CN-122003481-A - Ultrasensitive detection method using photoluminescent particles

CN122003481ACN 122003481 ACN122003481 ACN 122003481ACN-122003481-A

Abstract

The invention relates to a method for ultrasensitive in vitro detection and/or quantification of biological or chemical substances of interest in a sample by detecting luminescence emissions from inorganic photoluminescent nanoparticles, comprising at least the steps of arranging specific inorganic photoluminescent nanoparticles having vanadate or vanadate/phosphate matrices, which nanoparticles are coupled with a coupling agent for coupling to the substance of interest, contacting photoluminescent particles with the substance of interest under conditions for coupling the substance of interest with the coupling agent, exciting the matrix of inorganic photoluminescent nanoparticles by radiation having a wavelength of 240 nm to 330 nm, detecting the luminescence emissions of inorganic photoluminescent nanoparticles, and determining the presence and/or concentration of the substance of interest by interpretation of the measurement of the luminescence emissions of the particles.

Inventors

R. Quiner
Cedric buzger
C. Cardoni
R. Lestini
Antigone Alessandro
R. Viera Pereira
T. G.J. Gakuan

Assignees

巴黎综合理工学院
国家科学研究中心
国家健康与医学研究院（INSERM）

Dates

Publication Date: 20260508
Application Date: 20240809
Priority Date: 20230811

Claims (15)

