METHOD FOR MEASURING INTERACTION BETWEEN MOLECULES.

专利摘要:

公开号:BE1020732A5
申请号:E201300102
申请日:2013-02-15
公开日:2014-04-01
发明作者:Meike Roskamp；Vanessa Bonnard；Sylviane Boucharens
申请人:Pharmadiagnostics Nv；
IPC主号:

专利说明:

METHOD OF MEASURING INTERACTION BETWEEN MOLECULES FIELD OF THE INVENTION
The present invention relates to methods to measure the interaction between a first and a second molecule, for example a protein and an antibody, by conjugating one of these molecules with nanoparticles and by measuring the interaction between the first and the second molecule at changes in the optical properties of the nanoparticles. The present invention is further related to coating nanoparticles.
BACKGROUND OF THE INVENTION
Metal nanoparticles coated with molecules such as proteins can be used to determine binding events by monitoring a change in the optical properties of the particles, for example, by sensor measurements of the localized surface plasma resonance (Localized Surface Plasmon Résonance - LSPR). In the usual scheme, a recognition interface is built on the metal nanostructure. The specific binding of an analyte with the above-mentioned recognition interface is converted into an optical signal, e.g. a change in absorbance (wavelength, intensity) that is detected and analyzed.
Sensor measurement of the LSPR is based on the sensitivity of the localized plasmon absorbance of metal nanoparticles to changes in the dielectric properties of the contact medium.
In principle, LSPR can be used for the detection of antibody-ligand interactions, receptor-ligand interactions, enzyme-ligand binding and antibody-antigen association-dissociation kinetics.
In practice, however, it was observed that such methods where nanoparticles are used in a solution often do not provide the required accuracy.
For this reason, there remains a need in the art to provide methods that allow accurate determination of nanoparticle interactions between molecules.
SUMMARY OF THE INVENTION
The present invention relates to the methods for measuring the interaction between a first and a second molecule, by conjugating one of these molecules with nanoparticles and by measuring the interaction between the first and the second molecule on the basis of changes in the optical properties of the nanoparticles. The present invention offers the possibility of using nanoparticles in amounts sufficient for reliable detection, while avoiding, or at least reducing, depletion (depletion) of the ligand.
In a first aspect, the present invention provides a method for determining the interaction between a first and a second molecule, comprising: a) providing nanoparticles containing one or more metals: b) providing the aforementioned nanoparticles with one or more functional groups or linking the first molecule with a molecule that contains a metal-binding functionality; c) conjugating the above-mentioned first molecule with the above-mentioned nanoparticles, wherein the amount of the above-mentioned first molecule attached to the above-mentioned nanoparticles is less than 70%, and preferably between 10% and 70%, of the amount required for complete coverage of the above-mentioned nanoparticles with the above first molecule; d) incubating the above-mentioned nanoparticles with the above-mentioned second molecule; e) monitoring step d) by illuminating the above-mentioned nanoparticles with at least one excitation light source and monitoring one or more optical properties of the above-mentioned nanoparticles; and f) detecting a change in one or more optical properties of said nanoparticles wherein said change is a result of the presence of an interaction between said first molecule and said second molecule.
In certain embodiments, the nanoparticles contain gold, silver of copper. In certain embodiments, the nanoparticles are nanoparticles.
In certain embodiments, step f) comprises determining an association-dissociation equilibrium between the above-mentioned first and second molecule. In certain embodiments, step f) comprises the measurement of the above-mentioned one or more optical properties at two or more wavelengths between 350 and 1000 nm.
In certain embodiments, step b) comprises the provision of nanoparticles, having attached to their surface: one or more molecules containing a metal-binding functionality and a functional group selected from carboxyl, amino, azide, alkynyl, carbonyl and hydroxyl; and one or more molecules containing the above-mentioned metal-binding functionality and which does not contain the above-mentioned functional group selected from carboxyl, amine, azide, alkynyl, carbonyl and hydroxyl.
In certain embodiments, step c) comprises: c1) optionally, the selection of a suitable pH and an ionic strength for conjugation of the above-mentioned first molecule with the above-mentioned nanoparticles via a buffer test; c2) determining the amount of said first molecule required for the conjugation of said first molecule with said nanoparticles; c3) conjugation of the above-mentioned first molecule with the above-mentioned nanoparticles, based on the information obtained in step c2) and optionally c1).
In further embodiments, step c2) comprises a concentration titration of the above-mentioned nanoparticles with the above-mentioned first molecule, optionally at the pH and ionic strength selected in step c1).
In certain embodiments, step b) comprises coupling of the above-mentioned first molecule to a linker molecule that has a metal-binding functionality and step c) comprises conjugation of the above-mentioned first molecule with the above-mentioned nanoparticle via the above-mentioned left molecule. In further embodiments, the left molecule is a mercaptocarboxylic acid.
