Pre-functionalized nanoparticles using self-assembled monolayers and methods of making the same

Abstract

The present invention is in the field of pre-functionalized nanoparticles (NPs). It more particularly relates to the use of NPs pre-functionalized with self-assembled monolayers (SAMs), and also to the use of NPs functionalized with biomolecules, such that the NPs are stable in solution. These NPs can be used in a variety of applications, in particular as diagnostic tools, depletion tools of target molecules in solution, and therapeutic tools.

Description

[0001] This invention relates to the field of prefunctionalized nanoparticles (NPs). More specifically, it relates to NPs prefunctionalized using self-assembled monolayers (SAMs), and NPs functionalized using biomolecules, such that the NPs are stable in solution. The NPs can be used in a variety of applications, particularly as diagnostic tools, tools for depleting or concentrating target molecules (molecules of interest) in solution, and therapeutic tools.

[0002] Existing technology and disadvantages

[0003] Nanoparticles coated with a layer of SAM have been described in the literature. For example, Hurst KM et al. (Journal of Microelectromechanical Systems; Vol. 20, No. 2, April 2011) described immobilizing a SAM composed of p-aminophenyltrimethoxysilane (APhTS) and 3-mercaptopropyltrimethoxysilane (MPTS) onto nanoparticles (NPs) to stabilize the adhesion of the NPs to the silanized surface of the microelectromechanical system. However, such NPs are not suitable for functionalization using biomolecules.

[0004] Other existing technical literature describes nanoparticles covered with a thiolized monolayer.

[0005] Hurst et al. (2006) described gold nanoparticles carrying DNA molecules, and the aim of this study was to charge the maximum amount of DNA molecules on the surface of the nanoparticles. The authors demonstrated that the maximum carrying capacity could be achieved when the salinity conditions were controlled (0.7 M NaCl) and the DNA molecules contained polyethylene glycol (PEG) type spacers.

[0006] Wang et al. (RSC Adv. 2017, 7.3676-3679) described the use of a hybrid monolayer functionalized spherical gold nanoparticles containing PEG and thiolated DNA. The nanoparticles were prepared in two steps: functionalization with thiolated DNA molecules followed by thiolated PEG to cover the entire surface of the NP.

[0007] Li et al. (Langmuir 2015, DOI:10.1021 / acs.iangmuir.5b01680) described the use of DNA-functionalized nanorods (NRs). Since gold NRs are positively charged, they tend to link together in the presence of negatively charged DNA. The authors proposed grafting PEG and a surfactant onto the NR long molecules before adding thiol-modified DNA.

[0008] Reference US 2011 / 165077 describes the use of gold nanoparticles for imaging. Reporter molecules are grafted onto NP surfaces, and then these are covered to the saturation point with a PEG-SH protective layer; this layer prevents particle aggregation and allows for functionalization using specific probes.

[0009] Document US 2019 / 142966 describes the use of metallic NPs intended for cancer treatment. These NPs are coated with PEG-COOH molecules in one step, and then the carboxyl groups are functionalized with therapeutic and target molecules in the presence of SDS.

[0010] Document US 2016 / 243254 describes the preparation of nanoprobes as therapeutic diagnostic tools for cancer treatment. A first layer of thiolized PEG-COOH is grafted onto the NP surface; due to the high molecular weight of PEG-COOH, this first layer covers approximately 30% (representing maximum coverage) of the NP surface. A second layer of thiolized DNA-harpin molecules is added and sandwiched between the PEG molecules. In this construction, the combination of partial coverage and carboxyl functional groups favors the emergence of nonspecific interactions.

[0011] In the field of NPs intended for use as supports for immobilized biological probes, various constraints must be considered. First, pre-functionalized or functionalized NPs must be stable in solution, i.e., they must not aggregate. They must also allow specific probe / target molecule interactions.

[0012] Summary and advantages of the invention

[0013] This invention provides a solution to these problems by prefunctionalizing poly(NP) molecules (NPs) using a self-assembled monolayer (partially covering) formed from low molecular weight polyethylene glycol (PEG) molecules. The presence of this pre-layer on the NP surface allows for the preparation of solution-stable NPs, either alone or in combination with other molecules (functionalization). The unique feature of this monolayer is that it consists of molecules with low molecular weights (100 to 732 Daltons) and short molecular chains; the volume of the surface region is optimal, providing the following innovations for nanoparticles.

[0014] The structure of the elements grafted onto the NP surface is as follows: Figure 1 As shown.

[0015] The functionalization strategy according to the invention is a type of surface chemistry that can improve the stability of metal nanoparticles (e.g., gold) or core-shell hybrid nanoparticles in buffer solutions and complex media by controlling the number of ligands (biomolecules) immobilized on the surface of nanoparticles, by suppressing non-specific interactions, and by optimizing specific interactions. The technique involves pre-functionalization prior to immobilizing one or more biomolecules acting as probes. The biomolecules contain thiolated groups that allow them to be directly immobilized on the surface of the NP, or the immobilization of the biomolecules is performed in two steps: 1- immobilizing a linker molecule that has a thiol on one hand (for immobilizing it on the metal surface) and a reactive functional group on the other hand (for immobilizing the biomolecule), and then 2- adding a biomolecule (probe or active molecule) containing a group capable of reacting or interacting with the functional group present on the linker molecule. The method described herein is particularly applicable to spherical NPs, nanorods, nanocubes, nanotriangles, and nano-urchins.

[0016] Furthermore, this pre-functionalization allows for optimization of probe density, uniform probe distribution, and the absence of non-specific interactions. In particular, this invention enables the creation of NPs that utilize probe functionalization with low and optimized density to achieve high detection sensitivity. Multiple different probes can also be combined while maintaining controlled density and distribution for each probe. Invention Details

[0018] The first object of the present invention relates to a nanoparticle comprising a metal surface prefunctionalized with a self-assembled monolayer (SAM), the SAM being formed from a matrix of molecules having a thiol functional group at one end and an inert molecule at the other end, the nanoparticle being characterized in that the coverage W of the monolayer is 1.5% to 99%.

[0019] The unique feature of the self-assembled monolayer is that it is applied to the surface before NP functionalization and is unsaturated. It serves to protect the surface. This monolayer is formed from the molecule M of formula (I):

[0020] HS(CH2)n(OCH2CH2) m OH

[0021] in:

[0022] n represents the quantity of CH2, where 3 ≤ n ≤ 11

[0023] m represents the amount of ethylene glycol, where 1 ≤ m ≤ 12.

