Nanoparticles pre-functionalized with self-assembled monolayers and method for their preparation

Abstract

The present invention relates to the field of prefunctionalized nanoparticles (NPs). More particularly, the present invention relates to NPs prefunctionalized with self-assembled monolayers (SAMs) and functionalized with biomolecules such that the NPs are stable in solution. These NPs can be used in many applications, particularly as diagnostic tools, tools for depleting molecules of interest in solution, and therapeutic tools.

Description

Detailed Description of the Invention

[0001] The present invention relates to the field of pre-functionalized nanoparticles (NPs). More specifically, the present invention relates to NPs pre-functionalized with self-assembled monolayers (SAMs) and NPs functionalized with biomolecules, which are stable in solution. The NPs can be used in multiple applications, particularly as diagnostic tools, tools for depleting or concentrating molecules of interest in solution, and therapeutic tools.

[0002] Prior Art and Disadvantages Nanoparticles covered with molecular films of SAMs have been described in the literature. For example, Hurst KM et al. (Journal of Microelectromechanical Systems; Volume: 20, Issue: 2, April 2011) disclose the immobilization of SAMs consisting of p-aminophenyltrimethoxysilane (APhTS) and 3-mercaptopropyltrimethoxysilane (MPTS) on NPs to stabilize their adhesion to siliconized surfaces of microelectromechanical systems. However, such NPs are not suitable for functionalization with biomolecules.

[0003] Other prior art documents disclose nanoparticles coated with thiolated monolayers.

[0004] Hurst et al. (2006) described gold nanoparticles with charged DNA molecules, and the goal of this work was to achieve the maximum number of charged DNA molecules on the surface of the nanoparticles. The authors demonstrated that the maximum charge was achieved when the salt conditions were controlled (0.7 M NaCl) and the DNA molecules contained polyethylene glycol (PEG)-type spacers.

[0005] Wang et al. (RSC Adv. 2017, 7, 3676-3679) disclose spherical gold NPs functionalized with a mixed monolayer containing PEG and thiolated DNA. The nanoparticles are prepared in two steps: functionalization with thiolated DNA molecules and then coating the entire surface of the NPs with thiolated PEG.

[0006] Li et al. (Langmuir 2015, DOI: 10.1021 / acs.iangmuir.5b01680) describe nanorods (NRs) functionalized with DNA molecules. Because gold NRs are positively charged, they tend to aggregate together in the presence of negatively charged DNA. The authors propose latticing PEG and surfactant molecules onto the NRs before adding thiolated DNA.

[0007] Document US 2011 / 165077 discloses the use of gold nanoparticles for imaging. Reporter molecules are grafted onto the surface of the NPs, which are then covered with a protective PEG-SH molecular film up to the saturation point. This molecular film prevents the particles from agglomerating and allows them to be functionalized with specific probes.

[0008] Document US 2019 / 142966 discloses the use of metal NPs for the treatment of cancer: these NPs are coated with PEG-COOH molecules in a single step, and the carboxyl groups are then functionalized with therapeutic and targeting molecules in the presence of SDS.

[0009] US 2016 / 243254 describes the preparation of nanoprobes as theranostic tools in cancer treatment. A first molecular layer of thiolated PEG-COOH is grafted onto the surface of a nanoparticle. Due to the high molecular weight of PEG-COOH, this first molecular layer covers approximately 30% of the NP surface, which is the maximum coverage. A second molecular layer of thiolated DNA-harpin molecules is then added, sandwiched between PEG molecules. This configuration favors partial coverage combined with carboxyl functionality to allow for nonspecific interactions.

[0010] In the field of NPs intended to be used as supports for immobilizing biological probes, various constraints must be taken into account. First, pre-functionalized or functionalized NPs must be stable in solution, i.e., not aggregate. They must also allow specific probe / molecule interactions of interest.

[0011] DISCLOSURE AND ADVANTAGES OF THE INVENTION The present invention provides a solution to these challenges by pre-functionalizing NPs with a self-assembled monolayer formed from polyethylene glycol (PEG) molecules with a low molecular weight, providing partial coverage. The presence of this pre-layer on the surface of the NPs allows for the preparation of NPs that are stable in solution, either alone or in combination with other molecules (functionalization). This monolayer is specifically composed of molecules with a low molecular weight (between 100 and 732 daltons), whose molecular chains are short and whose bulk is optimal in the surface region, providing nanoparticles with their innovative properties, as described below.

[0012] An illustration of the configuration of the elements grafted on the surface of the NPs is shown in Figure 1 .

[0013] The functionalization strategy employed in this invention is surface chemistry, i.e., by inhibiting nonspecific interactions and optimizing specific interactions, allowing the control of the number of ligands (biomolecules) immobilized on the surface of nanoparticles, thereby improving the stability of metal nanoparticles, such as gold nanoparticles, or hybrid core-shell nanoparticles, in buffers and complex media. This technique involves prefunctionalization prior to the immobilization of one or more biomolecules acting as probes. Either the biomolecules contain thiolated groups that allow their direct immobilization on the surface of the NPs, or the immobilization of biomolecules is carried out in two steps: 1) the immobilization of linker molecules bearing a thiol on one side and a reactive functional group on the other (the former for their immobilization on the metal surface, the latter for the immobilization of biomolecules), and 2) the addition of biomolecules (probes or active molecules) containing groups capable of reacting or interacting with the functional groups present on the linker molecules. The method described here is particularly suitable for spherical NPs, nanorods, nanocubes, nanotriangles, and nanourchins.

[0014] Furthermore, this pre-functionalization allows for optimization of probe density, uniform distribution of probes, and the absence of non-specific interactions. In particular, the present invention allows for obtaining NPs functionalized with probes that are optimized at low density to achieve high detection sensitivity. It is also possible to combine multiple different probes while maintaining controlled density and distribution for each probe.

[0015] Detailed Description of the Invention A first object of the present invention relates to nanoparticles comprising a metal surface pre-functionalized with a self-assembled monolayer (SAM) formed by a matrix of molecules carrying a thiol functional group at one end and an inert end at the other, characterized in that the coverage W of said monolayer is between 1.5% and 99%.

[0016] The properties of the self-assembled monolayer are that it is applied to the surface of the NP before functionalization and that it is unsaturated. It serves to protect the surface. This monolayer is formed from molecules M of formula (I): HS(CH2)n(OCH2CH2) m OH where: n represents the number of CH2, and 3≦n≦11; m represents the number of ethylene glycols, and 1≦m≦12.

[0017] The molecular weight of molecule M is 100 to 732 daltons.