In certain embodiments, the first molecule is a protein and the surface of the aforementioned nanoparticles is provided with carboxyl groups. In further embodiments, the current method further comprises reacting free carboxyl groups on the surface of the aforementioned nanoparticles with a carboxyl blocking agent.
In a further aspect, the present invention provides a method for coating a metal nanoparticle with a compound, comprising: i) providing a liquid composition containing metal nanoparticles that are at least partially coated with cetyl trimethyl ammonium bromide (CTAB); ii) adding a thiol-polyethylene glycol to the above composition, thereby obtaining a liquid composition containing metal nanoparticles coated with thiol-polyethylene glycol; iii) purify the above composition obtained in step ii) by separating the above nanoparticles from free thiol-polyethylene glycol and free CTAB; iv) adding mercaptocarboxylic acid to the above-mentioned composition obtained in step iii), thereby obtaining a liquid composition containing metal nanoparticles coated with mercaptocarboxylic acid, and v) bringing the above-mentioned particles obtained in step iv) into contact with the above connection.
In certain embodiments, the liquid composition in steps i), ii), iii) and iv) is an aqueous composition.
In a further aspect, the present invention provides a kit comprising: - a medium containing a plurality of metal nanoparticles; instructions for using the above-mentioned nanoparticles in the method for determining an interaction between a first and a second molecule according to the present invention; - optionally, the above-mentioned first molecule; and - optionally, the second molecule mentioned above.
BRIEF DESCRIPTION OF THE FIGURES
The invention will now be described, inter alia with reference to the accompanying Images, which are only provided as an example and cannot be regarded as a limitation on the scope of the present invention.
Figure 1A shows a protein-titration curve for human Serum Albumin (HSA) and shows a graph of ARU (i.e., AOD ^ max + 80) / OD (Àmax)) relative to an amount of HSA per mL nanoparticle suspension.
Figure 1B shows the absorbance spectrum of identical nanoparticles conjugated with different amounts of a protein.
Figure 2 shows a graph of ARU relative to an amount of antibody per mL of nanoparticle-HSA conjugate suspension.
Figure 3 represents a plot of ARU relative to an amount of BSA_MUDA added to a suspension of mPEG-SH coated golden nanoparticles.
Figure 4 shows a conjugation of a first molecule (1) and a nanoparticle (2) according to a particular embodiment of the present invention.
Figure 5 shows a conjugation of a first molecule (1) and a nanoparticle (2) according to a particular embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described with respect to certain embodiments, but is not limited thereto, but only by the claims. Any reference marks in the claims cannot be interpreted as limiting their scope.
As used herein, the singular forms "one" and "the" include both the singular and plural forms, unless the context clearly dictates otherwise.
The terms "comprising", "includes" and "consisting of" as used herein are synonymous with "including", "understanding" of "containing", "contains", and have an inclusive character of an open end and do not exclude additional, unnamed parts, elements of method steps. When the terms "comprising", "includes" and "consisting of" refer to said parts, elements of method steps, they also include embodiments that "consist of" said mentioned parts, elements of method steps.
In addition, the terms first, second, third and the like in the description and in the claims are used to distinguish between similar elements and not necessarily to describe a sequential of chronological order, unless so specified. The terms are interchangeable under appropriate conditions and the embodiments described herein may be used in sequences other than those described herein.
The term "about", as used herein, when referring to a measurable value, such as a parameter, an amount, a duration, and the like, includes variations of +/- 10% of less, preferably + 1-5% of less , and more preferably +/- 1% of less, but preferably +/- 0.1% of less of the specified value, insofar as such variations are suitable for performing the same function. The value to which the "approximately" assertion refers is also specifically, and preferably, disclosed.
The designation of the numerical ranges by end points includes all numbers and fractions within the respective ranges, as well as the designated end points.
All documents stated in the present specification are hereby incorporated by reference in their entirety.
Unless otherwise specified, all terms used in the description, including technical and scientific terms, have the usual meaning as understood by someone with normal experience in the art. As a further guideline, this document contains definitions for the terms used in the description for a better understanding of the introduced concepts, provided herein. The terms of the definitions used herein are provided only for a better understanding of the invention.
As used herein, the term "localized surface plasmon resonance" or "LSPR" refers to methods that detect changes on or near the surface of metal nanoparticles. Normally these changes are detected by the detection of changes in one or more optical properties of the particles. When the metal surfaces of the nanoparticles are excited by electromagnetic radiation, they exhibit collective oscillations of their conduction electrons, known as localized surface plasmons (LSPs). When excited in this way, the nanoparticles behave like nanoscale antennas, which concentrate the electromagnetic field in very small volumes near the particles. In this way, exceptionally large improvements in electromagnetic intensity can be achieved. The nanoparticles used in the LSPR make the occurrence of resonance oscillations possible.
As used herein, the term "absorbance" refers to the extent to which a sample absorbs light or electromagnetic radiation in the UV, visual or near-infrared region of the spectrum. In LSPR, changes in the refractive index can be detected by monitoring changes in absorbance. After exposure of a sample, changes in the LSPR extinction band cause changes in the intensity and / or the wavelength of the maximum absorbance.