1. Method for ultrasensitive in vitro detection and/or quantification of biological or chemical substances (40) of interest in a sample (35), in particular a biological sample, by detecting the luminescent emission emitted by photoluminescent inorganic nanoparticles, said method comprising at least the following steps: (i) Providing photoluminescent particles (38) formed in whole or in part from photoluminescent inorganic nanoparticles having a vanadate or vanadate/phosphate matrix of formula (I): A 1-x Ln x VO 4(1-y) (PO 4 ) y (I) Wherein: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La) and mixtures thereof, in particular A represents Y; ln is selected from europium (Eu), dysprosium (Dy), thulium (Tm), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb) and mixtures thereof, in particular Ln represents Eu; 0< x <1, especially 0.02≤x≤0.5, especially 0.05≤x≤0.4, more especially x is 0.4, 0.2, 0.1 or 0.05, and 0.Ltoreq.y <1, in particular y is equal to 0, Coupling the photoluminescent inorganic nanoparticles to a reagent for direct (42) or indirect (44) coupling with a substance of interest; (ii) Contacting the photoluminescent particles (38) with the substance of interest (40) under conditions for coupling the substance of interest (40) with a coupling agent (42, 44); (iii) Exciting the matrix of photoluminescent inorganic nanoparticles of formula (I) with radiation having a wavelength of 240 nm to 330 nm, the radiation being emitted by a lighting device having a power of 50 mW to 500 mW; (iv) Detecting the luminescence emission, in particular time-resolved detection, of said photoluminescent inorganic nanoparticles, and (V) Determining the presence and/or concentration of the substance of interest by interpreting the measurement of the luminescence emission of the particles, The method has a detection sensitivity of less than or equal to 10 pM for the biological or chemical substance of interest in the sample.
2. The method of claim 1, which has a detection sensitivity of less than 1 pM, or even less than 0.1 pM, or even less than 0.01 pM, or even less than or equal to 1 fM, or even better still less than or equal to 0.1 fM (i.e. 100 aM), or even better still less than or equal to 0.01 fM (i.e. 10 aM) for the biological or chemical substance of interest in the sample, in particular without amplification and without the use of enzymes.
3. A method according to any one of the preceding claims, wherein the coupling agent is an agent (42) for targeting the substance of interest, the agent (42) being directly coupled with the substance of interest in step (ii), or the coupling agent is a molecule (44), the molecule (44) allowing binding of an agent for targeting the substance of interest, in particular binding of the agent for targeting the substance of interest to the substance of interest before contacting the photoluminescent particles with the substance of interest.
4. A method according to any of the preceding claims, wherein at least one nanoparticle is coupled with a plurality of coupling agents, the method comprising the step of coupling the nanoparticle with the coupling agents, comprising dissolving the nanoparticle in a proportion of coupling agents greater than the nanoparticle, in particular in a proportion of at least 2 coupling agents per nanoparticle, or even at least 10 coupling agents per nanoparticle, or even from 10 to 80 coupling agents.
5. A method as claimed in claim 4, wherein the photoluminescent particles each comprise a plurality of nanoparticles bound together, in particular by the coupling agent, to form nanoparticle aggregates, preferably in solution in the form of a colloidal dispersion in step (ii) of contacting with the substance of interest.
6. A method according to any one of the preceding claims, using at least two different types of photoluminescent particles, each type comprising nanoparticles according to formula (I) coupled with different coupling agents suitable for binding to different sites on the same analyte, in particular coupled with different oligonucleotides recognizing different regions of a nucleic acid type analyte.
7. The method according to any of the preceding claims, wherein the nanoparticles are each coupled with a plurality of coupling agents at several regions on the analyte, in particular with a plurality of oligonucleotides selected to bind to different regions on the nucleic acid.
8. Method according to any of the preceding claims, wherein the substance of interest of the sample is first immobilized on a surface of a support, which surface is passivated to prevent binding of the luminescent particles to the surface in the absence of the substance of interest in step (i), comprising in particular at least the following steps: (a) Providing a support, the surface of which is pre-passivated and functionalized with a reagent for capturing the substance to be detected/quantified, for example a monoclonal antibody, called capture antibody; (b) Contacting the sample to be analysed with the support from step (a) under conditions which favour binding of the substance to the capture agent, and (C) Contacting the photoluminescent particles coupled with at least one coupling agent with the support from step (b) so as to bind the particles directly or indirectly to the substance immobilized on the surface of the support.
9. A method according to any one of the preceding claims, using at least two types of nanoparticles doped with different rare earth metal ions, having different emission wavelengths, and coupled with different direct or indirect coupling agents, each coupled with one of two different substances of interest.
10. The method according to any of the preceding claims, which is performed using an apparatus comprising: A lighting device with a wavelength between 240 nm and 330 nm, better still between 260 nm and 330 nm, better still between 260 nm and 310 nm, better still between 270 nm and 290 nm, preferably of the Light Emitting Diode (LED) type (1), preferably with a power between 50 mW and 500 mW, better still between 50 mW and 150 mW, and -Means for detecting the intensity of light emitted by said nanoparticles in step (iii), in particular a single detector, for example of the photomultiplier type detecting only visible light, a photodiode, an avalanche photodiode, or a detector of the type of an array of photosensitive devices consisting of the 2D surface of the detection pixels, for example a CCD or EM-CCD camera or a CMOS camera.
11. The method according to any of the preceding claims, wherein the detection is time-resolved, the time-resolved detection being obtained by electronically or mechanically chopping the incident UV-B and/or UV-C beam.
12. The method according to any of the preceding claims, the nanoparticles being prepared by colloidal conversion of rare earth metal hydroxycarbonate particles, in particular by at least the following steps: (a) Preparing an aqueous solution (1) by mixing metavanadate, in particular ammonium metavanadate (NH 4 VO 3 ), and optionally phosphate, in an aqueous medium; (b) Preparing hydroxycarbonate nanoparticles of formula a 1-x Ln x 3+ CO 3 OH from precursors of elements a and Ln, in particular in salt form, in particular nitrate, and a source of bicarbonate ions, in particular excess, in particular urea, under conditions conducive to the formation of hydroxycarbonate nanoparticles by co-precipitation; (b') adding said aqueous solution (1) to said hydroxycarbonate nanoparticles in a colloidal suspension under conditions conducive to the formation of nanoparticles according to formula I by co-precipitation, and (C) Recovering the nanoparticle according to formula I.
13. A method according to any of the preceding claims, comprising in step (i), preferably before the nanoparticle is coupled with the coupling agent, mixing the nanoparticle with a protecting agent, then post-synthesis annealing at a temperature between 500 ℃ and 1500 ℃, more particularly between 800 ℃ and 1300 ℃, followed by removal of the protecting agent by a method suitable for the protecting agent, particularly by acid dissolution.
14. Use of a method as defined in any of the preceding claims for in vitro diagnostic purposes.
15. An in vitro diagnostic kit, in particular for performing the method according to any one of claims 1 to 13, comprising at least: -photoluminescent particles formed wholly or partly from photoluminescent inorganic nanoparticles having a vanadate or vanadate/phosphate matrix of formula (I): A 1-x Ln x VO 4(1-y) (PO 4 ) y (I) Wherein: A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La) and mixtures thereof, in particular A represents Y, Ln is selected from the group consisting of europium (Eu), dysprosium (Dy), thulium (Tm), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb) and mixtures thereof, in particular Ln represents Eu, 0< X <1, especially 0.02≤x≤0.5, especially 0.05≤x≤0.4, more especially x is 0.4, 0.2, 0.1 or 0.05, and 0.Ltoreq.y <1, in particular y is equal to 0, The particles are surface-functionalized with chemical groups provided by molecules, such as carboxyl, amino, mercapto, aldehyde or epoxy groups, molecules such as citric acid or polyacrylic acid, and/or particles are coupled to molecules, such as streptavidin, which are suitable for allowing the particles to be coupled to reagents for targeting a substance of interest, or The particles having been coupled with at least one agent for targeting a substance of interest, and -A detection and/or quantification system comprising at least: A lighting device with a wavelength between 240 nm and 330 nm, better still between 260 nm and 330 nm, better still between 260 nm and 310 nm, better still between 270 nm and 290 nm, preferably of the Light Emitting Diode (LED) type (1), preferably with a power between 50 mW and 500 mW, better still between 50 mW and 150 mW, and Means for detecting the intensity of the light emitted by said particles, and -Optionally, a support suitable for immobilizing a substance of interest of the sample.

