction opposite to the luminescence of the photoluminescent first agent, ii) exciting the photoluminescent first agent and the optically active second agent in the sample iii) measuring the luminous intensity of the sample at at least said first wavelength, and at least said second wavelength, and iv) estimating the presence and/or the quantity of oxidizing species by interpretation of said measured luminous intensities, and if applicable by reference to standard values.

2. The method as claimed in claim 1, in which, in step iv), a quantity of oxidizing species in the sample is determined by reading, on a standard sheet, the value corresponding to the pair of luminous intensities measured in step iii).

3. The method as claimed in claim 2, in which the standard sheet is a sheet (30) established beforehand by means of measurements performed with samples with a known quantity of oxidizing species.

4. The method as claimed in claim 1, in which step iii) is performed after a nonzero set waiting time after step ii).

5. The method as claimed in claim 1, in which the luminous intensity of the luminescence of the photoluminescent first agent increases with the quantity of oxidizing species.

6. The method as claimed in claim 1, in which the luminous intensity of emission of the optically active second agent is roughly constant with the quantity of oxidizing species.

7. The method as claimed in claim 1, in which the optically active second agent is photoluminescent.

8. The method as claimed in claim 1, in which the luminous intensity of emission of the optically active second agent decreases with the quantity of oxidizing species.

9. The method as claimed in claim 1, in which the first wavelength is representative of the oxidized form of the photoluminescent first agent and the second wavelength is representative of the reduced form of the optically active agent.

10. The method as claimed in claim 1, in which the optically active second agent is photoluminescent.

11. The method as claimed in claim 1, in which the photoluminescent first agent comprises at least partially reduced nanoparticles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y), where A is one from Y, Gd and La, 0x1 and 0y1.

12. The method as claimed in claim 12, comprising a step a), prior to step i), consisting of reducing nanoparticles to obtain the photoluminescent first agent.

13. The method as claimed in claim 12, in which, in step a), reduction is performed chemically, using a reducing agent.

14. The method as claimed in claim 13, in which the reducing agent contains NaBH.sub.4.

15. A method that can be used for analyzing oxidizing species in a sample, in particular in a biological sample, comprising the steps consisting of: i) providing photoluminescent nanoparticles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y), where A is one from Y, Gd and La, 0x1 and 0y1, said nanoparticles being reduced beforehand; iv) introducing said nanoparticles into the assay sample, v) exciting said nanoparticles, vi) measuring the luminous intensity emitted by the sample at at least one wavelength representative of an oxidized state or of a reduced state of said nanoparticles, and vii) estimating the presence and/or the quantity of oxidizing species by interpreting said measurement, if applicable by reference to a standard or calibration.

16. The method as claimed in claim 1 in which the particles are additionally coupled to one or more functionalized biomolecules and/or biomolecules known to be of interest selected from targeting molecules and stealth agents.

17. The method as claimed in claim 1, in which the sample is one from: a living organism, biological tissues, biological cells, a solution of biological molecules, a volume of gas.

18. A method of diagnosis, using a method as claimed in claim 1 for purposes of diagnosis.

19. A method of screening of the efficacy of an active substance with respect to a disorder, whether or not pathological, and associated with overexpression of one or more reactive oxygen species (ROS), using a method as claimed in claim 1.

20. The method as claimed in claim 19, in which the analysis is carried out on a volumetric sample.

21. A system for implementing the method as claimed in claim 1, comprising: a laser illumination device with one or two wavelengths for exciting the photoluminescent first agent and the optically active second agent in the sample, a spectrum splitter for filtering the emissions from the photoluminescent first agent and from the optically active second agent according to different wavelengths and thus forming two separate filtered images of the sample, and means for determining the luminous intensity of at least points of the two filtered images.

22. The system as claimed in claim 21, in which the means for determining the luminous intensity comprise a means for recording the images filtered according to the wavelengths.

23. The system as claimed in claim 22, in which the means for determining the luminous intensity comprise two photodetectors at different wavelengths.

24. The system as claimed in claim 21, further comprising a microscope functionally interposed between the sample and the spectrum splitter.

25. The system as claimed in claim 24, in which the microscope is one from a fluorescence microscope, a macroscope and a stereomicroscope.

26. The system as claimed in claim 21, further comprising a holder for the sample.

27. The system as claimed in claim 21, further comprising a separating plate functionally interposed between the illumination device and the sample, on the one hand, and between the sample and the spectrum splitter, on the other hand.

28. The system as claimed in claim 21, comprising a device for occultation, of the means for determining the luminous intensity, selectively activatable.

29. The method as claimed in claim 7, in which the optically active second agent comprises nanoparticles of LaPO.sub.4:Eu, LaPO.sub.4:Er, LaPO.sub.4:Nd, LaPO.sub.4:Tb, LaF.sub.3:RE, NaYF.sub.4:Yb, NaYF.sub.4:Er, CdSe/ZnS, CdTe/ZnS, KTP and/or BaTiO.sub.3.

30. The method as claimed in claim 10, in which the optically active second agent comprises nanoparticles of YAG:Ce, LaPO.sub.4:Ce.