The term "colloid" refers to a liquid composition of particles suspended in a liquid medium. The dimensions of the particles in representative colloids vary from one nanometer to one micrometer.
The term "sample," as used herein, refers to a liquid composition, wherein in certain embodiments, the liquid composition is an aqueous composition. While a person skilled in the art would understand that any type of sample can be used in the context of the present invention, non-limiting examples include biological samples, including patient and environmental samples, plasma, hybridoma supernatants, etc.
The term "C4-16 alkyl", as a group of a part of a group, refers to a hydrocarbyl radical of Formula CnH2n + i wherein n is a number ranging from 4 to 16.
Alkyl groups can be linear or branched and can be substituted as indicated herein. When subscript is used herein after a carbon atom, the subscript refers to the number of carbon atoms that the said group can contain. Thus, for example, C 6-12 alkyl means an alkyl of 6 to 12 carbon atoms. Examples of alkyl groups are octyl, decyl, undecyl and the chain isomers thereof.
The term "C4.16 alkylthior" refers to HS-RW, where Rw is C4-16 alkyl. Non-limiting examples of suitable C 4-6 alkylthiol include undecane-1-thiol, decane-1-thiol or octane-1-thiol.
The term "azide" refers to -N3. The term "amine" by itself or as part of another substituent refers to -NH2.
The term "alkynyl" refers to a branched or unbranched and cyclic or acyclic unsaturated hydrocarbon group that contains at least one triple bond. Non-limiting examples of alkyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl and the like.
The term "aqueous" as used herein means that more than 50 percent of the volume of the solvent is water. Aqueous compositions or dispersions may further contain other organic liquids that are miscible with water.
Reference in this specification to "one embodiment" or "an embodiment" means that a particular element, structure, or feature described in connection with the embodiment is part of at least one embodiment of the invention as contemplated herein. Thus, if the expressions "in one embodiment" of "in an embodiment" appear in different places in this specification, they do not necessarily all refer to the same embodiment, but possibly do. In addition, the particular elements, structures of properties can be combined in any suitable manner in one or more embodiments, as would be obvious to a person skilled in the art of this disclosure. Although some embodiments, described herein, contain some, but not the other elements that are part of other embodiments, combinations of elements of different embodiments are within the scope of the explanations provided herein, and form different embodiments, as it would be understood be experienced by people with experience. For example, in the following, any of the described embodiments may be used in any combination.
The present invention relates to the use of nanoparticles for determining the interaction between a first and a second molecule, in particular the provision of methods and means to increase the accuracy of the detection of such interactions. The present inventors have indeed established that the accuracy of the detection is highly dependent on the coating of the nanoparticles.
Typically, the binding of a ligand to a specific receptor or binding site can be characterized by the dissociation equilibrium constant (Kd). According to the law of mass action, the K a of the receptor or the ligand compound of the receptor or binding site is dependent on both the association (kon) and the dissociation (coffe) speed constants and determined as the ratio of coffe to k0i7.
Ko - kofl / kon
Standard methods for the analysis of the Ko parameter (saturation binding) normally assume that the concentration of free ligand is constant during the experiment and that the free ligand concentration does not differ significantly from the total added ligand concentration. In order to meet this restriction, the concentration of the receptor or binding site should be rather low compared to the Ko value. Although, in some experimental situations, where the receptors or binding site are present in high concentrations and have a high affinity for the ligand, this premise is incorrect and the free ligand concentration becomes depleted by binding to the receptors or binding sites. This is known as "depletion of the ligand" or ligand depletion (ligand depletion). Depletion of the ligand significantly impedes data analysis and can lead to the derivation of incorrect values for the binding parameters koff, kon and kd.
For this reason, when using bio-conjugated metal nanoparticle suspensions for the determination of ligand-receptor interactions, and more particularly a Ko and / or kon value, depletion of the ligand should be avoided. In practice, this means that less than 10% of the ligand should bind to the receptors or binding sites. Although depletion of the ligand can already be limited by reducing the concentration of nanoparticles containing the receptor, a minimum amount of nanoparticles is required for reliable detection, the ratio of surface area to volume of nanomaterials is very high compared to bulk materials (e.g. SPR sensors or wells in a microplate) and allows the presentation of a large number of binding sites per nanoparticle. For this reason, the reduction of the concentration of nanoparticles is often not sufficient to sufficiently limit depletion of the ligand, since the signal-to-noise ratio also decreases. The inventors have found a method to determine interactions between molecules without reducing the concentration of nanoparticles, which maintains an acceptable signal-to-noise ratio and reduces the effects of depletion of the ligand, thus enabling the determination of more accurate binding parameters.