[0018] Overall, a coverage W of at least 1.5% is essential to observe the effectiveness of the SAM molecular film. Low coverage allows for a high density of biomolecules on the NP surface, which may be the objective, for example, when two different biomolecules are present. On the other hand, coverage W can be increased to near the saturation point (e.g., 99%), allowing the attachment of single molecules; the only limitation of this approach is the detection limit, which is due to the lack of suitable sensitive tools. In any case, prefunctionalization of the NP surface allows for the immobilization of biomolecules in a controlled manner, i.e., ensuring a uniform average distance between the immobilized biomolecules.

[0019] In certain embodiments, the coverage W may be between 3% and 80%, such as between 10% and 50%, or between 5% and 30%, and may be 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98%, depending on the application.

[0020] Within the meaning of the present invention, "coverage" means the theoretical initial coverage, which corresponds to the molecular surface and is calculated according to the following formula (2): (nm 2 (abbreviation) × 100 / surface area of ​​nanoparticle Within the meaning of the present invention, "nanoparticles" (NPs) can be spherical nanoparticles (NSs), nanorods (NRs), cubic nanoparticles (NCs), nanotriangles (NTs), or nanourchins. These can be, in particular, spherical gold nanoparticles with a diameter of 10-80 nm, nanorods with an aspect ratio of 2.5-5, and nanocubes with a side length of 40 nm, or nanotriangles with a side length of 30-100 nm and a thickness of 10-40 nm. NPs can also be hybrid NPs of the same shape (spheres, nanorods, etc.), for example, comprising an oxide (e.g., iron oxide, silicon oxide) or metal core and a metal shell (e.g., gold). The metal surface of core-shell hybrid nanoparticles is similar to that of metal nanoparticles, and the methods for calculating their surface are identical.

[0021] The surface area of ​​a molecule depends on its shape and must be known to determine coverage.

[0022] For spherical nanoparticles, the surface area is given by: 4πr 2 where r is the radius of the nanoparticle.

[0023] For nanorods, the surface area is given by the following equation (4): 2πrl+2πr 2 where r is the radius of the nanorod and l is its length.

[0024] For nanocubes, the surface area is given by the following formula (5): 6a 2 where a is the side of the nanocube.

[0025] For nanotriangles, the surface area is given by the following equation (6): (ph / 2)+b where p is the perimeter of the base, h is the height of the slope, and b corresponds to the area of ​​the base.

[0026] The molecules present on the surface of the NPs for their pre-functionalization or for their functionalization may be chosen from M molecules as defined above, L-PEG and PEG-F molecules as defined below, as well as double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), RNA, proteins such as antibodies or peptides, aptamers, etc.

[0027] And to determine the coverage, it is also necessary to know the theoretical abbreviations of the molecules present on the surface of the NPs: Abbreviations M and PEG-F = 0.23 nm 2 [1] L-PEG abbreviation = 0.45 nm 2 [2] dsDNA abbreviation = 3.14 nm 2 .

[0028] In a more preferred embodiment, the nanoparticles pre-functionalized according to the invention are functionalized with at least one L-PEG molecule, the coverage X of which is at least 1%, preferably between 1% and 10%.

[0029] By "L-PEG molecule" is meant a molecule of thiolated polyethylene glycol having a long height and a high molecular weight.

[0030] The L-PEG molecule has the formula (7): SH-CH2CH2(CH2CH2O) q OF where: q represents the number of ethylene glycols, and 20≦q≦500; F represents a functional group such as CH3, OH, COOH or NH2.

[0031] The molecular weight of the L-PEG molecule is greater than or equal to 800 daltons.

[0032] Functionalization with L-PEG molecules is particularly aimed at stabilizing NPs in PBS solution, which can be combined with pre-functionalization with molecules M to prepare "ready-to-use" NPs onto which one or more biomolecules can be adsorbed.

[0033] Furthermore, if the F group is not CH3 or OH, the F group may interact and / or react with biomolecules to attach probes onto the surface of the NP.

[0034] In a particular embodiment of the invention, the pre-functionalized nanoparticles according to the invention are functionalized with at least one thiolated molecule, said thiolated molecule being a thiolated linker molecule having a thiol functional group at one end and an active or reactive group at the other end that can interact with a biomolecule, or biomolecules containing thiol functional groups, Either:

[0035] The nanoparticles can be further functionalized with L-PEG molecules as described above.

[0036] In a preferred embodiment, the thiolated linker molecule is a PEG-F molecule, where F is an activatable functional group such as COOH, and its coverage Y is at least 1%. The total coverage of the surface of the NPs can be partial or full (saturated), depending on the application. In a preferred embodiment, the total coverage is partial.

[0037] "PEG-F" refers to a molecule of thiolated polyethylene glycol.

[0038] The PEG-F molecule can be, for example, of the formula (8): HS(CH2) r (OCH2CH2) p OF where r represents the number of CH2 and is an integer of 3 or more; p represents the number of ethylene glycols, and 2≦p≦12; F represents a functional group such as CH3, COOH or NH2.

[0039] The molecular weight of the PEG-F molecule can be low or high, ranging up to over 800 kDa.

[0040] In another embodiment, the linker molecule may be an L-PEG molecule containing an activatable functional group.

[0041] In another preferred embodiment, the nanoparticles pre-functionalized according to the present invention are functionalized with thiolated biomolecules that act as probes (called "thiolated probes"), such as nucleic acids (DNA, RNA, PNA, etc.), proteins (antibodies, antigens, hormones, etc.), peptides, hormones, sugars, fatty acids, or indeed whole cells.

[0042] In another embodiment of interest, the thiolated biomolecule is a small molecule, such as a drug, sandwich, or complex, referred to herein as an "active molecule." These active molecules may be attached via activatable functional groups present on the PEG-F or L-PEG molecule.

[0043] When the thiolated molecule is double-stranded or single-stranded DNA, the coverage Z is at least 10%. The total coverage of the surface of the NPs can be partial or full (saturated), depending on the application. In a preferred embodiment, the total coverage is partial.

[0044] In a preferred embodiment, when PEG-F is short, L-PEG molecules are added at a rate of 1% to stabilize the NP.

[0045] Within the meaning of the present invention, a "thiolated probe" refers to a biomolecule that acts as a probe capable of specifically interacting with (capturing) a molecule of interest located in an aqueous medium (e.g., a buffer solution) or a complex medium (e.g., culture medium, biological medium, body fluid, and liquid matrices such as blood, plasma, serum, urine, tears, and cell extracts). The biomolecule functioning as a probe is immobilized on the support of the NP either by (i) a direct interaction between the thiolated group present on the biomolecule and the surface of the NP, or (ii) by having a thiolated molecule at one end and an active or reactive group at the other end that can react with (conjugate to) a binding molecule by forming a "strong" bond (e.g., a covalent bond such as an amide bond, chelation, or a strong affinity "tag" type bond such as a streptavidin-biotin bond). The thiolated molecule is referred to herein as a "linker molecule."