31. The method as claimed in claim 1, in which the photoluminescent first agent comprises at least partially reduced nanoparticles of A.sub.xEu.sub.1-xVO.sub.4, where A is selected from the group consisting of Y, Gd and La, and wherein 0x1.

32. The method as claimed in claim 13, in which step a) comprises a substep, subsequent to the chemical reduction, consisting of washing the reduced nanoparticles to remove the excess reducing agent.

33. A method as claimed in claim 15, in which, in step i), nanoparticles are chemically reduced using a reducing agent.

34. A method as claimed in claim 33, in which said reducing agent is NaBH.sub.4.

35. The method as claimed in claim 18 for diagnosis of physiological disorders, whether or not pathological, and associated with expression of one or more reactive oxygen species (ROS) that is not physiologically acceptable.

36. The method as claimed in claim 18, in which the analysis is carried out on a volumetric sample.

37. The system as claimed in claim 22, in which the recording means comprise a camera arranged functionally at the output of the spectrum splitter and connected to a computer recording the image recorded by the camera.

38. The system as claimed in claim 23, in which the means for determining the luminous intensity are of the photomultiplier or photodiode type.

Description

[0058] The invention will be better understood on reading the following description, given only for purposes of illustration, and nonlimiting, referring to the appended drawings, where:

[0059] FIG. 1 shows a functional diagram of a method for detecting oxidizing species;

[0060] FIG. 2 shows the level of luminous intensity of the luminescence of nanoparticles of Gd.sub.0.6Eu.sub.0.4VO.sub.4 after treatment with a solution of NaBH.sub.4 (1 mol.l.sup.1);

[0061] FIG. 3 shows intensity response curves of luminescence of reduced particles of Gd.sub.0.6Eu.sub.0.4VO.sub.4 and of YAG:Ce, respectively, in oxidizing media with a known concentration of hydrogen peroxide;

[0062] FIG. 4 shows an example of a sheet indicating a concentration of hydrogen peroxide in a sample as a function of a pair of measured luminous intensities;

[0063] FIG. 5 is a schematic representation of a system for implementing the method shown in FIG. 1;

[0064] FIG. 6 shows an example of observation of a sample, according to example 1, of mouse vascular smooth muscle cells in culture, under white light;

[0065] FIG. 7 shows an example of observation of the same sample, according to example 1, in which the nanoparticles indicated in the figure have been internalized and their luminescence is detected at wavelengths of 550 nm and 617 nm;

[0066] FIG. 8 shows the variation over time of the luminous intensity of photoluminescent agents in contact with the sample in FIG. 6 after injection of endothelin, according to example 1;

[0067] FIG. 9 shows the concentration of hydrogen peroxide in the sample in FIG. 6 obtained according to example 1, after injection of endothelin, determined from the sheet presented in FIG. 4;

[0068] FIG. 10 shows the variation of the luminescence of a solution in a vessel, according to example 2, containing a mixture of particles YAG:Ce and GdVO.sub.4:Eu after adding 2 mM of hydrogen peroxide (at t=0), detected at 550 nm and 617 nm on two photomultipliers;

[0069] FIG. 11 shows the photographs obtained by fluorescence microscopy and spectrum splitting of a sample according to example 3 of mouse vascular smooth muscle cells in culture;

[0070] FIG. 12 shows the variation, according to example 3, of the photoluminescences normalized by their initial value of the nanoparticles YAG:Ce (below 1) and GdVO.sub.4:Eu (above 1) obtained after stimulation by ET-1, with or without inhibition of the EGFRs; and

[0071] FIG. 13 shows the temporal variation of the absolute concentrations of hydrogen peroxide in the two cellular conditions (normal or EGFR inhibited) determined according to example 3.

DETAILED DESCRIPTION

[0072] The method for detecting oxidizing species 10 comprises a first step 12 of contacting the sample with a photoluminescent first agent and an optically active second agent.

[0073] As stated above, optically active agent means an agent that emits photons following excitation by photons, optionally of different wavelengths. Thus, a photoluminescent agent is an optically active agent of this kind. An agent for second harmonic generation is also an optically active agent in the sense of the present application. An example of such an agent for second harmonic generation is for example particles of KTPstanding for potassium titanyl phosphate (KTiOPO.sub.4). In the rest of the description, the optically active second agent is a photoluminescent second agent. Unless stated otherwise, what is described for the photoluminescent second agent also applies in the case when this second agent is optically active, notably an agent for second harmonic generation.

[0074] The photoluminescent first agent comprises nanoparticles doped with rare earths.

[0075] Here, nanoparticles means particles whose diameter is of the order of a nanometer, notably greater than 1 nm and/or less than 500 nm. Here, diameter means the largest dimension of the nanoparticle.

[0076] In a preferred embodiment, the photoluminescent second agent also comprises nanoparticles doped with rare earths.