Description

Ultrasensitive detection method using photoluminescent particles Technical Field The present invention relates to the fields of research, biological analysis and in vitro diagnostics. More specifically, the subject of the present invention is a method for ultrasensitive in vitro detection and/or quantification of biological or chemical substances of interest (e.g. biomarkers, proteins, peptides, hormones, antibodies, DNA, RNA and other compounds) in a sample (in particular a biological sample) by detecting the luminescent emission of photoluminescent inorganic nanoparticles with controlled optical and physicochemical properties. Background Detection and/or quantification of the concentration of biomarkers, antibodies, or DNA and RNA in biological samples (blood, serum, saliva, urine, cerebrospinal fluid, etc.) is very important for medical diagnosis. In the field of research, in vitro or ex vivo diagnostics, medical analysis and biological analysis, several methods have been proposed to detect and/or measure the presence of specific substances. Generally, these methods are based on the use of probes to detect and/or quantify the concentration in a solution. These probes are coupled to a recognition compound or targeting agent, enabling the probes to be attached to the molecular species to be analyzed. The recognition compound may be a molecule, DNA, aptamer, protein, or antibody. Probes that have been attached to the molecular species to be analyzed using a recognition compound can then be detected using one or more methods based on, for example, their luminescence, absorbance, chemical reactivity, radioactivity, and the like. The most commonly used biochemical assay, particularly for protein detection, is the enzyme-linked immunosorbent assay (ELISA), which generally relies on the use of horseradish peroxidase as an enzyme to cause a reaction with a substrate and to quantify the chemical reaction that occurs by measuring the absorbance of the reaction product in solution. The selection of the molecular recognition compound to which the probes are coupled determines the effectiveness of these probes. More specifically, the effectiveness of these methods depends on the specific affinity of the recognition compound for the target substance. For example, references [1] and [2] describe in detail the features of these mechanisms. Luminescent probes typically perform more sensitive detection than probes that detect by absorbance, because in the first case the measurement of light intensity is performed on a black background, while in the second case the change in light intensity is measured (measured on a bright background). Among the other assay methods currently proposed, mention may also be made of electrochemiluminescence immunoassays (ECLIA), fluorescence Immunoassays (FIA) and Radioimmunoassays (RIA). However, these methods have various drawbacks that limit their final detection sensitivity, in particular in terms of the luminescence characteristics of the probes (ECLIA, FIA) used, safety risks, expensive equipment, and the need for professional users (no automated machines) to perform RIA-type tests. In particular, currently available luminescent probes have several drawbacks that do not allow to fully exploit their potential as diagnostic probes. These include, for example, the photobleaching phenomenon in the case of organic fluorophores, which is reflected as the disappearance of fluorescence after an illumination-induced irreversible chemical change, or the emission scintillation phenomenon of semiconductor nanocrystals or "quantum dots", in which the probe periodically stops emitting, and is therefore unsuitable for generating a constant signal. Other disadvantages are caused, for example, by the width of the emission spectrum of the luminescent probe. In particular, an emission spectrum that is too broad makes it difficult to filter out any background signals that may be present and affects the signal quality, in particular the signal-to-noise ratio. In addition to the optical factors that contribute to the effectiveness of the probe in biological assays, the practicality and ease of use of the probe should also be considered. Thus, certain particles (e.g., in the case of semiconductor nanocrystals) lose their luminescent characteristics after freezing, which represents a disadvantage of bio-conjugation agent storage. The ease of coupling the probe to a molecular compound that allows for targeting of the desired molecule is also an aspect of consideration in selecting an appropriate probe. Thus, many particles, including semiconductor nanocrystals, are synthesized in organic solvents. As a result, their use in biological applications requires an additional surface preparation step to disperse these particles in water, a process that can be complex to implement and unstable over time [3]. They utilize chemical groups to allow functionalization coupled with molecular compounds that recognize target mo