[0024] The molecular weight of molecule M ranges from 100 to 732 Daltons.

[0025] In general, a minimum coverage W of 1.5% is required to observe the effect of the SAM layer; low coverage allows for high densities of biomolecules on the NP surface, which can be significant, for example, when two different biomolecules are present. In contrast, coverage W can be higher, close to the saturation point (e.g., 99%), allowing for the deposition of individual molecules; the only limitation of this approach due to the lack of appropriately sensitive tools is likely the limitation of detection. In any case, the fact that the NP surface is prefunctionalized allows for the immobilization of biomolecules in a controlled manner, i.e., ensuring that the average distance between the immobilized biomolecules is uniform.

[0026] In specific implementations, depending on the application, the coverage W can be from 3% to 80%, for example, from 10% to 50%, or from 5% to 30%, particularly 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 98%.

[0027] Within the meaning of this invention, "coverage" refers to the theoretical initial coverage; this corresponds to the surface area of ​​the molecule and is calculated according to the following formula (2):

[0028] (in nm) 2 (Imprint of expression) × 100 / surface area of ​​nanoparticles

[0029] Within the scope of this invention, "nanoparticles" (NPs) can be spherical nanoparticles (NS), nanorods (NR), cubic nanoparticles (NC), nanotriangular sheets (NT), or nano-sea urchins. These can be, in particular, spherical gold nanoparticles with a diameter of 10 nm to 80 nm, nanorods with an aspect ratio of 2.5 to 5, and nanocubes with a side length of 40 nm, or nanotriangular sheets with a side length of 30 nm to 100 nm and a thickness of 10-40 nm. NPs can also be hybrid NPs of the same shape (spheres, nanorods, etc.), including, for example, an oxide type (e.g., iron oxide, silicon oxide) or a metal core and a metal shell (e.g., gold). The metallic surface of core-shell hybrid nanoparticles is similar to the surface of metallic nanoparticles, and its surface is calculated using the same method.

[0030] The surface area of ​​a molecule depends on its shape, and this must be known to determine the coverage.

[0031] In the case of spherical nanoparticles, the surface area corresponds to the following equation (3): 4πr 2

[0032] Where r corresponds to the radius of the nanoparticle.

[0033] In the case of nanorods, the surface area corresponds to the following equation (4):

[0034] 2πrl+2πr 2

[0035] Where r corresponds to the radius of the nanorod and l corresponds to its length.

[0036] In the case of nanocubes, the surface area corresponds to the following equation (5):

[0037] 6a 2

[0038] Where 'a' corresponds to the side length of the nanocube.

[0039] In the case of nanotriangular sheets, the surface area corresponds to the following equation (6):

[0040] (ph / 2)+b

[0041] Where p corresponds to the perimeter of the base, h corresponds to the height of the slope, and b corresponds to the area of ​​the base.

[0042] The molecules present on the surface of NPs for their prefunctionalization or functionalization can be selected from the following molecules: such as molecule M as defined above, molecules L-PEG and PEG-F as defined below, as well as double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), RNA, proteins such as antibodies or peptides, aptamers, etc.

[0043] Therefore, in order to determine the coverage, it is also necessary to know the theoretical imprint of the molecules present on the NP surface:

[0044] The imprints of M and PEG-F are 0.23 nm. 2 [1]

[0045] L-PEG imprint = 0.45nm 2 [2]

[0046] dsDNA imprinting = 3.14nm 2

[0047] In an advantageous embodiment, the prefunctionalized nanoparticles according to the invention are functionalized using at least one L-PEG molecule, with a coverage X of at least 1%, preferably 1% to 10%.

[0048] "L-PEG molecule" refers to a highly long and high molecular weight thiolated polyethylene glycol molecule.

[0049] L-PEG molecules can have formula (7):

[0050] SH-CH2CH2(CH2CH2O) q OF

[0051] in:

[0052] q represents the amount of ethylene glycol, where 20 ≤ q ≤ 500;

[0053] F represents functional groups such as CH3, OH, COOH, NH2, etc.

[0054] The molecular weight of L-PEG molecules is greater than or equal to 800 Daltons.

[0055] Functionalization of L-PEG molecules is particularly significant for stabilizing NPs in PBS solutions. It can be combined with prefunctionalization using molecule M to prepare "ready-to-use" NPs capable of adsorbing one or more biomolecules.

[0056] Furthermore, when the group F is different from CH3 or OH, it can interact with and / or react with biomolecules to add probes to the NP surface.

[0057] In a specific embodiment of the present invention, the prefunctionalized nanoparticles according to the present invention are functionalized using at least one thiol molecule, wherein the thiol molecule is:

[0058] - A thiolized linker molecule containing a thiol functional group at one end and an active or reactive group capable of interacting with biomolecules at the other end.

[0059] - Or biomolecules containing thiol functional groups.

[0060] The nanoparticles can be further functionalized using L-PEG molecules as described above.

[0061] In the embodiment of interest, the thiolized linker molecule is a PEG-F molecule, where F is an activated functional group, such as COOH, and its coverage Y is at least 1%. The overall coverage of the NP surface can be partial or complete (saturated), depending on the application. In a preferred embodiment, the overall coverage is partial.

[0062] "PEG-F" refers to thiolated polyethylene glycol molecules.

[0063] PEG-F molecules can, for example, have the following formula (8):

[0064] HS(CH2) r (OCH2CH2) p OF, where:

[0065] r represents the quantity of CH2, and is an integer greater than or equal to 3;

[0066] p represents the amount of ethylene glycol, where 2 ≤ p ≤ 12, and

[0067] F represents functional groups such as CH3, COOH, and NH2.

[0068] The molecular weight of PEG-F molecules can be low or high, extending to over 800 kDa.

[0069] In an alternative embodiment, the linker molecule may be an L-PEG molecule containing an activatable functional group.

[0070] In another embodiment of interest, the prefunctionalized nanoparticles according to the invention are functionalized using thiolized biomolecules (referred to as "thiolized probes") that act as probes, such as nucleic acids (DNA, RNA, PNA, etc.), proteins (antibodies, antigens, hormones, etc.), peptides, hormones, sugars, fatty acids, or even the entire cell.

[0071] In another embodiment of interest, the thiolized biomolecules are small molecules, such as drugs (sandwiched or complexed), referred to herein as "active molecules". These active molecules can also bind via activatable functional groups present on PEG-F or L-PEG molecules.