For this reason, in a first aspect, the present invention provides a method for studying potential interactions between a first molecule and a second molecule with improved accuracy, particularly for the derivation of correct binding parameters. The methods of the invention include the use of nanoparticles that are conjugated to the first molecule and then brought into contact with the second molecule, the interaction between the first and the second molecule being determined by monitoring a change in optical properties . More specifically, the methods include the use of nanoparticles, wherein the amount of the first molecule adhered to the nanoparticles is less than 70% of the amount required for the complete coverage of the above-mentioned nanoparticles with the first molecule. With the current methods, the amount of the first molecule adhered to the nanoparticles can be controlled so that it is less than 70% of the amount required for complete coverage. In certain embodiments, the methods of the present invention include the following steps: a) providing nanoparticles containing one or more metals; b) providing the above-mentioned nanoparticles with one or more functional groups, or coupling the first molecule with a molecule that contains a metal-binding functionality; c) conjugating the above-mentioned first molecule with the above-mentioned nanoparticles, wherein the amount of the above-mentioned first molecule adhered to the above-mentioned nanoparticles is less than 70% of the amount required for complete coverage of the above-mentioned nanoparticles with the above-mentioned first molecule; d) incubating the above-mentioned nanoparticles with the above-mentioned second molecule; e) the monitoring of step d) by illuminating the above-mentioned nanoparticles with at least one excitation light source and the monitoring of one or more optical properties of the above-mentioned nanoparticles; and f) the detection of a change of one or more optical properties of said nanoparticles wherein said change is a result of the presence of an interaction between said first molecule and said second molecule.
In certain embodiments, the first and second molecules are an element of a specific known or intended binding pair or couple. Therefore, the second molecule (or potentially analogue ligand) can refer to a molecule that potentially interacts with the first molecule. Normally, both the first molecule and the second molecule are detection components that are elements of a binding couple such as antigen-antibody, receptor-ligand, enzyme-ligand, sugar-lectin, receptor-receptor binding agent, and others. In these embodiments, the methods of the present invention can serve to detect the interaction between the two elements of the binding pair. Relevant detection components include, but are not limited to, biomolecules, where the term "biomolecule" refers to any relevant organic or biochemical molecule, group, or species, e.g., that can specifically bind to a relevant analyte. Examples of biomolecules include, but are not limited to, peptides, proteins, amino acids and nucleic acids, small organic and inorganic molecules, ligands, etc. In certain embodiments, the first molecule is a protein.
In certain embodiments, the interaction measured between the first and second molecules is referred to as "binding." The term "bond" refers to two molecules that associate with each other in a non-covalent or covalent relationship.
The nanoparticles can be of any suitable shape and composition and can include, but are not limited to, nanoparticles, nanopherbs, nanopyramids, nanowires, nanoprismas, nanocubes, nanotetrapods, etc. Someone with expertise in the art will understand that other nanoparticles are also useful may be in the present invention.
In certain embodiments, the nanoparticles are nanoparticles. In further embodiments, the nanostars have an aspect ratio (i.e., length divided by width) ranging between 1.1 and 10, more particularly between 1.5 and 5. In certain embodiments, the nanostars have a width or diameter between 2 and 20 hm, more particularly between 5 and 18 nm, for example about 15 nm. In certain embodiments, the nanostars have a length between 4 and 60 nm, more particularly between 40 and 50 nm, for example approximately 48 nm.
The nanoparticles comprise or consist of one or more metals. In certain embodiments, the nanoparticles used in the context of the present invention comprise one or more metals selected from Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo , Tc, Ru, Rh, Pd, Ag, Cd, La, Hf, Ta, W, Re, Os, Ir, Pt, Au and / or Ac. In one embodiment, the nanoparticles comprise a metal selected from the group consisting of Au, Ag, Cu, Ta, Pt, Pd, and Rh. In certain embodiments, the aforementioned metal is selected from gold, silver and copper.
The nanoparticles provided in step a) of the methods according to the invention are normally provided as a colloid.
The solvents suitable for suspending the nanoparticles may depend on the nature of the surface of the nanoparticle. For example, the nanoparticles can be coated with a hydrophobic or hydrophilic coating. If the nanoparticles are provided with a hydrophobic coating, the solvent may consist of one or more solvents selected from toluene, hexane, heptane, pentane, cyclohexane, cyclopentane, chloroform, etc. If the nanoparticles are provided with a hydrophilic coating, solvent consist of one or more solvents selected from water, ethanol, butanol, isopropanol, acetone, etc. In certain embodiments, the particles are suspended in an aqueous medium.
In certain embodiments, the colloid comprises the nanoparticles in such a concentration that the colloid has an absorbance with Amax between 0.3 and 4, more particularly between 0.7 and 1.2, wherein Amax is the maximum absorbance of the nanoparticles between 350 and 1000 nm is.
In the methods of the present invention, the metal nanoparticles are optionally provided with one or more functional groups (referred to as step (b) above), preferably selected from amine, azide, alkynyl, carboxyl, hydroxyl and carbonyl. The functional groups can be used to conjugate the first molecule to the nanoparticles (as provided in step c) above) of the present method, and this will be further discussed below.