[0077] The photoluminescent first and second agents have different luminescence wavelengths. In other words, there is at least one first wavelength at which only the light emitted by the photoluminescent first agent is detectable and at least one second wavelength, different than the first, at which only the light emitted by the second agent can be detected. The photoluminescent agents are preferably selected in such a way that their line widths are small enough to be separated spectrally. This allows easier detection of the emissions from the photoluminescent first and second agents. To make this detection even easier and for greater accuracy, photoluminescent agents are selected whose maximum emission wavelengths are different and detection is performed at these maximum emission wavelengths.

[0078] Furthermore, the photoluminescent first agent is selected such that the intensity of its luminescence emitted at the detected wavelength (abbreviated hereinafter as: its luminescence) varies with the quantity of oxidizing species in whose presence it is. In the following, the case will be considered in which the luminous intensity of the luminescence of the photoluminescent first agent increases with the quantity of oxidizing species in whose presence it is.

[0079] The photoluminescent agents may be deposited so as to obtain a low surface density on or in a sample, which may for example be received on a glass slide. Low surface density means a surface density below 1 m.sup.2.

[0080] However, the photoluminescent agent or agents considered in the methods according to the invention may also advantageously be introduced directly in a volumetric sample, for example a volume of liquid sample or in the bulk of a sample.

[0081] Photoluminescent First Agent

[0082] The photoluminescent first agent advantageously comprises nanoparticles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y), where A is one from Y and Gd and La, 0x1 and 0y1.

[0083] In particular, in the following, the particular case is considered in which the photoluminescent first agent is formed by nanoparticles of Gd.sub.0.6Eu.sub.0.4VO.sub.4.

[0084] According to a preferred variant, these nanoparticles are reduced, before being brought into contact with the analysis sample.

[0085] The reduced nanoparticles of a photoluminescent first agent of this kind may notably be obtained in a preliminary step 14 consisting of reducing nanoparticles to obtain the photoluminescent first agent.

[0086] As detailed hereinafter, reduction may be effected:

[0087] physically, notably by laser, electronic or gamma irradiation, for example; or

[0088] chemically, using a reducing chemical agent.

[0089] According to a first embodiment, before being brought into contact with the analysis sample, the nanoparticles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y) are reduced physically, in particular by laser excitation.

[0090] In the context of this embodiment, the nanoparticles, when they are brought into contact with the analysis sample, already contain europium in the form Eu.sup.2+. For example, in the case when particles of Gd.sub.0.6Eu.sub.0.4VO.sub.4 are used, the Eu.sup.3+ ions of the particles may be reduced reversibly to Eu.sup.2+ ions in step 14 of the method of the invention.

[0091] It is to be understood that this reduced species of the nanoparticles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y) may also be generated in situ, i.e. in the assay sample, for example by photoreduction of nanoparticles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y) in which the species Eu is essentially in the form Eu.sup.3. This approach is notably described in Casanova et al., Nat Nanotech (2009).

[0092] However, the embodiment consisting of employing nanoparticles, of Gd.sub.0.6Eu.sub.0.4VO.sub.4 for example, as the starting reactant, already incorporating Eu in the reduced form is preferred for its efficiency and simplicity. Moreover, this variant does not affect the integrity of any biological cells present in the assay sample.

[0093] According to another embodiment, before they are brought into contact with the analysis sample, the nanoparticles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y) are reduced chemically, using a reducing agent.

[0094] This substep of chemical reduction may be followed by a further substep, consisting of washing the reduced nanoparticles, to remove excess reducing agent. For example, the particles are diluted in a solution containing a reducing agent, then centrifuged, and then dispersed again in fresh solution without a reducing agent (pure water for example), thus allowing successive washing operations.

[0095] The reducing agent employed may notably be NaBH.sub.4. A last step may then consist of washing the reduced nanoparticles, to remove excess reducing agent.

[0096] For example, the nanoparticles of Gd.sub.0.6Eu.sub.0.4VO.sub.4 may be reduced reversibly with sodium borohydride (NaBH.sub.4) in step 14.

[0097] FIG. 2 shows a comparison between the levels of luminescence of a solution of nanoparticles of Gd.sub.0.6Eu.sub.0.4VO.sub.4 under laser illumination of wavelength 466 nm, before, immediately, 7 h and 2 days after treatment with 1M of NaBH.sub.4.

[0098] The inventors have shown that the particles obtained following reduction with a chemical agent are capable of detecting oxidizing species, for example H.sub.2O.sub.2, in a similar fashion to the detection of H.sub.2O.sub.2 by particles with Eu.sup.3+ ions reduced beforehand to Eu.sup.2+ following laser excitation.

[0099] Thus, according to another of its aims, the invention relates to a method that can be used for analyzing oxidizing species in a sample, in particular in a biological sample, comprising the steps consisting of: [0100] i) providing photoluminescent nanoparticles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y), where A is one from Y, Gd and La, 0x1 and 0y1, said nanoparticles being reduced beforehand, in particular chemically using a reducing agent, in particular by NaBH.sub.4; [0101] ii) introducing said nanoparticles into the assay sample, [0102] iii) exciting said nanoparticles, [0103] iv) measuring the luminous intensity emitted by the sample at at least one wavelength representative of the oxidized form or of the reduced form of said nanoparticles, and [0104] iv) estimating the presence and/or the quantity of oxidizing species by interpreting said measurement, if applicable by reference to a standard or calibration.