[0072] When the thiolized molecule is double-stranded or single-stranded DNA, the coverage Z is at least 10%. The overall coverage of the NP surface can be partial or complete (saturated), depending on the application. In a preferred embodiment, the overall coverage is partial.

[0073] In a preferred embodiment, when PEG-F is short, L-PEG molecules are added at a ratio of 1% to stabilize NP.

[0074] Within the meaning of this invention, a "thiolized probe" refers to a biomolecule that acts as a probe capable of specifically interacting with (capturing) target molecules in aqueous media (e.g., buffer solutions) or complex media (culture media, biological culture media, body fluids, liquid matrices such as blood, plasma, serum, urine, tears, cell extracts, etc.). The biomolecule acting as the probe is immobilized on the support of the NP as follows: (i) through direct interaction between the thiolized group present on the biomolecule and the surface of the NP, or (ii) by means of immobilization at one end of the thiolized molecule, which has an active or reactive group at the other end, capable of reacting with (binding) the binding molecule by forming a "strong" bond (e.g., a covalent bond such as an amide bond, chelation, or a "tag" type strong affinity such as a streptavidin-biotin bond); the thiolized molecule is referred to herein as a "connector molecule".

[0075] Once the substrate is protected by the protective layer SAM as described above, the additional step of depositing the thiolization probe is performed immediately or just before use, or hours, days, or months later.

[0076] These thiolation probes can be:

[0077] • The incubation time required for direct thiolization probes depends on the concentration, which is typically low (micromolar level, or has a low concentration);

[0078] • Thiolized molecules possessing reactive amine or carboxyl groups, carbon-carbon double or triple bonds, epoxy groups, click chemistry, etc.

[0079] • Thiolized molecules that have active groups that interact with complementary molecules to form sandwich structures (e.g., biotin-streptavidin-biotin).

[0080] A second object of the present invention relates to a method for preparing nanoparticles comprising a metal surface prefunctionalized using a self-assembled monolayer (SAM), the SAM being formed from a solution of a molecule having a thiol functional group at one end and an inert end, the method comprising the following steps:

[0081] - Add a molecule M capable of forming a self-assembled monolayer to achieve a coverage of 1.5% to 99% W.

[0082] - Stir for at least 5 minutes.

[0083] This method allows for the preparation of prefunctionalized NPs that are stable in water and PBS. NPs prepared in this way can be stored at 4°C for several months until use.

[0084] In this method, a surfactant may be added. The surfactant used may be selected from all surfactants known to those skilled in the art, particularly SDS, CHARS, NP40, Tween20 (SDS: sodium dodecyl sulfate, CHARS: 3-[(3-cholanopropyl)dimethylamino]-1-propanesulfonate), etc. Those skilled in the art are familiar with the use of surfactants to prepare NP in solution.

[0085] In a specific embodiment of the present invention, an L-PEG molecular solution is further added to an NP solution prefunctionalized with a SAM layer to achieve a coverage of 1% to 10% Y, and then incubated while stirring for at least 5 minutes, typically 5 minutes to 1 hour.

[0086] The NP, prefunctionalized with molecule M and optionally functionalized with L-PEG molecules, can be further functionalized with thiolated molecules and / or biomolecules. In an alternative embodiment, the L-PEG molecule can be added after the PEG-F molecule.

[0087] This preparation method includes the following steps:

[0088] - Add a thiolation molecule, selected from thiolation linker molecules or thiolation biomolecules capable of interacting with biomolecules.

[0089] - Stirring, for example, from 5 minutes to 24 hours.

[0090] - If the thiolated molecule is a biomolecule, then place it in a suitable buffer condition.

[0091] - Remove excess thiol molecules, for example, by centrifugation.

[0092] -Redisperse in a suitable solution (distilled water, buffer solution containing surfactant, etc.).

[0093] - If the thiolized molecule is an activatable molecule, add an activator (e.g., ECD / NHS or PDEA), incubate for at least 5 minutes, centrifuge and redisperse in a buffer solution, then incubate with a solution of the known biomolecule to be immobilized.

[0094] - If the thiolized molecule is a thiolized linker molecule, then optionally a solution of a biomolecule capable of interacting with the groups of the linker molecule is subsequently added.

[0095] This method allows for the preparation of different types of functionalized NPs based on the steps performed:

[0096] -M prefunctionalized NP

[0097] -M+L-PEG prefunctionalized NP

[0098] -M+PEG-F prefunctionalized NP

[0099] -M+L-PEG+PEG-F prefunctionalized NP

[0100] -M+thiolation of prefunctionalized NPs in biomolecules (e.g., DNA)

[0101] -M+L-PEG+thiolized biomolecules prefunctionalized NPs, etc., each type of biomolecule is suitable as a probe.

[0102] Implementation methods are as follows Figure 1 As shown.

[0103] The advantage of the method starting with the preparation of NPs prefunctionalized with molecule M at varying coverage is that the NPs are stable during the functionalization steps, which can be performed in water or in a buffer solution. The molecule M is a small, short, low-molecular-weight, inert molecule. Prefunctionalization using molecule M ensures that functionalization is uniform across the entire surface of the NP, regardless of the NP's size and shape. Furthermore, it ensures that the immobilized biomolecules do not denature and are positioned towards the solution rather than towards the NP surface. Additionally, molecule M prevents nonspecific interactions.

[0104] A third object of the present invention relates to the use of the nanoparticles according to the present invention.

[0105] Functionalized NPs can be used in a variety of applications well known to those skilled in the art in the fields of medicine, agriculture and food, and the environment.

[0106] They are particularly useful for detecting target molecules in solutions, and are commonly used in diagnostic tests.

[0107] Therefore, the present invention relates to a method for detecting target molecules in solutions, particularly complex media, comprising the following steps:

[0108] -Contact the solution with at least one functionalized nanoparticle as defined above.

[0109] - Detect a specific signal when a biomolecule interacts with one of the components of a solution using SPR, strip test, or any other suitable method.

[0110] The functionalized NP according to the invention can also be used to deplete specific molecules, whether these are target molecules intended for recycling or actually unwanted molecules intended for elimination.

[0111] Therefore, the present invention relates to a method for detecting target molecules present in solutions, particularly complex media, the method comprising the following steps:

[0112] a) Contact the solution containing the target molecule with functionalized nanoparticles as defined above.

[0113] b) Incubate the solution in the presence of the nanoparticles.

[0114] c) Recover nanoparticles.

[0115] d) Optionally, repeat steps a) through c) until the solution of the target molecule is exhausted.