The nanoparticles used in the methods of the present invention are conjugated to a first molecule. It was found that the conjugation density is crucial to ensure the required accuracy for the optical detection of the interaction between the first and the second molecule, in particular in the determination of association / dissociation kinetics. For this reason, the methods of the present invention require control over the binding density of the first molecule to the nanoparticles, by conjugating the first molecule to the nanoparticles in such a way that the nanoparticles are not completely covered with the first molecule. Therefore, in a next step (corresponding to step c) described above) of the methods of the present invention, the first molecule is conjugated to the nanoparticles (provided in step b)), wherein the amount of the first molecule is conjugated to the nanoparticles less than 70% of the amount required for complete coverage of the above-mentioned nanoparticles with the above-mentioned first molecule. In certain embodiments, the amount of the first molecule conjugated to the nanoparticles is more than 10% of the amount required for complete coverage. This offers the possibility to use nanoparticles in amounts sufficient for reliable detection of interactions between the first and second molecules, while depletion of the ligand can be avoided, or at least reduced,. In certain embodiments, the amount of the first molecule conjugated to the nanoparticles is more than 15% of the amount required for complete coverage. In certain embodiments, the amount of the first molecule conjugated to the nanoparticles is more than 20% of the amount required for complete coverage.
The term "full coverage," as used herein, refers to the maximum amount of the first molecule that can be conjugated or attached to a nanoparticle as a single layer around the above nanoparticle. Full coverage can be achieved by exposing the nanoparticles to a large excess of the first molecule, in conditions suitable for coating the nanoparticles. In certain embodiments, the amount of the first molecule conjugated to the nanoparticles is less than 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25% of the amount required for complete cover. In certain embodiments, the amount of the first molecule conjugated to the nanoparticles is between 10% and 70%, preferably between 20% and 70%, more preferably between 20% and 60%, even more preferably between 30 and 50% of the quantity required for full coverage. The optimum amount of the first molecule can be determined via titration experiments and can depend on the characteristics of the nanoparticles, such as dimensions and shape, and the first and / or second molecule. In certain embodiments, the amount of the first molecule conjugated to the nanoparticles is between 30 and 50%, preferably between 40 and 50%, of the amount required for full coverage, wherein the nanoparticles are nanopots with a length between 40 and 60 nm and a diameter between 10 and 20 nm.
The first molecule is normally absorbed on / attached to / linked to / linked to the surface of the nanoparticle, which is commonly referred to herein as "conjugated to" or "conjugated to" by incubating the nanoparticles in a solution containing the first molecule under conditions that allow the adhesion of the first molecule to the surface of the nanoparticles. Specifically, the first molecule can be conjugated to the nanoparticles by various methods, for example: I) by incubating the nanoparticles that are provided with one or more functional groups with the above-mentioned first molecule, wherein the above-mentioned one or more functional groups are suitable for the first molecule covalently bind; II) by linking the first molecule to a left molecule that has metal-binding functionality, followed by conjugation of the first molecule to the nanoparticles via the left molecule. Any of these methods can be used in a strategy to reduce the number of binding sites proposed per nanoparticle.
Therefore, the step of functionalizing the nanoparticles (corresponding to step b) described above) of the current method in certain embodiments requires providing the surface of nanoparticles (provided in step a)) with one or more functional groups suitable for covalently bind the first molecule. In certain embodiments, the above functional groups are selected from carboxyl, amine, azide, alkynyl, carbonyl or hydroxyl.
Methods for the functionalization of nanoparticles are well known to those skilled in the art and may include, for example, the attachment of a left molecule to the surface of the nanoparticle, wherein the above-mentioned left molecule contains a first portion linked to the nanoparticle (e.g. via a metal-binding functionality) and also a second portion that is a functional group capable of forming a covalent bond with the first molecule.
There is a variety of linker molecules known to persons skilled in the art and usually includes bi-functional molecules. Generally, left molecules comprise a spacer group that ends at one end with a metal-binding functionality and at the other end with a functional group that is capable of covalently binding the first molecule.
Relevant spacer groups may consist of aliphatic and unsaturated hydrocarbon chains, spacers containing hetero atoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may contain hetero atoms. The spacer group is preferably as short as possible because it has been established that the optical detection of the interaction of a biomolecule with a second molecule improves with a reduced distance of the interaction from the surface of the nanoparticle. Although, a spacer group that is too short can be insufficient to stabilize the nanoparticles in suspension. For optimum results, the spacer group should preferably contain a hydrocarbon chain with 6 to 18 and preferably 6 to 16 carbon atoms, for example 11 carbon atoms.