[0105] It was not in any way obvious that reduction of the particles of A.sub.xEu.sub.1-x(VO.sub.4).sub.y(PO.sub.4).sub.(1-y) with a chemical agent, for example NaBH.sub.4, might give oxidizable nanoparticles capable of detecting oxidizing species, for example H.sub.2O.sub.2.

[0106] Without wishing to be bound to any theory, the inventors have shown that the mechanism of reduction is not the same in both cases.

[0107] In the case of reduction by laser excitation, the result of laser excitation resonant with the electronic transitions of the Eu.sup.3 ions is the appearance of Eu.sup.2+ ions, which makes the particles oxidizable and capable of detecting oxidizing species. However, in the case of reduction of the nanoparticles by a chemical agent, there is no persistent significant change in the number of Eu.sup.3+ ions, and reduction with a chemical agent probably involves a phenomenon of quenching acting on the emission of the Eu.sup.3+ ions, with reoxidation of the particle allowing the return of luminescence.

[0108] Optically Active Second Agent

[0109] The optically active and preferably photoluminescent second agent is selected in such a way that its luminescence (or the luminous intensity of its luminescence) is constant or, preferably, also varies with the quantity of oxidizing species.

[0110] Thus, according to a first variant of the method for detecting oxidizing species 10 described above, the intensity of luminescence of the optically active, notably photoluminescent, second agent is roughly constant in the presence of oxidizing species.

[0111] Roughly constant means that the variation of luminous intensity is between plus or minus 10%, preferably plus or minus 5%. In this case, the optically active second agent may notably comprise or consist of nanoparticles of LaPO.sub.4:Eu, LaPO.sub.4:Er, LaPO.sub.4:Nd, LaPO.sub.4:Tb, LaF.sub.3:RE (RE=rare earth), NaYF.sub.4:Yb, NaYF.sub.4:Er, YAG:Eu, fluorescent semiconducting nanocrystals CdSe/ZnS, CdTe/ZnS, or particles displaying properties of emission by second harmonic generation such as KTP and/or BaTiO.sub.3. This variant of the method also makes it possible to detect the presence of oxidizing species in a test sample.

[0112] According to a second preferred variant of the method for detecting oxidizing species 10 described above, the intensity of luminescence of the optically active second agent varies as a function of the quantity of oxidizing species, in a direction opposite to the variation of luminescence observed at said first accredited length of the photoluminescent first agent.

[0113] Thus, in the rest of the description, it is considered that the luminescence of the photoluminescent second agent decreases with the quantity of oxidizing species, in whose presence it is. For example, the photoluminescent second agent may consist of a solution containing nanoparticles of YAG:Ce and/or LaPO.sub.4:Ce. In the rest of the description, it is considered that the photoluminescent second agent is formed by nanoparticles of YAG:Ce.

[0114] FIG. 3 illustrates the emission intensity response curves 50-56 and 58-64 of the reduced particles of Gd.sub.0.6Eu0.sub.0.4VO.sub.4 and of YAG:Ce, respectively, in an oxidizing medium with a known concentration of hydrogen peroxide, with a concentration equal to 0 M, 5 M, 10 M and 50 M, respectively. Each curve 50-64 is the average from monitoring about 10 nanoparticles detected individually.

[0115] Determination of the Presence or Absence of Oxidizing Species According to the Invention

[0116] The method for determining the presence of oxidizing species 10 continues with a step 16, consisting of exciting the photoluminescent first and second agents in the sample. For this purpose, the sample may be subjected to double luminous excitation, notably by laser. In the case of the examples of photoluminescent agents considered in the present description, this double excitation of the sample may for example be performed at wavelengths of 396 nm (for Gd.sub.0.6Eu0.sub.0.4VO.sub.4) and 488 nm (for YAG:Ce), by means of a laser diode and an argon laser, respectively.

[0117] The method for detecting oxidizing species 10 then continues with a step 18 consisting of measuring the luminous intensity emitted by the sample at at least one first wavelength corresponding to a luminescence wavelength of the photoluminescent first agent, and at at least one second wavelength, corresponding to a luminescence wavelength of the photoluminescent second agent, the first and second wavelengths being different. In the example considered here, the luminous intensity emitted by the sample is measured at the two luminescence wavelengths of Eu.sup.3+ and Ce.sup.3+, namely 617 nm and 550 nm, respectively.

[0118] In fact, the presence of oxidizing species in the sample causes, for example, oxidation of the Eu.sup.2+ and Ce.sup.3+ ions to Eu.sup.3+ and Ce.sup.4+ ions, respectively. This is reflected in a drop in the luminous intensity of emission of the particles based on cerium at a wavelength of 550 nm and an increase in the luminous intensity of the particles based on europium at a wavelength of 617 nm.