[0116] For a long time, the use of functionalized nanoparticles (NPs) in cancer treatment has been envisioned to enable them to target target cells and / or graft therapeutic compounds. In radiotherapy, NPs, which can be activated by X-rays, represent a significant advance in radiotherapy practices aimed at controlled destruction of tumors. NPs can also be used in photothermal therapy, one of the most promising applications of gold nanoparticles in combating cancer. The idea is to inject gold nanoparticles into the patient's bloodstream. Due to their structure, these nanoparticles tend to become fixed in cancerous tissue, but they can be more specifically directed towards the tumor by coating them with specific molecules (functionalization). They are then "heated" using a laser. The heat released by the NPs causes irreversible damage to the cancer cells. This technology is currently being tested in multiple clinical trials for head and neck cancer, and even lung and prostate cancer.

[0117] Therefore, this invention relates to the use of the functionalized NP according to the invention in the treatment of cancer, particularly by radiotherapy or photothermal therapy. Controlled photothermal therapy can cause therapeutic molecules previously grafted onto nanoparticles to salt out without affecting cells or target organs.

[0118] This invention relates to the use of a functionalized NP according to the present invention for medical imaging.

[0119] The properties of solutions containing NPs can vary. They can be simple media (water, PBS, model media) or complex media (body fluids, wastewater, effluents, etc.). More generally, they can also be a matrix in which NPs can be dispersed or dispersed upon contact with them. This matrix can be a gel, sand, or any flat surface on which the target molecules may be contained.

[0120] In specific embodiments, the solution is a complex biological medium, such as cell extracts, bacterial culture extracts, biological human samples selected from serum, blood, urine, amniotic fluid, tears, etc., and aquatic media selected from wastewater, polluted water, or water that may be such, seawater, water from aquariums, etc. Attached Figure Description

[0121] Figure 1 : A view of NP functionalization techniques. A) Prefunctionalization using SAM composed of molecule M (represented by a short black curve); B) M + L-PEG (represented by a long light gray curve); C) M + PEG-F or biomolecule (represented by a medium-length medium gray curve); D) M + PEG-F or biomolecule + L-PEG. F indicates a functional group.

[0122] Figure 2Absorption spectra of NS prefunctionalized with molecule M in PBS with or without surfactant, based on coverage. 20 nm spherical nanoparticles; stirring time = 4 hours, M (n = 11, m = 4).

[0123] Figure 3 Absorption spectra of NR prefunctionalized with molecule M in PBS in the presence of surfactant, based on coverage. Nanorod λ = 800 nm; stirring time = 30 min; M (n = 11, m = 4); W = 8%.

[0124] Figure 4 After centrifugation, the absorption spectra of NS prefunctionalized with molecule M were obtained in PBS based on the coverage. 20 nm nanoparticles; coverage 1.6% to 80%; stirring time = 5 min, M (n = 11, m = 4), PBS + 0.005% surfactant.

[0125] Figure 5 Absorption spectra of NS prefunctionalized with molecule M in PBS in the presence of surfactant, based on coverage. 20 nm nanoparticles; coverage 1.6% to 80%; stirring time = 4 h, M (n = 11, m = 4), PBS + 0.005% surfactant.

[0126] Figure 6 Absorption spectra of NS prefunctionalized with molecule M in PBS in the presence of surfactant, based on coverage. 40 nm commercial nanoparticles (reference: 741981 Sigma Aldrich); coverage 0%, 1.6%, and 16%; stirring time = 4 h, M (n = 11, m = 12), PBS + 0.005% surfactant.

[0127] Figure 7 Absorption spectra of NS prefunctionalized with molecule M and NP prefunctionalized with molecule M and L-PEG (W = 5%) dispersed in PBS, with different L-PEG coverages (X = 0%, 1%, 10%). 40 nm nanoparticles (reference: 741981 Sigma Aldrich), incubation time L-PEG = 5 min, M (n = 11, m = 4), L-PEG molecular weight = 6000, F = CH3.

[0128] Figure 8 Absorption spectra of NS dispersed in PBS with thiol-functionalized molecules bearing COOH functional groups (Y = 1.6%), exhibiting different molecular M coverages (W = 0%, 1.6%, and 16%). 20 nm nanoparticles. M (n = 11, m = 4) and PEG-COOH (r = 11, p = 6).

[0129] Figure 9 Absorption spectra of NS dispersed in PBS with different L-PEG coverage (X = 0% or 10%), prefunctionalized with molecular M and functionalized with PEG-COOH (W = 5%, Y = 5%); NS size = 20 nm. M (n = 11, m = 4), L-PEG molecular weight = 6000, and F = CH3, PEG-COOH (r = 11, p = 6).

[0130] Figure 10 Absorption spectra of NS with different L-PEG coverage (X = 0% or 10%), prefunctionalized with molecular M and functionalized with PEG-COOH (W = 5%, Y = 5%), dispersed in PBS; NS size = 80 nm commercial (refer to 742023 Sigma Aldrich). M (n = 11, m = 4), L-PEG molecular weight = 6000, F = CH3, PEG-COOH (r = 11, p = 6).

[0131] Figure 11 Absorption spectra of NS prefunctionalized with molecular M and DNA functionalized (W = 0% or 1.6%; Z = 12.5%) dispersed in PBS; M (n = 11, m = 4); NS size = 20 nm.

[0132] Figure 12 Absorption spectra of NS with different L-PEG coverage (X = 0% or 10%), prefunctionalized with molecular M (W = 10%) and DNA-functionalized (z = 25%), dispersed in PBS, F = CH3; NS size = 20 nm. Commercial (Ref. 741965 Sigma Aldrich).

[0133] Figure 13 Absorption spectra of NS with different L-PEG coverage (Y = 0% or 10%), prefunctionalized with molecular M (W = 10%) and DNA-functionalized (Z = 25%), dispersed in PBS, F = CH3; NS size = 80 nm, commercially available (reference 742023 Sigma Aldrich).

[0134] Figure 14 Absorption spectra of NS prefunctionalized with molecule M in PBS + 0.005% surfactant according to incubation time; NS 20 nm; stirring time = 5, 30, 120, 240 and 1440 min, M (n = 11, m = 4), W = 40%.

[0135] Figure 15Absorption spectra of NS with molecular M prefunctionalized and DNA functionalized (W=4% and Z=50%), corresponding to 3 different experiments. M (n=11, m=4), NP 20nm.