Potential functional groups capable of covalently binding the first molecule include nucleophilic functional groups (amines, alcohols, thiols, azides, hydrazides), electrophilic functional groups (alkynes, carboxyl, aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reaction, forming disulfide compounds, or bonds to metals. Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonyl imidazoles, nitrophenyl esters, trifluoroethyl esters, glycidyl ethers, vinyl sulfones, and maleimides. Specific linker molecules that may find use in the present bifunctional molecules include compounds such as mercaptocarboxylic acids such as 11-mercaptoundecanoic acid, 11- [2- (2-azide-ethoxy) -ethoxy] -undecane-1-thiol, azidobenzoyl hydrazide, N- [4- ( p-azidosalicylamine) butyl] -3- [2'-pyridyldithio] propionamide), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyl tartrate, INI-maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobidimid-4-azidobidimidylidazid-4-azidobidimidyl-4-azidobidimidyl-4-azidobidyl] 1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl] amino benzoate, glutaraldehyde, and succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate, 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid, N-hydroxysuccinimide ester (SMCC), and the like.
In certain embodiments, one or more functional groups provided on the surface of the nanoparticle comprise carboxyl groups. Carboxyl groups are particularly useful for protein binding because an activated carboxyl group can react with an amine group of a protein, thereby forming an amide bond. Accordingly, in certain embodiments, the nanoparticles are coated with one or more (linker) molecules that comprise a carboxyl group and a metal-binding functionality. In certain embodiments, the metal-binding functionality is a sulfhydryl. Sulphhydryl groups bind strongly to a metal surface, in particular to gold surfaces. In further embodiments, the nanoparticles are at least partially coated with a mercaptocarboxylic acid. The sulfhydryl group of the mercaptocarboxylic acid can form a (coordination) bond with a metal atom from the surface of the nanoparticle, while the carboxyl group can be used to bind molecules such as proteins. In certain embodiments, the mercaptocarboxylic acid is a molecule of formula (I):
(I] wherein n is an integer from 6 to 16. In certain embodiments, the mercaptocarboxylic acid is an 11-mercaptoundecanoic acid.
In certain embodiments, one or more functional groups provided on the nanoparticles allow a coupling mechanism, as used in Click Chemistry. For example, the functional groups may comprise an azide or an alkyne, thereby allowing an azide alkyne Huisgen cycloaddition, using a room temperature Cu catalyst, as is known by those skilled in the art. Accordingly, in certain embodiments, the nanoparticles are at least partially coated with a (left) molecule of formula (IIIa), (IIIb), (IIIa) and / or (IIIb):
wherein na, nb, ng and ni are independently an integer from 6 to 16 and nh and nj are independently an integer from 1 to 5.
An azide functional group also offers the possibility of Staudinger ligation. Staudinger ligation normally requires reaction between an azide group with a phosphine or a phosphate group. Accordingly, in certain embodiments, the nanoparticles are at least partially coated with a molecule of formula (lia) or (Nb) as described above, wherein na and ng are independently an integer from 6 to 16.
In certain embodiments, it is possible that the first molecule as such does not contain a functional group suitable for covalently binding the functional groups provided on the nanoparticles. For this reason, the methods of the present invention may require a further step comprising providing at least one functional group on the first molecule prior to the conjugation of the first molecule to the nanoparticles.
If the first molecule is conjugated to the nanoparticles through the functional groups as described above, the amount of functional groups provided on the surface of the nanoparticle determines the maximum amount of the first molecule that can be conjugated to the nanoparticles. Thus, by limiting the amount of functional groups, it is possible to ensure that less than complete coverage of the nanoparticles is achieved by the first molecule, as required in the methods of the present invention. In certain embodiments, this is ensured by contacting the nanoparticle with different linker molecules, one bearing the reactive functional group, while the other not bearing the reactive functional group, so that the nanoparticle is coated with a mixture of reactive and non-reactive linkers . Therefore, in certain embodiments, the nanoparticles used in the methods of the present invention are generated in such a way that they include: one or more linker molecules comprising a functional group and a metal-binding functionality; and one or more molecules comprising the above-mentioned metal-binding functionality and not including the above-mentioned functional group. These one or more molecules are also referred to herein as "non-left molecules".
The metal-binding functionality ensures that the linker and non-linker molecules can be attached to the surface of the metal nanoparticle, while the functional group of the linker ensures that the first molecule can be covalently bound to the nanoparticles. Since the non-left molecules do not include the functional group, the adhesion of a sufficient amount of these molecules to the surface of the nanoparticle ensures that the amount of proteins conjugated to the nanoparticles is less than 70% of the amount required for full coverage of the above nanoparticles with that protein. Such a coating can be obtained by exposing the nanoparticles with a mixture of the left and non-left molecules. Alternatively, the coating can be obtained by completely or almost completely coating the nanoparticles with left molecules, and partially exchanging the coating with non-left molecules, or vice versa. For this reason, the selection of the linker comprising non-functional groups is ideally selected so that a) it can be partially exchanged with a linker with a reactive functional group and b) it does not interfere with subsequent reactions.