[0046] Once the substrate is protected by the protective molecular film SAM described above, a further step of thiolated probe attachment follows, either immediately before or after use, or hours, days, or months later.

[0047] These thiolated probes are Direct thiolated probes, which generally require incubation times dependent on low concentrations (micromolar or lower), Thiolated molecules with reactive amine or carboxyl groups, double or triple carbon-carbon, epoxy resin, click chemistry, etc. thiolated molecules with active groups that interact with complementary molecules to form sandwiches, such as biotin-streptavidin-biotin, It could be.

[0048] A second object of the present invention relates to a method for preparing nanoparticles comprising a metal surface pre-functionalized with a self-assembled monolayer (SAM) formed by a solution of molecules carrying a thiol functional group at one end and an inert end at the other, comprising the following steps: Adding molecules M capable of forming a self-assembled monolayer to achieve a coverage W of 1.5% to 99%; Stir for at least 5 minutes. Includes.

[0049] This type of method allows the preparation of pre-functionalized NPs that are stable in water and PBS. NPs prepared in this way can be stored at 4 °C for several months before use.

[0050] During this process, it is possible to add a surfactant. The surfactant used can be selected from all surfactants known to those skilled in the art, in particular SDS, CHARS, NP40, Tween 20 (SDS: sodium dodecyl sulfate, CHARS: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), etc. Those skilled in the art are familiar with the use of surfactants for the preparation of NPs in solution.

[0051] In a particular embodiment of the invention, a solution of L-PEG molecules is further added to the solution of NPs pre-functionalized with a SAM molecular film to achieve a coverage Y of 1-10%, followed by incubation under stirring for a minimum of 5 minutes, typically between 5 minutes and 1 hour.

[0052] NPs pre-functionalized 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 may be added after the PEG-F molecule.

[0053] This type of preparation method comprises the following steps: adding a thiolated molecule selected from a thiolated linker molecule capable of interacting with a biomolecule or a thiolated biomolecule, Stir for 5 minutes to 24 hours, for example. if the thiolated molecule is a biomolecule, placing the thiolated molecule in buffer conditions suitable for the thiolated molecule; removing excess thiolated molecules, for example by centrifugation; Redisperse in an appropriate solution (distilled water, buffer solution containing surfactant, etc.) If the thiolated 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 before incubating with a known solution of the biomolecule to be immobilized; if the thiolated molecule is a thiolated linker molecule, then optionally adding a solution of a biomolecule capable of interacting with the group of the linker molecule, Includes.

[0054] This method can produce different types of functionalized NPs depending on the steps performed: NP Reserved Function M NP pre-functionalized M+L-PEG NP spare function M+PEG-F NP Pre-functionalized M+L-PEG+PEG-F NP pre-functionalized M+ thiolated biomolecule (e.g., DNA) NP pre-functionalized M+L-PEG+thiolated biomolecules with any type of biomolecule suitable for use as a probe This allows the preparation of

[0055] An embodiment is shown in FIG.

[0056] The advantage of starting with the preparation of NPs pre-functionalized with molecule M at different coverage rates is that the NPs are stable during the subsequent functionalization step, which can be carried out in water or buffer solution. The molecule M is a small, inert molecule with a low molecular weight. Pre-functionalization with molecule M ensures uniform functionalization across the entire surface of the NPs, regardless of their size and shape. Furthermore, it ensures that the immobilized biomolecules do not denature and are oriented toward the solution, not toward the surface of the NPs. Furthermore, molecule M prevents nonspecific interactions.

[0057] A third subject of the invention relates to the use of the nanoparticles according to the invention.

[0058] The functionalized NPs can be used in a number of applications in the medical, agri-food, and environmental fields that are well known to those skilled in the art.

[0059] In particular, they can be used to detect molecules of interest in solution, typically in diagnostic tests.

[0060] The present invention therefore relates to a method for detecting a molecule of interest in a solution, in particular in a complex medium, comprising the following steps: contacting the solution with at least one functionalized nanoparticle as described above; detecting a specific signal when a biomolecule interacts with one of the components of said solution by SPR, a strip test or any other suitable method; Includes.

[0061] Functionalized NPs according to the present invention can also be used to deplete specific molecules, whether these are target molecules intended to be recovered or undesirable molecules intended to be actually eliminated.

[0062] The present invention therefore relates to a method for detecting a molecule of interest present in a solution, in particular in a complex medium, comprising the following steps: a) contacting the functionalized nanoparticles with a solution containing a molecule of interest; b) incubating said solution in the presence of said nanoparticles; c) recovering said nanoparticles; d) optionally repeating steps a) to c) until the solution of the molecule of interest is exhausted; Includes.

[0063] The use of functionalized NPs to enable their targeting and / or grafting of therapeutic compounds in target cells has long been envisioned for cancer treatment. In radiation therapy, these NPs, which can be activated by X-rays, could represent a major advance in the practice of radiation therapy aimed at controlling tumor destruction. NPs can also be used in photothermal therapy, one of the most promising applications of gold nanoparticles for fighting cancer. The concept is to inject gold nanoparticles into the patient's blood circulation. Due to their structure, these nanoparticles tend to become anchored in cancerous tissues, but coating them with specific molecules (functionalization) can make them more specifically targeted to tumors. They are then "heated" using laser light. The heat released from the NPs causes irreversible damage to cancer cells. This technology is currently being tested in multiple clinical trials targeting head and neck cancers, as well as lung and prostate cancers.

[0064] The present invention therefore relates to the use of functionalized NPs according to the invention in the treatment of cancer, in particular by radiotherapy or photothermal therapy. Controlled photothermal therapy may allow the salting out of therapeutic molecules pre-grafted onto the nanoparticles without affecting the cells or target organs.

[0065] The present invention relates to the use of NPs functionalized according to the present invention for medical imaging diagnostics.

[0066] The solution to which the NPs are added can vary in nature. It can be a simple medium (water, PBS, model medium) or a complex medium (body fluids, wastewater, effluent, etc.). It can also, more generally, be a matrix in which or in contact with which the NPs can be dispersed. This matrix can be a gel, sand, or any flat surface that may contain the molecule of interest on its surface.

[0067] In certain embodiments, the solution is a complex biological medium such as a cell extract, a bacterial culture extract, a biological human sample (selected from serum, blood, urine, amniotic fluid, tears, etc.), an aqueous medium (selected from wastewater, polluted water or water (which may be seawater, water from an aquarium, etc.)).