[0136] Figure 16 Absorption spectra of the supernatant from the first wash of NS prefunctionalized with molecule M and DNA functionalized (W = 4% and Z = 50%), corresponding to 3 independent experiments. M (n = 11, m = 4), NS 20 nm.

[0137] Figure 17 Absorption spectra of NS prefunctionalized with molecule M after preparation and storage at 4 °C for 1 day, 3 months, and 11 months. NS 20 nm, M (n = 11, m = 4), where W = 32%.

[0138] Figure 18 Absorption spectra of NS prefunctionalized with molecule M and functionalized with thiol-modified DNA after storage at 4°C for 1 day and 2 months. M (n = 11, m = 4), where W = 0% and 16%, Z = 12.5%.

[0139] Figure 19 Absorption spectra of NPs prefunctionalized with molecular M and functionalized with thiol-modified DNA (W = 32% and Z = 25%) after storage at 4°C for 1 day and 3 months. NS size = 20 nm, M (n = 11, m = 4).

[0140] Figure 20 UV-Vis spectra of NPs prefunctionalized with molecule M and functionalized with thiol-modified DNA (W = 30%, Z = 12.5%) before the addition of cell extract (solid line) and after incubation for 1 hour in cell extract diluted 100-fold (dashed line). NS size = 20 nm, M (n = 11, m = 4).

[0141] Figure 21 Absorption spectra of cell extract diluted 100 times and the sum of the three supernatants after centrifugation (dashed line). NS size = 20 nm, M (n = 11, m = 4) (W = 30%, Z = 12.5%).

[0142] Figure 22 Left image: Fluorescence image of Cy5, used to locate complementary fluorescent DNA; Right image: Contrast image of the gel, used to locate nanoparticles. NS size = 20 nm, M (n = 11, m = 4) (W = 0% and 16%, Z = 12.5%). Example

[0143] the term:

[0144] • Thiolized molecules: "Thiolized linker molecules capable of interacting with biomolecules" or "thiolized biomolecules"

[0145] W is the coverage of the prefabricated layer formed by molecules M.

[0146] ·X is the L-PEG coverage

[0147] Y represents PEG-F coverage.

[0148] Z represents the coverage of thiolated DNA.

[0149] Example 1: A general scheme for the prefunctionalization / functionalization of nanoparticles

[0150] Prior to any prefunctionalization, it is preferable to remove excess surfactants (e.g., citrate) from the nanoparticle (NP) solution (diluted in water). To achieve this, the NP solution is pre-centrifuged, the supernatant is removed, and the NP cake is redispersed in an equal volume of distilled water. The centrifugation speed and time will be adjusted according to the size of the NPs.

[0151] Examples of preparation of functionalized spherical NPs

[0152] The following steps are performed starting with NP solutions (sizes ranging from 10 nm to 80 nm in diameter):

[0153] a) Add molecule M to achieve a coverage of 1.5% to 99% W.

[0154] b) Stir for 5 minutes to 4 hours.

[0155] c) Optionally, L-PEG molecules are added to achieve a coverage of 1% to 10% Y.

[0156] d) Stir for 5 to 60 minutes.

[0157] e) Add thiolated molecules (thiolated molecules capable of interacting with biomolecules, or thiolated biomolecules) to achieve at least 1% coverage Y or Z = at least 10%.

[0158] f) Stir for 5 minutes to 24 hours.

[0159] g) If the thiolated molecule is a biomolecule, place it in a suitable buffer condition.

[0160] h) Centrifuge to remove excess thiol molecules.

[0161] i) Redisperse in a suitable solution (distilled water, buffer solution containing surfactant, etc.).

[0162] Example 2: Prefunctionalization of the surface of gold nanoparticles using molecule M improved the stability of the nanoparticles in PBS solution.

[0163] a) Case of spherical nanoparticles

[0164] A solution of molecule M (W = 24%) was added to the nanoparticle solution. The mixture was stirred for several hours (at least 4 hours), and then 0% and 0.005% surfactant, phosphate buffer solution at pH 7.4, and NaCl were added. The NPs were then dispersed in PBS (10 nM phosphate, pH = 7.4, and 0.1 M NaCl) with or without 0.005% surfactant.

[0165] Data related to the stability of nanoparticles, such as Figure 2 As shown.

[0166] It was observed that in the absence of molecule M, the nanoparticles were unstable and aggregated. Conversely, the presence of molecule M allowed the nanoparticles to remain suspended. According to... Figure 2 The absorption spectrum showed that the stability of the nanoparticles was improved in the presence of surfactant in PBS because the plasma band was refined.

[0167] b) The case of nanorods

[0168] A solution of molecule M and 0.005% surfactant was added to the nanorod (NR) solution. The mixture was stirred for 30 minutes, and then a phosphate buffer solution at pH 7.4 and NaCl were added. The NPs were then dispersed in PBS (10 nM phosphate, pH 7.4, and 0.1 M NaCl) in the presence of 0.005% surfactant.

[0169] Data related to the stability of nanoparticles, such as Figure 3 As shown, the nanoparticles were observed to be unstable and aggregated in the absence of molecule M. Conversely, the presence of molecule M allowed the nanoparticles to remain suspended in PBS + 0.005% surfactant.

[0170] All other experiments were conducted using spherical NPs.

[0171] Example 3: Nanoparticles prefunctionalized with molecule M can be washed and redispersed in PBS solution containing surfactant.

[0172] Add the solution of molecule M to the nanoparticle solution. Stir the mixture for 5 minutes or 4 hours, centrifuge, and redisperse in a 1x PBS solution containing 0.005% surfactant.

[0173] "PBS 1x" refers to a solution containing 137mM NaCl, 10mM phosphate, 2.7mM KCl, and a pH of 7.4.

[0174] Various solutions were tested:

[0175] a) 20 nm spherical nanoparticles were incubated in a solution of molecule M for 5 minutes.

[0176] Data related to the stability of nanoparticles, such as Figure 4 As shown.

[0177] b) 20 nm spherical nanoparticles were incubated in a solution of molecule M for 4 hours.

[0178] Data related to the stability of nanoparticles, such as Figure 5 As shown.

[0179] c) 40 nm spherical nanoparticles were incubated in a solution of molecule M for 4 hours.

[0180] Data related to the stability of nanoparticles, such as Figure 6 As shown.

[0181] These results clearly demonstrate that the stability of the nanoparticles increases with the coverage of the molecular M solution.

[0182] Example 4: Functionalization with L-PEG molecules improved the stability of nanoparticles prefunctionalized with molecule M in PBS solution.