Examples of suitable linker molecules are described above. The one or more non-left molecules are preferably structurally similar to the left molecule. In certain embodiments, the non-left molecules are identical to the left molecule, except that they do not include the functional group. This normally results in a similar affinity and exchange characteristics of the left and non-left molecules on the surface of the nanoparticle. In certain embodiments, the non-left molecules have a reduced length compared to the left molecule. This reduces steric hindrance by the non-left molecules upon reaction of the functional group of the left molecules with the first molecule. Although, in certain embodiments, the non-left molecule (s) has a length similar to or longer than the left molecule. This can promote the stability of the coating.
In certain embodiments, the non-linker molecule (s) comprise one or more functional groups that improve other characteristics of the nanomaterial, such as solubility and / or stability. In certain embodiments, the presence of the non-linker molecules improves the stability of the nanoparticle suspensions. For example, if the non-linker molecules comprise sulfate, hydroxyl or polyethyleneglycol (PEG) elements, the stability of the nanoparticle colloids in aqueous media can be improved.
In certain embodiments, the functional group of the left molecule (s) is selected from carboxyl, azide, alkynyl, amine, carbonyl, and hydroxyl. In certain embodiments, the functional group is selected from carboxyl, azide and alkynyl. In certain embodiments, the functional group is a carboxyl. In certain embodiments, the above-mentioned metal-binding functionality is a sulfhydryl. In certain embodiments, the one or more linker molecules comprise a mercaptocarboxylic acid of formula (I) above such as 11-mercaptoundecanoic acid, and the one or more non-linker molecule (s) is / are a composition of formula (IV) and / or (V):
wherein R1 and R2 are independently selected from sulfate, hydroxyl, hydrogen or methoxy; wherein nc is such a number that the molecular weight of the compound (IV) is between 100 Da and 10 kDa, more particularly between 100 Da and 1 kDa; and nd is an integer from 6 to 16.
In specific embodiments, nc is an integer from 1 to 10. Normally, a relatively shorter linker molecule is chosen for the mixed single layers, but if the linker is sufficiently flexible (e.g., PEG), longer linker may be used.
Less than complete coverage can also be achieved by reacting only a certain fraction of the functional groups provided on the surface of the nanoparticle with the first molecule. The required amount of the first molecule to achieve the desired coverage can be found by a titration experiment (see below). However, the unreacted functional groups still present on the surface of the nanoparticle can cause non-specific bonds and reduced stability of the nanoparticle conjugate, for example during purification and buffers. Accordingly, in certain embodiments, the unreacted functional groups present on the nanoparticles are reacted with (an excess of) a bleeding reagent after conjugation of the nanoparticles with the first molecule. The blocking reagent reacts with the remaining unreacted functional groups present on the nanoparticles, thereby avoiding non-specific bonds.
Alternatively, less than complete coverage can also be obtained by blocking a certain fraction of the functional groups provided on the nanoparticles with a blocking reagent, after which the non-blocked functional groups are reacted with (an excess of) the first molecule. In order to determine the optimum amount of the blocking reagent required for blocking a certain portion of the functional groups provided on the nanoparticles, a concentration titration can be performed.
The blocking reagent is normally chosen such that it does not interact with the substances that will be tested with the conjugated nanomaterial. In certain embodiments, the blocking reagent is further selected to contribute to good solubility of the conjugated nanoparticles in one or more solvents, for example by adding charge, hydrophilic or steric hindrance. The solubility of the conjugated nanoparticles in polar solvents can be increased by providing blocking reagents containing one or more ethylene glycol groups, and / or blocking reagents containing one or more polar functional groups such as a sulfate, hydroxyl or methoxy.
If the functional group is a carboxyl, then the blocking reagent is a carboxyl blocking reagent. The carboxyl blocking reagent can contain an amine group so that it reacts with the remaining activated carboxyl groups. In certain embodiments, the (carboxyl) blocking reagent is a compound of formula (VI) or (VII)
wherein R 3 and R 4 are independently selected from sulfate, hydroxyl, hydrogen, and methoxy; ne is such a number that the molecular weight of composition (VI) is between 89 Da and 10 kDa, preferably between 1 kDa and 10 kDa, for example 1 kDa; and nf is such a number that the molecular weight of composition (IV) is between 30 Da and 500 Da. In certain embodiments, nf is an integer from 1 to 16, more particularly from 1 to 8.
As described above, the choice of blocking reagent may depend on various factors, such as the substances that will be tested with the protein-conjugated nanomaterial. In certain embodiments, the carboxyl blocking reagent is selected from Bovine Serum Albumin (BSA), Ovalbumin, and an amine polyethylene glycol of formula (VI) as described above. These blocking reagents are particularly useful if the first molecule is a Human Serum Albumin (HSA). In certain embodiments, any of the functional groups provided on the nanoparticles can be activated prior to the reaction with the first molecule. If the functional group is a carboxyl, the carboxyl can be activated by one or more carboxyl-activating groups. Useful carboxyl-activating groups include, but are not limited to, carbodiimide reagents. In certain embodiments, the activation of the carboxyl groups includes the addition of an N-hydroxysuccinimide (NHS) such as sulfo-NHS, together with a coupling reagent such as ethyl (dimethylaminopropyl) car carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC), preferably EDC. In certain embodiments, the activation includes the addition of sulfo-NHS together with EDC. This is also referred to in this document as "sulfo-NHS / EDC coupling".