[0119] In the embodiment in which the second agent possesses a constant luminescence regardless of the concentration of oxidizing species to be analyzed, its accredited luminous signal may be used directly as a reference scale. Thus, this signal makes it possible to demonstrate either a change in the luminous intensity of the wavelength or of one of the wavelengths representative of the oxidized form or of the reduced form of the photoluminescent first agent, or a lack of variability of this intensity. A change in luminous intensity is representative of the presence of oxidizing species. Conversely, lack of variability of this luminous intensity indicates absence of oxidizing species in the sample to be analyzed.

[0120] In another embodiment, the method comprises a step 20 of comparison of the measured luminous intensities, at the scale of the individual nanoparticle, of the luminescence of each of the photoluminescent agents against standard values, in order to determine the quantity of oxidizing species in the sample tested. This step is notably necessary in the case when the luminous intensity of the photoluminescent second agent also varies with the quantity of oxidizing species in the analysis sample. The quantity of oxidizing species may notably be a concentration of oxidizing species in the sample tested.

[0121] Here, the standard values may be determined from a sheet 30 as illustrated in FIG. 4. This sheet represents a set of points corresponding to a triplet:

[0122] luminous intensity of the luminescence of the photoluminescent agent comprising the particles based on europium,

[0123] luminous intensity of the luminescence of the photoluminescent agent comprising the particles based on cerium, and

[0124] concentration of oxidizing species (H.sub.2O.sub.2 in the case shown in FIG. 3).

[0125] This sheet 30 may notably be obtained at least partially starting from curves 50-64 in FIG. 3. Here, in fact, as the two photoluminescent agents have an opposite luminescence response as a function of the quantity of oxidizing species, each pair of curves 50-64 corresponding to one and the same concentration of oxidizing species makes it possible to determine a point of the sheet 30. As a variant, sheet 30 may be interpolated based on the data set of curves 50-64 where a pair of normalized luminous intensities corresponds uniquely to a concentration of oxidizing species.

[0126] Thus, advantageously, in the case of different, monotonic dependences of the luminescences of the two agents with the concentration of oxidants, it is possible to construct a sheet uniquely determining a concentration of oxidant based on the instantaneous luminescences of the two agents. Several types of luminescence response may be envisaged for the two agents. However, a preferred variant is the use of two agents having variations of luminescence with opposite response to the oxidants.

[0127] As a variant of this sheet 30, the standard values may be obtained from a table or from computer software, which may also interpolate, from measured points, a concentration of oxidizing species, of a pair of measured intensities.

[0128] In all cases, this comparison of the pair of measured luminous intensities against standard values makes it possible to dispense with measurement of the derivative of the luminous intensities, which is necessary for the method described in Casanova et al., Nat. Nanotech (2009). The method thus offers better temporal resolution.

[0129] The luminous intensity measured at the luminescence wavelengths of the Eu.sup.3+ and Ce.sup.3+ ions therefore makes it possible to determine a point of the sheet 30 corresponding to a unique concentration of oxidizing species present in the sample tested. The uniqueness of this concentration is ensured by the different, monotonic variations of the luminescence intensity of the photoluminescent agents as a function of the quantity of oxidizing species with which they are present.

[0130] The method for detecting oxidizing species according to the invention is therefore particularly advantageous for determining quickly, and relatively simply, a quantity of oxidizing species in a sample from a double measurement of luminous intensity.

[0131] This method allows a ratiometric measurement. Notably, in the case when the luminescence of the photoluminescent first agent varies as a function of the quantity of oxidizing species, but the luminous intensity emitted by the optically active second agent is roughly constant regardless of the concentration of oxidizing species, it may be advantageous to use the ratio between the intensities of emission of the first and second agents for determining the concentration of oxidizing species.

[0132] The method is, moreover, particularly suitable for detecting reactive oxygen species (or ROS) in the bulk. In fact, such detection depends neither on the concentration of photoluminescent agents, nor on the volume of the sample probed. The method may notably be employed for monitoring the production of oxidizing species in living tissues (sampled ex vivo or in vivo).

[0133] It will be recalled that the ROS are known to be produced abnormally in pathologies such as certain cancers, neurodegenerative diseases and cardiovascular diseases. The method described may therefore complement the numerous studies conducted in this field and thus help to respond to the public health challenges posed by these pathologies.

[0134] The method proposed is also advantageously adaptable to variable concentrations of oxidizing species, notably by modifying the luminous intensity of excitation of the photoluminescent agents. The oxidizing species that can be detected using the method include notably, but not exclusively, H.sub.2O.sub.2, NO and ClO. The method makes it possible to determine an absolute concentration of oxidizing species in the sample. It offers better spatial and temporal resolution than the known methods. It does not cause any side effects, and is biocompatible. It may be used for assaying oxidizing species in cells or biological tissues, but also for detecting and/or assaying oxidizing species in a solution of biological molecules.

[0135] The method 10, described above, may notably be implemented by means of a system 100 for determining the presence of oxidizing species as illustrated in FIG. 1. In FIG. 1, the system for determining the presence of oxidizing species in a sample 102 comprises firstly a laser illumination device 104 formed here from two different laser sources 106, 108, configured to emit beams at different wavelengths. In the example considered above in which the photoluminescent agents are Gd.sub.0.6Eu0.sub.0.4VO.sub.4 and YAG:Ce, the first laser source 106 is configured to emit a first laser beam 110 at a wavelength of 488 nm, whereas the second laser source 18 emits a second laser beam 112 with a wavelength of 396 nm.