[0068] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Overview of technological methods for the functionalization of NPs. A) Prefunctionalization with SAM of molecule M (short black curve); B) M + L-PEG (long light gray curve); C) M + PEG-F or biomolecule (medium gray curve); D) M + PEG-F or biomolecule + L-PEG. F denotes a functional group. Figure 2: Absorbance spectra of NSs pre-functionalized with molecules M as a function of coverage in PBS in the presence or absence of surfactant. 20 nm spherical nanoparticles; stirring time = 4 h, M (n = 11, m-4). Figure 3: Absorbance spectra of NRs pre-functionalized with molecules M as a function of coverage in PBS in the presence of surfactant. Nanorods λ = 800 nm; stirring time = 30 min, M (n = 11, m = 4), W = 8%. Figure 4: Absorbance spectra of NSs pre-functionalized with molecule M as a function of coverage in PBS after centrifugation. 20 nm nanoparticles; coverage 1.6–80%; stirring time = 5 min, M (n = 11, m = 4), PBS + 0.005% surfactant. Figure 5: Absorbance spectra of NSs pre-functionalized with molecule M as a function of coverage in PBS in the presence of surfactant. 20 nm nanoparticles; coverage 1.6–80%; stirring time = 4 h; M (n = 11, m = 4), PBS + 0.005% surfactant. Figure 6: Absorbance spectra of NSs pre-functionalized with molecule M as a function of coverage in PBS in the presence of surfactant. 40 nm commercial nanoparticles (ref: 741981 Sigma Aldrich); coverage 0, 1.6 and 16%; stirring time = 4 h, M (n = 11, m = 12), PBS + 0.005% surfactant. Figure 7: Absorbance spectra of NSs pre-functionalized with molecule M and NPs pre-functionalized with molecule M and functionalized with L-PEG (W=5%) dispersed in PBS with different L-PEG coverages (X=0, 1, 10%). 40 nm nanoparticles (ref: 741981 Sigma Aldrich), incubation time L-PEG=5 min, M (n=11, m=4), L-PEG molecular weight=6000, F=CH3. Figure 8: Absorbance spectra of NS functionalized with thiolated molecules bearing COOH functional groups (Y = 1.6%) dispersed in PBS with different coverages of molecules M (W = 0, 1.6, and 16%). 20 nm nanoparticles. M (n = 11, m = 4) and PEG-COOH (r = 11, p = 6). Figure 9: Absorbance spectra of NSs dispersed in PBS with different L-PEG coverages (X = 0 or 10%), pre-functionalized with molecule M, and functionalized with PEG-COOH depending on the presence of L-PEG functionalization (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). Figure 10: Absorbance spectra of NSs dispersed in PBS with different L-PEG coverage (X = 0 or 10%), pre-functionalized with molecule M, and functionalized with PEG-COOH depending on the presence of L-PEG functionalization (W = 5%, Y = 5%). NS size = commercial 80 nm (ref: 742023 Sigma Aldrich), M (n = 11, m = 4), L-PEG molecular weight = 6000, F = CH3, PEG-COOH (r = 11, p = 6). Figure 11: Absorbance spectra of NSs pre-functionalized with molecule M and bearing functionalized DNA (W = 0 or 1.6%; Z = 12.5%); M (n = 11, m = 4), NS size = 20 nm. Figure 12: Absorbance spectra of NSs pre-functionalized with molecule M (W = 10%) and functionalized with DNA (z = 25%) dispersed in PBS with different L-PEG coverage (X = 0 or 10%). F = CH3; NS size = 20 nm. Commercially available (ref: 741965 Sigma Aldrich). Figure 13: Absorbance spectra of NSs pre-functionalized with molecule M (W = 10%) and functionalized with DNA (Z = 25%) dispersed in PBS with different L-PEG coverage (Y = 0 or 10%), F = CH3; NS size = 80 nm commercially available (ref: 742023 Sigma Aldrich). Figure 14: Absorbance spectra of NSs pre-functionalized with molecule M in PBS + 0.005% surfactant as a function of incubation time; 20 nm NSs; stirring time = 5, 30, 120, 240 and 1440 min, M (n = 11, m = 4), W = 40%. Figure 15: Absorbance spectra of NS pre-functionalized with molecule M and functionalized DNA (W = 4% and Z = 50%) corresponding to three different experiments. M (n = 11, m = 4), NP 20 nm. Figure 16: Absorbance spectra of the supernatant of the first wash of NS pre-functionalized with molecule M and carrying functionalized DNA (W=4% and Z=50%), corresponding to three different experiments. M (n=11, m=4), NS 20 nm. Figure 17: Absorbance spectra of NS pre-functionalized with molecule M after 1 day, 3 months, and 11 months of preparation and storage at 4 °C. NS 20 nm, M (n = 11, m = 4), W = 32%. Figure 18: Absorbance spectra of NS pre-functionalized with molecule M and functionalized with thiolated DNA after 1 day and 2 months of storage at 4 °C. M (n = 11, m = 4), W = 0 and 16%, Z = 12.5%. Figure 19: Absorbance spectra of NPs (W = 32% and Z = 25%) pre-functionalized with molecule M and functionalized with thiolated DNA after 1 day and 3 months of storage at 4 °C. NS size = 20 nm, M (n = 11, m = 4). Figure 20: UV-visible spectrum of NPs (W = 30%, Z = 12.5%) pre-functionalized with molecule M and functionalized with thiolated DNA after 1 h incubation in 100-fold diluted cell extract (dotted line) and before addition of cell extract (solid line). NS size = 20 nm, M (n = 11, m = 4). Figure 21: Absorbance spectra of the cell extract diluted 100 times and the sum of the three supernatants after centrifugation (dotted line). NS size = 20 nm, M (n = 11, m = 4) (W = 30%, Z = 12.5%). Figure 22: Left image: Cy5 fluorescence image to identify the location of complementary fluorescent DNA; Right image: Gel contrast image to identify the location of nanoparticles. NS size = 20 nm, M (n = 11, m = 4) (W = 0 and 16%, Z = 12.5%).

[0069] [Example] (term) Thiolated molecule: "thiolated linker molecule capable of interacting with a biomolecule" or "thiolated biomolecule" W is the coverage of the preliminary film formed from molecules M.

[0070] X is the coverage of L-PEG.

[0071] Y is the coverage of PEG-F.

[0072] Z is the coverage of thiolated DNA.

[0073] Example 1: General protocol for pre-functionalization / functionalization of nanoparticles Before any pre-functionalization, it is preferable to remove excess surfactant (e.g., citrate) in the nanoparticle (NP) solution (diluted in water). To achieve this, the NP solution is centrifuged first, the supernatant is removed, and the NP cake is redispersed in an equal volume of distilled water. The speed and time of centrifugation are adjusted depending on the size of the NPs.

[0074] (Example of preparation of functionalized spherical NPs) Starting from the NP solution (sizes of 10-80 nm in diameter), carry out the following steps.