[0183] a) Individual prefunctionalization

[0184] For individual prefunctionalization, add a solution of molecular M with W = 1.6% to 80% to the nanoparticle solution. Stir the mixture for several minutes or hours (5 minutes to 24 hours), centrifuge, and redisperse in an appropriate solution.

[0185] b) Prefunctionalization followed by functionalization using L-PEG molecules.

[0186] For prefunctionalization followed by functionalization with L-PEG molecules, a solution of molecule M was added to the nanoparticle solution to achieve a coverage of W = 5%. After stirring for at least 5 minutes, L-PEG solution was added to achieve coverages of 0%, 1%, and 10%. The mixture was stirred again for 15 minutes, then centrifuged and redispersed in PBS (1x) solution.

[0187] Data related to the stability of nanoparticles, such as Figure 7 As shown.

[0188] These results indicate that adding L-PEG starting at X = 1% allows for the stabilization of NPs in PBS that are covered by a low layer of monolayer molecule M (in this case, W = 5%). Indeed, the plasma bands were refined upon using L-PEG, confirming the improved stability.

[0189] Example 5: Prefunctionalization of nanoparticles using molecule M improves the stability of nanoparticles functionalized with thiol-modified molecules in PBS solution.

[0190] a) Functionalization using thiol-modified carboxyl molecules without pre-functionalization.

[0191] Add PEG-COOH solution to the nanoparticle solution to achieve a coverage Y of 1.6%. Stir the mixture for 30 minutes, centrifuge, and redisperse in H2O or PBS 1x + 0.005% surfactant.

[0192] b) Prefunctionalization followed by functionalization using thiolated carboxyl molecules.

[0193] A solution of molecule M was added to the nanoparticle solution to achieve a theoretical initial coverage Y of 1.6% to 80%. After stirring for 5 minutes, a PEG-COOH solution was added to achieve a coverage Y of 1.6%. The mixture was stirred for 30 minutes, centrifuged, and redispersed in distilled water or PBS 1x + 0.005% surfactant.

[0194] Data related to the stability of nanoparticles, such as Figure 8 As shown.

[0195] These results indicate that NPs functionalized with PEG-COOH alone aggregate in PBS. The fact that NPs are prefunctionalized using a solution of molecule M improves their stability in solution, particularly starting from a coverage of W = 1.6%. Stability is further improved by increasing the incubation time and W, i.e., by increasing the density of molecule M on the NP surface (in this case, W = 16% on the curve, reflecting the highest stability).

[0196] Example 6: Functionalization with L-PEG molecules improved the stability of NPs prefunctionalized with molecule M and functionalized with thiol-modified molecules in PBS solution.

[0197] Add a solution of molecule M to the NP solution to achieve a coverage of W = 5%. Then add an L-PEG solution to achieve a coverage of X of 0% or 10%. Then add a PEG-COOH solution to achieve a coverage of Y = 5%. Stir the mixture, centrifuge, and redisperse it in distilled water or PBS 1x + 0.005% surfactant.

[0198] Data related to nanoparticle stability for 20nm spherical NPs, such as Figure 9 As shown, for an 80nm spherical NP, Figure 10 As shown.

[0199] Note that the addition of 10% L-PEG increases the stability of prefunctionalized and thiolized NPs (in this case, PEG-COOH), especially when the coverage of molecule M and thiolized molecule is low (5% coverage each).

[0200] Example 7: Prefunctionalization of molecule M improves the stability of NPs functionalized with thiol-modified molecules in PBS solution: thiol-modified DNA molecules.

[0201] a) Functionalization using thiol-modified DNA molecules without pre-functionalization.

[0202] Add the DNA solution to the nanoparticle solution to achieve a coverage of at least 12.5% ​​Z. Stir the mixture for 1 hour, then add 0.005% surfactant, phosphate buffer, and NaCl. Stir the mixture for 18 hours, centrifuge, and redisperse in PBS 1x.

[0203] b) Prefunctionalization followed by functionalization using thiolated DNA molecules.

[0204] Solution of molecule M was added to the NP solution to achieve 1.6% and 80% coverage. Then, DNA solution was added to achieve at least 12.5% ​​coverage (Z). After stirring for 1 hour, 0.005% surfactant, phosphate buffer, and NaCl were added. The mixture was stirred for 18 hours, centrifuged, and redispersed in PBS 1x.

[0205] Data related to the stability of nanoparticles, such as Figure 11 As shown.

[0206] It was noted that DNA-functionalized NPs aggregated in PBS. However, starting from a coverage of 1.5% W, prefunctionalization with molecule M improved stability. Stability could be further improved by increasing the incubation time and W, i.e., by increasing the density of molecule M on the NP surface (data not shown).

[0207] Example 8: L-PEG functionalization improves the stability of NPs prefunctionalized with molecule M and functionalized with thiol-modified DNA in PBS solution.

[0208] A solution of molecule M was added to the nanoparticle solution to achieve a coverage of 1.6% and 80% W. Then, L-PEG solution was added to achieve a coverage of 1% to 10% X after incubation for 30 minutes. The mixture was stirred for 15 minutes. Then, SH-DNA solution was added to achieve at least 12.5% ​​of the theoretical initial coverage. After adding the DNA solution, 0.005% surfactant, phosphate buffer (pH 7.4, final concentration 10 mM), and NaCl (final concentration 0.1 M) were added. The mixture was stirred for 18 hours, centrifuged, and redispersed in PBS 1x.

[0209] Data related to nanoparticle stability for 20nm NPs, such as Figure 12 As shown, for an 80nm NP, as Figure 13 As shown.

[0210] An improvement in NP stability was observed upon the addition of L-PEG molecules. Notably, the presence of the L-PEG layer enabled the stability of 80 nm NPs in PBS solution with a low level of molecular M (W = 10%).

[0211] Example 9: Effect of incubation time of NP in solution containing molecule M on stability

[0212] A solution of molecule M was added to the nanoparticle solution. The mixture was stirred for several minutes or hours (5 minutes to 24 hours), and then 0.005% surfactant, phosphate buffer solution at pH 7.4, and NaCl were added. NP was then dispersed in PBS (10 nM phosphate, pH 7.4, and 0.1 M NaCl) in the presence of 0.005% surfactant.

[0213] Data related to the stability of nanoparticles, such as Figure 14 As shown.

[0214] Note that coverage increases as a function of time, reaching its maximum between 120 and 240 minutes of incubation. Continuing incubation beyond this duration has no further effect on W.