[0136] The system 100 next comprises a dichroic mirror 114 for combining the first and second laser beams 110, 112, and guiding them toward an optical lens 116 for focusing the laser beams 110, 112 on a zone of the test sample 102. This zone may for example correspond to an area of some mm.sup.2. It should be noted here that the sample 102 may be arranged on or in a sample holder 103. Notably, in the case when the sample is gaseous, this sample holder 103 takes the form of a closed space to prevent dispersion of the test sample 102. The sample holder 103 may also take the form of a cell or cuvette, in particular in the case when the sample 102 is in the form of solution.

[0137] The test sample is brought into contact with the photoluminescent first agent and the optically active second agent, here photoluminescent. As in the example considered previously, the photoluminescent agents in this case take the form of two different types of particles:

[0138] particles of YAG:Ce (designated * in FIG. 5), and

[0139] reduced particles of Gd.sub.0.6EuO.sub.0.4VO.sub.4 (designated o in FIG. 5).

[0140] The first and second laser beams 110, 112 excite the photoluminescent first and second agents in the sample 102. The photoluminescent agents, of different types, respond to the excitation of one of the two laser beams, preferably selectively (i.e. exclusively to one or the other), by emitting in two different wavelength ranges. Two different wavelength ranges means two ranges of wavelengths comprising at least one wavelength not included in the other wavelength range. For example, here, the particles of YAG:Ce respond to the excitation of the first laser beam at 488 nm, by emitting at a first wavelength of 550 nm. The reduced particles of Gd.sub.0.6EuO.sub.0.4VO.sub.4 respond to the excitation of the second laser beam at 396 nm by emitting at a second wavelength of 617 nm.

[0141] The lens 116 makes it possible to focus the emission of the two luminescent agents 118 including the two response wavelengths of the photoluminescent agents onto a spectrum splitter 120, optionally via the dichroic mirror 114 through which it passes. The dichroic filter 114 then performs the role of a separating plate functionally interposed between the laser sources 106, 108 and the sample 102, on the one hand, and between the sample 102 and the spectrum splitter 120, on the other hand, by reflecting the laser sources and by being transparent to the emission of the luminescent agents.

[0142] As a variant, the lens is incorporated in an optical device, notably a microscope, functionally interposed between the sample 102 and the spectrum splitter 120. This microscope may notably be a, preferably wide-field, fluorescence microscope, a macroscope or a stereomicroscope.

[0143] The spectrum splitter 120 makes it possible to obtain, from a single incoming beam 118, two output images 122, 124 filtered at two different wavelengths, here the response wavelengths of the photoluminescent agents in contact with the sample 102. In other words, the spectrum splitter 120 makes it possible to filter the emissions of the photoluminescent agents according to different wavelengths and thus form two separate filtered images of the test sample 102.

[0144] For this purpose, the spectrum splitter 120 in this case comprises a dichroic mirror 126. First 130 and second 132 filters of respective wavelength corresponding to the response wavelengths of the photoluminescent agents are placed respectively in the path of the beam 122 reflected by the dichroic mirror 126 and in the path of the beam 124 passing through the dichroic mirror 126. This allows spatial separation of the beams of different wavelengths for detection on two separate sensors or on two different regions of a single sensor, such as a camera 134.

[0145] The system 100 further comprises means for determining the luminous intensity associated with the two filtered images. Here, these means comprise a camera 134, for example an EMCCD camera (EMCCD: Electron Multiplying Charge Coupled Device) connected to a recorder, for example a computer, for detecting the two filtered images, each on a respective region 136, 138, corresponding to the response of each of the two photoluminescent agents.

[0146] Alternatively, the means employed for determining the luminous intensity comprise two photodetectors at different wavelengths, notably of the photomultiplier or photodiode type. These photodetectors make it possible to measure the total luminous intensity arriving from the whole of the illuminated sample without spatial resolution.

[0147] It should be noted here that the system 100 makes it possible to detect particles of each type simultaneously and individually. In the case when a full-field device is used, such as the camera 134, the particles may moreover be detected individually and localized spatially, because of the low surface density of the sample. Thus, monitoring at the scale of each single particle, of these two luminescence emissions and of their variations allows quantitative, local (notably with a localization precision of 40 nm) and time-resolved measurement, with an acquisition rate of the order of 100 ms, notably 33 ms, of the oxidizing species in the sample. The temporal resolution of the measurement is in fact then given by the acquisition rate of the camera 134.

[0148] The powers of the excitation lasers may, moreover, be modulated to change the range of concentrations of oxidizing species detected. The method and the system for detecting oxidizing species may thus notably be employed in many applications, such as detection of ROS in inflammations or certain tumors.

EXAMPLES

Example 1

[0149] An example of an experiment conducted for detecting oxidizing species produced in a mammalian cell following stimulation by a protein is described in detail below.