[0075] a) Adding molecules M to achieve a coverage W of 1.5% to 99%; b) Stir for 5 minutes to 4 hours. c) optionally adding L-PEG molecules to achieve a coverage Y of 1% to 10%; d) Stirring for 5 to 60 minutes; e) adding thiolated molecules (either thiolated linker molecules capable of interacting with biomolecules or thiolated biomolecules) to achieve a coverage Y of at least 1% or Z=at least 10%; f) Stirring for 5 minutes to 24 hours; g) if the thiolated molecule is a biomolecule, placing the thiolated molecule in buffer conditions appropriate for the thiolated molecule; h) centrifugation to remove excess thiolated molecules; i) Redisperse in an appropriate solution (distilled water, a buffer solution containing a surfactant, etc.).

[0076] Example 2: Pre-functionalization of gold nanoparticle surfaces with molecule M improves the stability of these nanoparticles in PBS solution a) Spherical nanoparticles A solution of molecule M (W = 24%) is added to the nanoparticle solution. The mixture is stirred for several hours (at least 4 hours), and then 0 and 0.005% surfactant, phosphate buffer pH 7.4, and NaCl are added. The NPs are then dispersed in PBS (phosphate 10 nM, pH = 7.4, and 0.1 M NaCl) in the presence or absence of 0.005% surfactant.

[0077] The stability data of the nanoparticles are shown in FIG.

[0078] It is observed that without molecule M, the nanoparticles are not stable and aggregate. In contrast, the presence of molecule M allows the nanoparticles to remain in suspension. According to the absorbance spectrum in Figure 2, an improvement in the stability of the nanoparticles in PBS in the presence of surfactant is observed, as the plasmon band is refined.

[0079] b) Nanorods A solution of molecule M, followed by 0.005% surfactant, is added to a solution of nanorods (NRs). The mixture is stirred for 30 minutes, and then phosphate buffer solution at pH 7.4 and NaCl are added. The NPs are then dispersed in PBS (phosphate 10 nM, pH 7.4, and 0.1 M NaCl) in the presence of 0.005% surfactant.

[0080] Data on the stability of the nanoparticles are shown in Figure 3. It is observed that without molecule M, the nanoparticles are not stable and aggregate. In contrast, the presence of molecule M allows the nanoparticles to remain suspended in PBS + 0.005% surfactant.

[0081] The remaining experiments were carried out with spherical NPs.

[0082] Example 3: Nanoparticles pre-functionalized with molecule M can be washed and redispersed in PBS solution in the presence of surfactants The solution of molecule M is added to the solution of nanoparticles. The mixture is stirred for 5 minutes or 4 hours, centrifuged and redispersed in a solution of PBS 1× in the presence of 0.005% surfactant.

[0083] "PBS 1x" means a solution containing 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and having a pH of 7.4.

[0084] Various protocols were tested: a) 20 nm spherical nanoparticles were incubated in a solution of molecule M for 5 minutes.

[0085] The data on nanoparticle stability are shown in FIG.

[0086] b) 20 nm spherical nanoparticles were incubated in a solution of molecule M for 4 hours.

[0087] The data on nanoparticle stability are shown in FIG.

[0088] c) 40 nm spherical nanoparticles were incubated in a solution of molecule M for 4 hours.

[0089] The data on nanoparticle stability are shown in FIG.

[0090] These results clearly show that the stability of the nanoparticles increases with the coverage of the solution of molecule M.

[0091] Example 4: Functionalization with L-PEG molecules improves the stability of nanoparticles pre-functionalized with molecule M in PBS solution a) Only spare function provided For pre-functionalization only, a solution of molecule M with W = 1.6–80% was added to the nanoparticle solution. The mixture was stirred for a few minutes or hours (5 min–24 h), centrifuged, and redispersed in an appropriate solution.

[0092] b) Functionalization using pre-functionalized L-PEG molecules For functionalization with L-PEG molecules after pre-functionalization, a solution of molecule M is added to the nanoparticle solution to achieve a coverage of W = 5%. After stirring for at least 5 min, L-PEG solutions are added at coverages of 0, 1, and 10%. The mixture is stirred again for 15 min, then centrifuged and redispersed in a solution of PBS (1x).

[0093] The nanoparticle stability data are shown in FIG.

[0094] These results show that, starting from X = 1%, the addition of L-PEG allows NPs coated with a monolayer of molecule M in PBS to be stabilized at low coverages (in this case, W = 5%). Indeed, the plasmon band is refined with L-PEG, confirming the improved stability.

[0095] Example 5: Pre-functionalization of nanoparticles with molecule M improves the stability of the nanoparticles functionalized with thiolated molecules in PBS solution a) Functionalization with thiolated carboxyl molecules (without pre-functionalization).

[0096] A solution of PEG-COOH was added to the nanoparticle solution to achieve a coverage Y of 1.6%. The mixture was stirred for 30 min, centrifuged, and redispersed in either HO or PBS 1× + 0.005% surfactant.

[0097] b) Functionalization with thiolated carboxyl molecules after prefunctionalization A solution of molecule M was added to the nanoparticle solution to achieve a theoretical initial coverage Y of 1.6%–80%. After stirring for 5 min, a solution of PEG-COOH was added to achieve a coverage Y of 1.6%. The mixture was stirred for 30 min, centrifuged, and redispersed in either distilled water or PBS 1× + 0.005% surfactant.

[0098] The data on nanoparticle stability are shown in FIG.

[0099] These results show that NPs functionalized only with PEG-COOH aggregate in PBS. Pre-functionalizing the NPs with a solution of molecules M allows us to improve the stability of the NPs in solution, especially from a coverage of W = 1.6%. By increasing the incubation time and W, i.e., by increasing the density of molecules M on the surface of the NPs, the stability is further improved (in this case, the curve reflecting the highest stability at W = 16%).

[0100] Example 6: Functionalization with L-PEG molecules improves the stability of NPs pre-functionalized with molecule M and functionalized with thiolated molecules in PBS solution A solution of molecule M was added to the NP solution to achieve a coverage of W = 5%. Next, a solution of L-PEG was added to achieve a coverage of X = 0 or 10%. A solution of PEG-COOH was then added to achieve a coverage of Y = 5%. The mixture was stirred, centrifuged, and redispersed in either distilled water or PBS 1x + 0.005% surfactant.

[0101] Data on nanoparticle stability are shown in Figure 9 for 20 nm spherical NPs and Figure 10 for 80 nm spherical NPs.

[0102] It should be noted that the addition of 10% L-PEG increases the stability of NPs that have been pre-functionalized and further functionalized with a thiolated molecule (PEG-COOH in this case), especially at low coverages of molecule M and thiolated molecule (5% coverage each).