[0215] Example 10: Reproducibility of a method for stabilizing nanoparticles

[0216] To confirm the reproducibility of the protocol, these steps were repeated three times on different days. Sample stability was characterized by UV-Vis spectroscopy. Regarding the grafting of thiolated DNA, the first supernatant during the washing step to remove excess DNA was analyzed by UV-Vis spectroscopy.

[0217] Results regarding the stability of functionalized NP solutions are as follows: Figure 15As shown, and regarding the results of the supernatant obtained during the first step of washing away unabsorbed DNA, as... Figure 16 As shown.

[0218] Note that the UV-Vis spectrum contains superimposed fine plasmonic bands, indicating that the nanoparticles NP, prefunctionalized with molecule M and functionalized with DNA, are stable, as evidenced by the identical absorbance intensity of the supernatant indicating that the same amount of DNA has been attached to the NP. In summary, these results confirm the reproducibility of the sample preparation.

[0219] Example 11: Stability of NPs prefunctionalized with molecule M in water for several months

[0220] As described above, NP at 20 nm was prefunctionalized using molecule M and then stored at 4 °C in the dark for several months. Stability was studied by UV-Vis spectroscopy. Typically, 1 ml of solution was extracted and analyzed using a UVIKON spectrometer. Spectra obtained at different times were compared.

[0221] Data related to the stability of nanoparticles, such as Figure 17 As shown.

[0222] A good correlation was observed in the absorption spectra of the NPs prefunctionalized with molecule M after 1 day and 11 months of storage. These three spectra showed fine plasmon bands without any shift, confirming that the prefunctionalized NPs remained stable in water after 11 months of storage at 4°C.

[0223] Example 12: Prefunctionalization of NPs using molecule M and DNA-functionalization resulted in stability of NPs in PBS solution for several months.

[0224] As described above, the 20 nm NP was prefunctionalized using molecule M and then functionalized using DNA, followed by storage at 4 °C and in the dark for several months. Stability was assessed using UV-Vis spectroscopy. Typically, 1 ml of solution was extracted and analyzed using a UVIKON spectrometer. Spectra obtained at different storage times were compared.

[0225] Data related to the stability of nanoparticles, such as Figure 18 and 19 As shown.

[0226] First, it is noted that the presence of molecule M ensures the stability of NPs both after preparation and during storage. Comparison of spectra obtained after 1 day and 2–3 months of storage shows a good correlation between the spectra of pre-functionalized and DNA-functionalized NPs. Both spectra show two fine plasmon bands without any shift, confirming the stability of NPs in PBS after 2–3 months of storage at 4°C.

[0227] Example 13: Stability of prefunctionalized and DNA-functionalized NPs in complex media.

[0228] 10 μl of cell extract was added to 1 ml of NP solution prefunctionalized with molecule M and DNA-functionalized with 1 nM thiol-modified DNA (100-fold dilution for complex media). The mixture was stirred for 1 hour, then centrifuged and redispersed in PBS 1x. The stability of NP in the supernatant and the absorbance intensity of the cell culture medium were characterized by UV-Vis spectroscopy. According to Beer-Lambert's law, absorbance intensity is related to concentration in solution. If the absorbance intensity of the cell extract is similar to that of the supernatant, it indicates that the biomolecules present in the cell extract are not absorbed nonspecifically at the NP.

[0229] Data related to the stability of nanoparticles, such as Figure 20 and 21 As shown.

[0230] The absorption spectra of the samples before and after 1 hour of incubation were compared. Notably, no aggregation occurred in the complex medium, indicating good particle protection. To determine the presence of nonspecific adsorption, the particles were washed by centrifugation to remove the cell extract. The sum of the three supernatants after 1 hour of incubation was superimposed on the spectrum of the initial cell extract. The superimposed spectra indicate that the proteins derived from the cell extract showed little or no specific adsorption on the functionalized NP surface.

[0231] Example 14: Prefunctionalization of NPs with molecule M improves hybridization in complex media

[0232] 1 μL of 100 nM complementary fluorescent DNA (Cy5) was added to 200 μL of 1.5 nM NP solution, which was used for DNA functionalization with or without pre-functionalization using molecule M. Therefore, the total complementary DNA concentration was 500 pM. Hybridization was performed in three different media at 37 °C for 2 hours: PBS 1x, cell extract, and human serum. The complex media were diluted 100-fold. To distinguish between fluorescent DNA, DNA-functionalized NP, and pre-functionalized NP prior to DNA functionalization, samples were deposited on agarose gels for electrophoresis. Electrophoresis allowed for the separation and differentiation of hybridized nanoparticles and free complementary DNA in solution.

[0233] Hybridization of complementary fluorescent chains with two types of nanoparticles (NPs functionalized only with DNA molecules, or NPs pre-functionalized with monolayer molecules M followed by DNA-functionalized NPs) was investigated in three different media: PBS, cell extract diluted 100-fold, and human serum diluted 100-fold. The nanoparticles used were synthesized two months prior to use and stored at 4°C.

[0234] The results of the experiment are as follows: Figure 22 As shown.

[0235] Only the nanoparticles were observed to be visible, as individual fluorophores were not excited. The migration distance of the NPs using DNA-functionalized nanoparticles, as well as the migration distance of NPs pre-functionalized before DNA functionalization, appeared unaffected in PBS and cell extracts. In contrast, in human serum, the migration distance differed very slightly for NPs pre-functionalized before functionalization, but the change was more significant for NPs using DNA functionalization (without molecule M), suggesting possible non-specific absorption on these nanoparticles (visible as a slowdown in migration speed due to increased NP size). In the left part of the figure, only fluorescence emission (black bands in the gel) was observed, indicating the location of complementary DNA in the gel. Comparison Figure 22 The intensity and position of the bands can be correlated with the location of the DNA and the position of the NP in the gel, and thus with the hybridization effectiveness. While the hybridization efficiency between pre-functionalized and unfunctionalized nanoparticles appeared unaffected in PBS, a significant difference was observed in complex media. In fact, hybridization appears to be affected for samples without the molecule M (i.e., DNA only), as the fluorescence corresponding to free DNA is significantly stronger (less complementary DNA hybridizes with DNA linked to the NP). These results suggest that pre-functionalized samples have higher hybridization effectiveness in complex media. It can be hypothesized that in complex media, non-specific uptake of proteins on DNA-functionalized (but unpre-functionalized) NPs prevents hybridization between complementary DNA and DNA linked to the NP.