[0150] Firstly, mammalian cells were prepared. For this purpose, mouse vascular smooth muscle cells (primary culture) were cultured in RPMI (Roswell Park Memorial Institute medium) containing 10% of fetal calf serum and antibiotics, in the present case penicillin and streptomycin. When confluence reaches 80%, the cells are taken and deposited on glass slides. In this particular case, 1.510.sup.4 cells are typically arranged on a glass slide. The cells are then cultured on the slides for forty-eight hours in the complete culture medium, and then in a medium without serum for twenty-four hours.

[0151] In addition, a stable colloidal solution of Gd.sub.0.4EuO.sub.0.6VO.sub.4 with a concentration roughly equal to 1 mM is prepared. The solution is then centrifuged for five minutes at an acceleration of 5000 g in order to precipitate the particles. The supernatant is removed and the pellet is resuspended in an aqueous solution of sodium borohydride (NaBH.sub.4) of concentration 1 M. Reaction of the NaBH.sub.4 with the particles and with water produces dihydrogen in the form of bubbles. Therefore care is taken not to confine the reaction mixture. After some minutes, the solution is centrifuged again, for five minutes, at an acceleration of 5000 g. The supernatant is removed and the pellet is resuspended in distilled water. The washing operation is repeated twice in order to remove any residual reducing ions.

[0152] A stable solution of YAG:Ce (4%) of about 1 mM is prepared, with water as the main solvent. These particles are synthesized by the glycothermal route in the form of dispersions in ethanol. Washing cycles are carried out: the necessary centrifugations may be effected for 5 minutes at an acceleration of 5000 g and then the particles are resuspended in the solvent selected for the measurements, water in the case described here.

[0153] In addition, 10 L of suspension of YAG:Ce and 15 L of suspension of reduced and washed particles of Gd.sub.0.4EuO.sub.0.6VO.sub.4 are mixed in a commercial solution of hypertonic medium (LifeTechnologies). The cells are incubated with this solution for ten minutes and then two minutes in a hypotonic solution containing 70% of RPMI and 30% of sterile distilled water. Then the cells are incubated in their complete medium for thirty to sixty minutes.

[0154] Then a slide with cells loaded with nanoparticles is placed on a sample holder and 1 mL of buffered physiological medium (HBSS/1 mM HEPES) is added. The slide is then mounted on an epifluorescence microscope for observation of individual particles (Olympus IX71, Objective x63 NA=1.4). Then the cells are observed in transmission of white light, as illustrated in FIG. 6, and under dual illumination (laser sources at 488 nm and 396 nm), as illustrated in FIG. 7. A cell having a suitable morphology is selected, i.e. a cell that is alive and adherent. After fifty seconds of observation, the initial medium is replaced with physiological medium containing 270 nM of endothelin-1 (ET-1) and the luminescence of individual nanoparticles is observed for several minutes on a full-field sensor (EM-CCD ImageEM camera, Hamamatsu). FIG. 8 illustrates the response curves 200, 202 corresponding to the particles based on Eu and Ce, respectively. We observe an increase in luminous intensity of the particles based on Eu and a decrease in luminous intensity of the particles based on Ce.

[0155] Next, the variations in intensity of the particles observed (doped with Ce or Eu) over time are determined using image analysis or calculation software (for example Matlab). Each curve obtained is normalized, taking the first values measured for reference. The concentrations of hydrogen peroxide are found from the sheet 30 in FIG. 4. The curve 204 illustrated in FIG. 9 is thus obtained, as well as the corresponding smoothed curve 206, which gives the variation of the concentration of H.sub.2O.sub.2 in the sample tested, as a function of time.

Example 2

[0156] FIG. 10 illustrates another example of use of the method according to the invention, in which the sample to be analyzed is contained in a vessel. The two agents, YAG:Ce and GdVO.sub.4:Eu respectively, are put in the vessel in solution. They are excited by specific sources. After adding 2 mM of hydrogen peroxide (at t=0), the luminescence of each of the agents, detected at 550 nm and 617 nm, is collected on two photomultipliers, to form the two curves 208, 210, respectively.

Example 3

[0157] The vascular smooth muscle cells coat the walls of blood vessels. Stimulation of them by a powerful vasoconstrictor such as endothelin-1 (ET-1) causes them to contract and induces an increase in arterial pressure. The cell signaling process responsible for this response is known to induce production of hydrogen peroxide (oxidizing species). Moreover, the membrane receptors EGFR (Epidermal Growth Factor Receptor) are also known to participate in this process. However, their contribution and in particular their effect on the production of oxidants are still unknown. This is because there are no methods for quantitative detection that are fast enough for characterizing this phenomenon.

[0158] In this example, the transactivation of the EGFR receptors in response to the vasoconstrictor ET-1 was studied by the method of the invention.

[0159] For this, the same protocol as in example 1 was followed, namely culture of vascular smooth muscle cells, preparation of the suspension of reduced particles YAG:Ce and Gd.sub.0.4EuO.sub.0.6VO.sub.4, and incubation of the cultured cells with the suspension.