[0103] Example 7: Pre-functionalization with molecule M improves the stability of NPs functionalized with thiolated molecules in PBS solution: the case of thiolated DNA molecules a) Functionalization with thiolated DNA molecules (without pre-functionalization) A solution of DNA was added to the nanoparticle solution to achieve a coverage Z of at least 12.5%. The mixture was stirred for 1 hour, and then 0.005% surfactant, phosphate buffer, and NaCl were added. The mixture was stirred for 18 hours, centrifuged, and redispersed in PBS 1x.

[0104] b) Functionalization using pre-functionalized thiolated DNA molecules A solution of molecule M was added to the NP solution to achieve coverages of 1.6 and 80%. A solution of DNA was then added to achieve a coverage Z of at least 12.5%. 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.

[0105] The data on nanoparticle stability are shown in FIG.

[0106] Note that DNA-functionalized NPs aggregate in PBS. However, pre-functionalization with molecule M improves stability starting from a coverage W of 1.5%. By increasing the incubation time and W, i.e., by increasing the density of molecule M on the surface of the NPs, stability can be further improved (data not shown).

[0107] Example 8: L-PEG functionalization improves the stability of NPs pre-functionalized with molecule M and functionalized with thiolated DNA in PBS solution A solution of molecule M was added to the nanoparticle solution to achieve a coverage of 1.6 and 80% W. A solution of L-PEG was then added after 30 minutes of incubation to achieve a coverage of 1-10% X. The mixture was stirred for 15 minutes. A solution of SH-DNA was then added to achieve a theoretical initial coverage of at least 12.5%. After the addition of the DNA solution, 0.005% surfactant, pH 7.4 phosphate buffer (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.

[0108] Data on nanoparticle stability are shown in Figure 12 for 20 nm NPs and Figure 13 for 80 nm NPs.

[0109] An improvement in the stability of NPs is observed when L-PEG molecules are added. In particular, the presence of a molecular film of L-PEG makes it possible to stabilize 80 nm NPs in a PBS solution with a low level of molecular weight (W = 10%).

[0110] Example 9: Effect of incubation time of NP in solution containing molecule M on stability A solution of molecule M is added to the nanoparticle solution. The mixture is stirred for several minutes or several hours (5 minutes to 24 hours), and then 0.005% surfactant, phosphate buffer pH 7.4, and NaCl are added. The NPs are then dispersed in PBS (phosphate 10 nM, pH 7.4, and 0.1 M NaCl) in the presence of 0.005% surfactant.

[0111] The data on nanoparticle stability are shown in FIG.

[0112] Note that coverage increases as a function of time, reaching a maximum between 120 and 240 minutes of incubation. Continuing incubation beyond this period has no further effect on W.

[0113] [Example 10] Reproducibility of the method for stabilizing nanoparticles To confirm the reproducibility of the protocol, the protocol was repeated three times on different days. The stability of the samples was characterized by UV-visible spectroscopy. Regarding the grafting of thiolated DNA, the first supernatant was analyzed by UV-visible spectroscopy during the washing step to remove excess DNA.

[0114] The results are shown in Figure 15 for the stability of the solution of functionalized NPs and in Figure 16 for the supernatant obtained during the first step of washing off the non-adsorbed DNA.

[0115] The UV-visible spectra contain fine superimposable plasmon bands, which suggest that the nanoparticles NP pre-functionalized with molecule M and functionalized with DNA are stable, as is the identical absorbance intensity of the supernatant, which suggests that the same amount of DNA is bound to the NP. In conclusion, these results confirm the reproducibility of the sample preparation.

[0116] Example 11: NPs pre-functionalized with molecule M are stable in water for several months As described above, 20 nm NPs were pre-functionalized with molecule M and then stored at 4 °C in the dark for several months. Stability was investigated by UV-visible spectroscopy. Typically, 1 mL of solution was removed and analyzed using a UVIKON analyzer. Spectra obtained at different times were compared.

[0117] The nanoparticle stability data are shown in FIG.

[0118] A good correlation of the absorbance spectra of NPs pre-functionalized with molecule M is observed after 1 day and 11 months of storage. The three spectra show a fine plasmon band without any displacement of the plasmon band, confirming that the pre-functionalized NPs are still stable in water after 11 months of storage at 4 °C.

[0119] Example 12: NPs pre-functionalized with molecule M and functionalized with DNA are stable in PBS solution for several months As described above, 20 nm NPs were pre-functionalized with molecule M and functionalized with DNA, and then stored at 4 °C in the dark for several months. Stability was assessed by UV-visible spectroscopy. Typically, 1 mL of solution was removed and analyzed using a UVIKON analyzer. The spectra obtained after different storage times were compared.

[0120] Data on nanoparticle stability are shown in Figures 18 and 19.

[0121] First, it should be noted that the presence of molecule M makes it possible to stabilize the NPs after preparation and during storage. These results show a good correlation of the spectra of NPs pre-functionalized and functionalized with DNA compared to the spectra obtained after 1 day and 2-3 months of storage. The two spectra show two fine plasmon bands without any displacement of the plasmon band, confirming that the NPs are still stable in PBS after 2-3 months of storage at 4 °C.

[0122] Example 13: Pre-functionalized and DNA-functionalized NPs are stable in complex media 10 μl of cell extract was added to 1 ml of a solution of NPs pre-functionalized with molecule M at 1 nM (dilution of complex medium is ×100) and functionalized with thiolated DNA. The mixture was stirred for 1 hour, then centrifuged and redispersed in PBS 1×. The stability of the NPs and the absorbance intensity of the cell culture medium in the supernatant were characterized by UV-visible spectroscopy. According to the Beer-Lambert law, the absorbance intensity is related to the concentration in the solution. If the absorbance intensity of the cell extract is similar to that of the supernatant, this indicates that the biomolecules present in the cell extract are not absorbed nonspecifically by the NPs.

[0123] Data on nanoparticle stability are shown in Figures 20 and 21.

[0124] The absorbance spectra of the samples before and after 1 hour of incubation were compared. Note that no aggregation occurred in the complex medium, suggesting that the particles were well protected. To determine whether there was nonspecific adsorption, the particles were washed by centrifugation to remove the cell extract. The combined spectrum of the three supernatants after 1 hour of incubation was overlaid with the spectrum of the initial cell extract. It was possible to observe that the spectra were superimposed, suggesting little or no specific adsorption of proteins from the cell extract to the surface of the functionalized NPs.

[0125] Example 14: Prefunctionalization of NP with molecule M improves hybridization in complex media One microliter of 100 nM complementary fluorescent DNA (Cy5) was added to 200 μl of 1.5 nM NPs, functionalized with DNA with or without pre-functionalization with molecule M. Thus, the total concentration of complementary DNA was 500 pM. Hybridization was incubated for 2 hours at 37 °C in three different media (PBS 1x, cell extract, and human serum). The complex media were diluted 100-fold. To distinguish between fluorescent DNA, DNA-functionalized NPs, and NPs pre-functionalized before DNA functionalization, the samples were placed on an agarose gel for electrophoresis. Electrophoresis allows for the separation and differentiation of hybridized nanoparticles and free complementary DNA in solution.