[0236] Example 15: Evaluation of the detection of target complementary DNA in different media

[0237] Following the hybridization efficiency between the complementary target fluorescent DNA (Cy5) and DNA immobilized on nanoparticles prefunctionalized using molecule M, the detection of single-stranded target DNA (cDNA) is permitted. Once the DNA has hybridized, it is subjected to two gel electrophoresis processes, one before and one after centrifugation of the NPs. Centrifugation allows for the concentration of the NPs, enabling improved visualization of the complementary fluorescent DNA on the NP surface.

[0238] The hybridization protocol followed was as follows: 50 pM to 1 nM fluorescently complementary DNA was added to a 2 nM NP suspension (NP size = 20 nm, M (n = 11 and m = 4), W = 16% and Z = 12.5%). The mixture was incubated in PBS or commercial human serum (diluted 100) (Thermofisher, Normal Human Serum, reference 31876) at 37°C for 2 hours.

[0239] Gel migration of NPs was performed (data not shown). The "Nanoparticle Images" section shows the location of the bioconjugated NPs, and the "Fluorescence Images" section shows the location of the fluorescently complementary DNA. Controls correspond to free fluorescently complementary DNA. Different concentrations were charged from 50 pM to 1 nM.

[0240] These results confirm that hybridization occurred because the fluorescence was correlated with the position of the nanoparticles. Fluorescence was observed in PBS or human serum at target DNA concentrations up to 50 pM. Even at low concentrations of target DNA, the presence of complex media did not prevent hybridization.

[0241] in conclusion: Hybridization of DNA probes on NPs allows for the detection of complementary DNA ranging from 50 pM to 1 nM in PBS or human serum.

Claims

1. A nanoparticle comprising a metal surface prefunctionalized with a self-assembled protective monolayer, said self-assembled protective monolayer being formed from a matrix of a molecule M of formula (1), wherein formula (1) is: HS(CH2)n(OCH2CH2)m OH, in: n represents the number of CH2 groups, where 3 ≤ n ≤ 11 m represents the number of ethylene glycols, where 1 ≤ m ≤ 12. The molecule M has a thiol functional group at one end and is inert at the other end, characterized in that the coverage W of the monolayer is 1.5% to 80%. The nanoparticles are further functionalized using at least one L-PEG molecule of formula (7), which is: HS-CH2CH2(OCH2CH2). q OF, in: q represents the number of ethylene glycols, where 20 ≤ q ≤ 500; F indicates that CH3 or the COOH functional group can be activated. The coverage X of the L-PEG molecules is 1-10%.

2. A nanoparticle comprising a metal surface prefunctionalized with a self-assembled protective monolayer, said self-assembled protective monolayer being formed from a matrix of a molecule M of formula (1), wherein formula (1) is: HS(CH2)n(OCH2CH2)m OH, in: n represents the number of CH2 groups, where 3 ≤ n ≤ 11 m represents the number of ethylene glycols, where 1 ≤ m ≤ 12. The molecule M has a thiol functional group at one end and is inert at the other end, characterized in that the coverage W of the monolayer is 1.5% to 80%. The nanoparticles are further functionalized using at least one thiol molecule, wherein the thiol molecule is: -The PEG-F linker molecule of formula (8), or - Biomolecules containing thiol functional groups. Equation (8) is: HS(CH2) r (OCH2CH2) p OF, where: r represents the number of CH2 and is an integer from 3 to 11. p represents the number of ethylene glycols, where 2 ≤ p ≤ 12, and F indicates that CH3 or the COOH functional group can be activated. The PEG-F linker molecule has a thiol functional group at one end and an active or reactive group capable of interacting with biomolecules at the other end, and the coverage Y of the PEG-F linker molecule is 1% to 5%.

3. The nanoparticles as described in claim 2, characterized in that, It is further functionalized using at least one biomolecule that binds to the PEG-F linker molecule.

4. The nanoparticles as described in claim 2, characterized in that, The biomolecule containing thiol functional groups is thiolized DNA with a coverage of at least 10% Z.

5. The nanoparticles as described in claim 1 or 2, wherein the nanoparticles are spherical nanoparticles, nanorods, cubic nanoparticles, or nanotriangular sheets.

6. The nanoparticles as claimed in claim 1 or 2, wherein the nanoparticles are core-shell nanoparticles.

7. The nanoparticles as described in claim 1 or 2, wherein the nanoparticles are nano-sea urchins.

8. A method for preparing nanoparticles comprising a metal surface prefunctionalized using a self-assembled protective monolayer, said self-assembled protective monolayer being formed from a solution of a molecule having a thiol functional group at one end and an inert end, said method comprising the steps of: - Add molecule M of formula (1) as defined in claim 1 to achieve a coverage of 1.5% to 80% W. - Stir for at least 5 minutes. - Further includes the step of adding a solution of L-PEG molecules of formula (7) as defined in claim 1 to achieve a coverage of 1% to 10% X.

9. A method for preparing nanoparticles comprising a metal surface prefunctionalized using a self-assembled protective monolayer, the self-assembled protective monolayer being formed from a solution of a molecule having a thiol functional group at one end and an inert end, the method comprising the steps of: - Add molecule M of formula (1) as defined in claim 2 to achieve a coverage of 1.5% to 80% W. - Stir for at least 5 minutes. - Further includes the following steps: adding a PEG-F connector molecule selected from the biomolecule of formula (8) as defined in claim 2 that is capable of interacting with a biomolecule or a thiolized molecule of a biomolecule containing a thiol functional group as defined in claim 2.

10. A method for detecting a target molecule in a solution, said method not being a method for disease diagnosis or treatment, comprising the following steps: - To contact the solution with at least one functionalized nanoparticle as defined in any one of claims 2 to 7. - Detect specific signals when the biomolecule interacts with the target molecule in solution by surface plasmon resonance or test paper examination.

11. A method for detecting a target molecule present in a solution, the method not being a method for disease diagnosis or treatment, the method comprising the following steps: a) Contacting a solution containing the target molecule with at least one functionalized nanoparticle as defined in any one of claims 2 to 7. b) Incubate the solution in the presence of the nanoparticles. c) Recover the nanoparticles. d) Optionally repeat steps a) to c) until the solution of the target molecule is exhausted.

12. The nanoparticles as described in any one of claims 1 to 7, which are used in radiotherapy and phototherapy for cancer.

13. The nanoparticles as described in any one of claims 1 to 7, used in medical imaging.

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