[0160] The presence of these particles was observed by fluorescence microscopy and spectrum splitting, as indicated in example 1 (FIG. 11). A mixture of nanoparticles (GdVO.sub.4:Eu reduced chemically beforehand and YAG:Ce) is internalized in vascular smooth muscle cells deposited on a glass slide (170 m) and the slide is mounted on an epifluorescence microscope for observation of individual particles (Olympus IX71, Objective x63 NA=1.4) equipped with a spectrum splitter allowing the photoluminescence of each type of particle to be projected on two different regions of an ultra-sensitive camera (EM-CCD, Evolve 512 Roper Scientific).

[0161] The stimulation of normal cells and of cells where the EGFRs have specifically been inhibited by the presence of the agent AG1478 (application 30 min before stimulation and addition to the medium for observation under the microscope) was performed with a concentration of 270 nM of endothelin-1. Immediately after stimulation, the luminescence signals emitted by the different particles in the cells were collected. Thus, FIG. 12 shows the photoluminescences normalized by their initial value of the nanoparticles YAG:Ce (below 1) and GdVO.sub.4:Eu (above 1) obtained after stimulation by ET-1 (with or without inhibition of the EGFRs).

[0162] Next, owing to calibration as described above, the temporal variation of the absolute concentrations of hydrogen peroxide was deduced (FIG. 13) in the two cellular conditions (normal or EGFRs inhibited). This step makes it possible to quantify the contribution of the EGFR pathway to the overall signaling process. This contribution reaches 50%, since the inhibited cells produce half as much hydrogen peroxide as the normal cells. It also makes it possible to quantify the effect of this inhibition on the kinetics of production of hydrogen peroxide, in contrast for example to Bouzigues et al., Chem. Biol., 2014; 21:647-56, by revealing that the transactivation of the EGRs takes place in less than 1 second and controls said kinetics of production to times <1 min.

[0163] Of course, the present invention is not limited to just the examples described above; many variants are accessible by a person skilled in the art, within the scope of the accompanying claims.

[0164] Thus, for example, the optically active second agent may consist of any type of photostable agent, such as semiconducting nanocrystals, for example.

[0165] Moreover, the particles may be functionalized and coupled to one or more biomolecules of interest notably for the purpose of targeting either specific cellular compartments in the case of measurements at the cellular level, or specific cell types, for example cancer cells, in the case of measurements at the level of tissues or in vivo (in living organisms).

[0166] More precisely, the biologically active molecules are targeting molecules, i.e. molecules that will allow specific targeting of the particle according to the invention to an organ, a body fluid (for example blood), a cellular type (for example platelets, lymphocytes, monocytes, tumor cells, etc.) or a cellular compartment. Thus, this specific targeting may be accomplished using monoclonal or polyclonal antibodies, or protein or polypeptide ligands of cellular receptors. As nonlimiting examples, we may mention the following receptor/ligand pairs: TGF/TGFR, EGF/EGFR, TNF/TNFR, interferon/interferon receptor, interleukin/interleukin receptor, GMCSF/GMCSF receptor, MSCF/MSCF receptor, and GCSF/GCSF receptor. We may also mention, as ligands, toxin fragments or detoxified toxins and their cellular receptors. Regarding the antibodies, they will be selected as a function of the antigen or antigens against which the antibody is directed.

[0167] In another embodiment, the biologically active molecule or molecules are stealth agents, such as polyethylene glycol (PEG) or dextran, for endowing the particles with stealth characteristics in the body and thus increasing their circulation time in the blood.

[0168] In a particular embodiment, the particles according to the invention may be functionalized with targeting molecules and stealth molecules as defined above.

[0169] Regardless of the embodiment, the biologically active molecules may be attached to the surface of the particle or if applicable to the preparation layer, directly or via a layer bearing functional groups, by covalent or noncovalent bonds. Attachment of these biologically active molecules is performed by the conventional techniques of oxidation, halogenation, alkylation, acylation, addition, substitution or amidation of the surface of the particle, of the preparation layer and/or of the layer bearing functional groups, with the biologically active molecules.

[0170] The preparation layer is applied directly on the particle, either by covalent binding or by absorption. This preparation layer may be hydrophilic or hydrophobic. In a particular embodiment, this preparation layer is amorphous.

[0171] Coupling between the particles and a biomolecule is described for example in application WO-A-2012/010811. Furthermore, application WO-A-2013/123197 describes coating of the particles, which may lead to particles coupled to biomolecules.

[0172] Moreover, in order to improve the signal to noise ratio, it is possible to use delayed detection of the signal emitted by the europium-doped particles, which have a long lifetime of the excited state compared to most fluorophores. With the aid of a device for occultation, notably mechanical or electronic, of the means for determining the luminous intensity, selectively activatable, this approach consists of detecting, after a laser pulse for excitation of the first and second agents, only the photons emitted after a set time. This set time may be selected so as not to detect the photons coming from other emitters, for example by autofluorescence, and only detect photons emitted by the YVO.sub.4:Eu nanoparticles.