[0126] Hybridization of complementary fluorescent strands with two types of nanoparticles, either NPs functionalized with DNA molecules alone or NPs pre-functionalized with a monolayer of molecule M and then functionalized with DNA molecules, was investigated in three different media: PBS, 100-fold diluted cell extract, and 100-fold diluted human serum. The nanoparticles used were synthesized 2 months before use and stored at 4°C.

[0127] The results of this experiment are shown in FIG.

[0128] Only the nanoparticles are observed to be visible, since only the fluorophore is not excited. The migration distance of nanoparticles functionalized with DNA, as well as that of NPs pre-functionalized before functionalization with DNA, appears unaffected in PBS and cell extracts. In contrast, in human serum, the migration distance changes slightly for NPs pre-functionalized before functionalization, but more significantly for NPs functionalized with DNA (without molecule M). This suggests the possibility of nonspecific adsorption on these nanoparticles (evident by the slowdown in migration due to the increasing size of the NPs). In the left part of the figure, only fluorescence emission is observed (black band in the gel), indicating the location of the complementary DNA in the gel. Comparing the band intensity and band position in Figure 22, it is possible to correlate the location of the DNA with the location of the NPs in the gel, and therefore the effectiveness of hybridization. While the effectiveness of hybridization between pre-functionalized and non-pre-functionalized nanoparticles appears unaffected in PBS, clear differences are observed in complex media. Indeed, for samples without molecule M (i.e., DNA only), hybridization appears to be affected, since the fluorescence corresponding to free DNA is significantly stronger (less complementary DNA hybridized to the DNA bound to the NPs). These results suggest that the effectiveness of hybridization for prefunctionalized samples is greater in complex media. It is possible to hypothesize that in complex media, nonspecific adsorption of proteins on NPs functionalized with DNA (but not prefunctionalized) prevents hybridization between complementary DNA and the DNA bound to the NPs.

[0129] Example 15: Evaluation of detection of target complementary DNA in various media The detection of single strands of the target DNA (cDNA) becomes possible according to the effectiveness of hybridization between the complementary fluorescent DNA (Cy5) of the target and the DNA immobilized on the nanoparticles pre-functionalized with molecule M. Once the DNA is hybridized, two gel electrophoresis runs were performed, one before and one after centrifugation of the NPs. Centrifugation allows for the concentration of the NPs, which allows for improved visualization of the complementary fluorescent DNA on the surface of the NPs.

[0130] The hybridization protocol is as follows: 1 nM of complementary fluorescent DNA (50 pM) was added to a suspension of 2 nM NPs (NP size = 20 nm, M (n = 11 and m = 4), W = 16% and Z = 12.5%). The mixture was incubated for 2 h at 37 °C either in PBS or in commercial human serum (dilution 100) (Thermofisher, Normal Human Serum, ref. 31876).

[0131] Gel migration of the NPs was performed (data not shown). The "Nanoparticle Image" section shows the position of the bioconjugated NPs, and the "Fluorescence Image" section shows the position of the complementary fluorescent DNA. The control corresponds to free complementary fluorescent DNA. Different concentrations were charged, ranging from 50 pM to 1 nM.

[0132] These results confirm that hybridization has occurred, as fluorescence correlates with nanoparticle location. Fluorescence is observed for a range of target DNA concentrations up to 50 pM in PBS or human serum. The presence of complex media does not prevent hybridization, even at low concentrations of target DNA.

[0133] (Conclusion) Hybridization of DNA probes onto NPs allows detection of 50 pM of complementary DNA at 1 nM in PBS or human serum. [Brief explanation of the drawings]

[0134] [Figure 1]Overview of technological methods for functionalization of NPs. [Figure 2] Absorbance spectra of NSs prefunctionalized with molecule M as a function of coverage in PBS in the presence or absence of surfactant. [Figure 3] Absorbance spectra of NRs pre-functionalized with molecule M as a function of coverage in PBS in the presence of surfactant. [Figure 4] Absorbance spectra of NSs pre-functionalized with molecule M as a function of coverage in PBS after centrifugation. [Figure 5] Absorbance spectra of NSs pre-functionalized with molecule M as a function of coverage in PBS in the presence of surfactant. [Figure 6] Absorbance spectra of NSs pre-functionalized with molecule M as a function of coverage in PBS in the presence of surfactant. [Figure 7] Absorbance spectra of NSs pre-functionalized with molecule M and NPs pre-functionalized with molecule M and functionalized with L-PEG (W = 5%) dispersed in PBS with different L-PEG coverages (X = 0, 1, 10%). [Figure 8] Absorbance spectra of NSs functionalized with thiolated molecules bearing COOH functional groups (Y = 1.6%) dispersed in PBS with different coverages of molecules M (W = 0, 1.6, and 16%). [Figure 9] Absorbance spectra of NSs dispersed in PBS with different L-PEG coverage (X = 0 or 10%), pre-functionalized with molecule M, and functionalized with PEG-COOH depending on the presence of L-PEG functionalization (W = 5%, Y = 5%). [Figure 10] Absorbance spectra of NSs dispersed in PBS with different L-PEG coverage (X = 0 or 10%), pre-functionalized with molecule M, and functionalized with PEG-COOH depending on the presence of L-PEG functionalization (W = 5%, Y = 5%). [Figure 11]Absorbance spectra of NS pre-functionalized with molecule M and bearing functionalized DNA (W = 0 or 1.6%; Z = 12.5%). [Figure 12] Absorbance spectra of NSs pre-functionalized with molecule M (W = 10%) and functionalized with DNA (z = 25%) dispersed in PBS with different L-PEG coverages (X = 0 or 10%). [Figure 13] Absorbance spectra of NSs pre-functionalized with molecule M (W = 10%) and functionalized with DNA (Z = 25%) dispersed in PBS with different L-PEG coverages (Y = 0 or 10%). [Figure 14] Absorbance spectra of NSs pre-functionalized with molecule M in PBS + 0.005% surfactant as a function of incubation time. [Figure 15] Absorbance spectra of NS pre-functionalized with molecule M and functionalized DNA (W = 4% and Z = 50%) corresponding to three different experiments. [Figure 16] Absorbance spectra of the supernatant of the first wash of NS pre-functionalized with molecule M and with functionalized DNA (W=4% and Z=50%), corresponding to three different experiments. [Figure 17] Absorbance spectra of NSs pre-functionalized with molecule M after 1 day, 3 months, and 11 months of preparation and storage at 4 °C. [Figure 18] Absorbance spectra of NS pre-functionalized with molecule M and functionalized with thiolated DNA after 1 day and 2 months of storage at 4 °C. [Figure 19] Absorbance spectra of NPs pre-functionalized with molecule M and functionalized with thiolated DNA (W = 32% and Z = 25%) after 1 day and 3 months of storage at 4 °C. [Figure 20] UV-visible spectra of NPs (W = 30%, Z = 12.5%) pre-functionalized with molecule M and functionalized with thiolated DNA after 1 h of incubation in 100-fold diluted cell extract (dotted line) and before addition of cell extract (solid line). [Figure 21]Absorbance spectra of cell extracts diluted 100-fold and the sum of the three supernatants after centrifugation (dotted line). [Figure 22] Left image: Cy5 fluorescence image to identify the location of the complementary fluorescent DNA; right image: gel contrast image to identify the location of the nanoparticles.

Claims

1. Formula (1): HS(CH ２ )n(OCH ２ CH ２ ) nanoparticles comprising a metal surface pre-functionalized with a self-assembled protective monolayer formed by a matrix of molecules M of mOH, where: n is CH ２ where n is equal to 11, m represents the number of ethylene glycols, and 4≦m≦12; The molecule has a thiol functional group at one end and an inert end at the other end; The coverage W of the monolayer is 1.5% to 50%; the nanoparticles are further functionalized with at least one L-PEG molecule of formula (7): HS-CH 2 CH 2 (OCH 2 CH 2 ) q OF; where: q represents the number of ethylene glycols, and 20≦q≦500; F represents CH 3 or an activatable COOH functional group; The nanoparticles, wherein the coverage X of the L-PEG molecules is at least 1%.

2. and further functionalized with at least one thiolated molecule, said thiolated molecule comprising: Formula (8): HS(CH ２ ) ｒ (OCH ２ CH ２ ) ｐ a PEG-F linker molecule of O-F, where: r is CH ２ represents the number of, and is an integer of 3 or more, p represents the number of ethylene glycols, and 2≦p≦12; F is CH ３ or an activatable COOH functional group, a PEG-F linker molecule having a thiol functional group at one end and an active or reactive group at the other end that can interact with a biomolecule; or 3. The nanoparticle of claim 1, wherein the nanoparticle is a biomolecule comprising a thiol functional group.

3. Nanoparticles comprising a metal surface pre-functionalized with a self-assembled protective monolayer formed by a matrix of molecules M of formula (1): HS(CH 2 )n(OCH 2 CH 2 )mOH, where: n represents the number of CH2, n is equal to 11, m represents the number of ethylene glycols, and 4≦m≦12; The molecule has a thiol functional group at one end and an inert end at the other end; The coverage W of the monolayer is 1.5% to 50%; The nanoparticles are further functionalized with at least one thiolated molecule, the thiolated molecule comprising: Formula (8): HS(CH ２ ) ｒ (OCH ２ CH ２ ) ｐ a PEG-F linker molecule of O-F, where: r is CH ２ represents the number of, and is an integer of 3 or more, p represents the number of ethylene glycols, and 2≦p≦12; F is CH ３ or an activatable COOH functional group, a PEG-F linker molecule having a thiol functional group at one end and an active or reactive group at the other end that can interact with a biomolecule; or a nanoparticle, which is either a biomolecule containing a thiol functional group;

4. 4. The nanoparticles according to claim 2 or 3, characterized in that the thiolated molecule is a PEG-F linker molecule of formula (8), wherein F is an activatable COOH functional group, and the coverage Y is at least 1%.

5. 5. The nanoparticles of claim 4, further functionalized with at least one biomolecule attached to the PEG-F linker molecule of formula (8).

6. Nanoparticles as described in claim 2 or 3, characterized in that the biomolecule containing a thiol functional group is thiolated DNA and its coverage rate Z is at least 10%.

7. The method of claim 6, wherein the thiolated molecule is a PEG-F linker molecule of formula (8), and the nanoparticle is further functionalized with at least one L-PEG molecule of formula (7): HS-CH 2 CH 2 (OCH 2 CH 2 ) q O-F; where: q represents the number of ethylene glycols, and 20≦q≦500; F represents CH 3 or an activatable COOH functional group; The nanoparticles of claim 3, wherein the coverage X of the L-PEG molecules is at least 1%.

8. The nanoparticles according to any one of claims 1 to 7, wherein the nanoparticles are spherical nanoparticles, nanorods, cubic nanoparticles, nanotriangles, core-shells, or nanourtines.

9. 1. A method for preparing nanoparticles comprising a metal surface pre-functionalized with a self-assembled protective monolayer formed by a solution of molecules having a thiol functional group at one end and an inert end at the other end, said method comprising the steps of: Adding a molecule M of formula (1) as defined in claim 1 to achieve a coverage W of 1.5% to 50%; Stirring for at least 5 minutes; adding a solution of L-PEG molecules of formula (7) as defined in claim 1 to achieve a coverage X of 1% to 10%.

10. 10. The method of claim 9, further comprising the step of adding a thiolated molecule selected from the PEG-F linker molecules of formula (8) defined in claim 2, which is capable of interacting with a biomolecule or a biomolecule containing a thiol functional group.

11. A method for preparing nanoparticles comprising a metal surface pre-functionalized with a self-assembled protective monolayer formed by a solution of molecules having a thiol functional group at one end and an inert end at the other end, said method comprising the steps of: Adding a molecule M of formula (1) as defined in claim 3 to achieve a coverage W of 1.5% to 50%; Stirring for at least 5 minutes; Attaching a thiolated molecule selected from the PEG-F linker molecules of formula (8) as defined in claim 3, wherein the thiolated molecule is capable of interacting with a biomolecule or a biomolecule containing a thiol functional group.

12. A method for detecting a molecule of interest in a solution, in particular in a complex medium, comprising the following steps: (i) contacting at least one functionalized nanoparticle according to claim 2 or 3 or at least one functionalized nanoparticle according to claim 5 or 6 with the solution containing the molecule of interest, when the thiolated molecule is the biomolecule containing a thiol functional group; (ii) detecting by SPR or strip test a specific signal when the biomolecule of the at least one functionalized nanoparticle interacts with one of the components of the solution.

13. After step (i), the following step: a) incubating the solution in the presence of the nanoparticles; b) recovering said nanoparticles; 13. The method of claim 12, further comprising: c) optionally repeating steps a)-b) until the solution of the molecule of interest is exhausted.

14. A pharmaceutical composition comprising nanoparticles according to any one of claims 1 to 8 for use in the treatment of cancer, in particular in radiotherapy, phototherapy, and in the preparation of medicines and in medical imaging.

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