Alternative carboxyl-activating groups include phosphonium reagents such as benzotriazolyloxy-tris (dimethylamine) phosphonium hexafluorophosphate (BOP) and the like, uranium or carbonium reagents such as O- (benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU), N-hydroxy succinimide (NHS), benzotriazol-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and the like; 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquunoline (EEDQ); 1-methyl-2-chloropyridinium iodide (Muikaiyama's reagent) and the like.
If the functional group is an azide, potential blocking reagents are molecules that contain a phosphine or alkyne group. If the functional group is an alkyne or phosphine, potential blocking reagents are molecules that contain an azide group. Examples of such molecules are modified proteins that do not interact strongly with the second molecule.
An alternative method for coupling the first molecule to the nanoparticles is by linking the first molecule to a left molecule that has a metal-binding functionality in step b), followed by the conjugation of the first molecule to the nanoparticles via the left molecule in step c ) (method II referred to above). The use of a linker with a metal-binding functionality eliminates the need to provide nanoparticles with functional groups for the binding of the first molecule. The first molecule is then conjugated to the nanoparticles via the left molecule, more specifically via the metal-binding functionality of the left molecule. This is achieved by incubating the nanoparticles with (an excess of) the first molecule with the left molecule attached to it. In certain embodiments, the left molecule comprises a carboxyl group and a metal-binding functionality. The carboxyl group allows the binding of the left molecule to amine functions present on the proteins. In certain embodiments, the left molecule is a mercaptocarboxylic acid, e.g., mercaptoundecanoic acid.
In certain embodiments, the metal nanoparticles, e.g., gold nanoparticles, are coated with a thiol PEG of formula (IV) as described above, prior to incubation with (an excess of) the first molecule with the left molecule attached thereto. The thiol-PEG molecules are then at least partially exchanged for the first molecule, via the left molecule. In certain embodiments, the use of nanoparticles coated with neutral thiol-PEG molecules such as methoxyl-PEG-thiol is particularly useful for avoiding the agglutination of the nanoparticles during conjugation with the first molecule; since the use of the EDC / Sulfo-NHS coupling method on carboxyl-functionalized nanomaterials that are negatively charged for some proteins that are highly positively charged can be challenging due to agglutination of the nanoparticles.
In certain embodiments, the step of conjugating the above-mentioned first molecule to the above-mentioned nanoparticles comprises the step of determining the amount of the first molecule required for the desired coverage of the nanoparticles. In further embodiments, step c) of the current method comprises: c1) optionally, selecting a suitable pH and ionic strength for conjugation of said first molecule with said nanoparticles via a buffer test; c2) determining the amount of said first molecule necessary for conjugation of said first molecule to said nanoparticles; c3) conjugation of the above-mentioned first molecule to the above-mentioned nanoparticles, based on the information obtained in step c2) and optionally c1).
Step c2) is normally performed by a concentration titration, in which the nanoparticles are mixed with different amounts of protein and analyzed. The titration can be performed at the pH and ionic strength selected in step c1). The titration can be monitored via the measurement of the absorbance of the nanoparticles, preferably at two or more wavelengths.
In certain embodiments, step c2) includes the addition of different amounts of the first molecule to a fixed amount of nanoparticles and the recording of absorbance spectra. The ratio OD (Amax + 80) / OD (Amax) (ARU) can then be graphically represented versus the amount of the first molecule added. The conjugation of the first molecule to the nanoparticles leads to a difference in the refractive index around the nanoparticles and thereby to a redshift of the Amax, which can be detected by reading an absorbance spectrum. By increasing the amount of the first molecule conjugated to the nanoparticles, the redshift also increases. After reaching the maximum amount of the first molecule that can be conjugated to the nanoparticles, the spectrum no longer changes, resulting in a plateau in the display of OD (Amax + 80) / OD (Amax) (ARU) versus the amount of the first molecule. The lowest amount of protein on the plateau is the minimum amount of the first molecule needed to completely cover the surface of the nanoparticle with the first molecule (or at least the LSPR-sensitive part of the nanomaterial). In order to obtain a lower number of binding sites than that corresponding to a complete coverage of the nanoparticles with the first molecule, a lower amount of the first molecule than the maximum amount can be used for conjugation. The optimum amount of the first molecule to be used for conjugation is a compromise between two tendencies to ensure optimum sensitivity. Usually the ideal amount of protein to be used is the amount corresponding to 30 - 60% of the plateau.
If the nanoparticles are provided as a colloid, the colloid should preferably be purified after conjugation of the first molecule to the nanoparticles, in particular when the conjugation of the first molecule to the nanoparticles occurs by incubating the nanoparticles in a solution that contains an excess of the first molecule. The excess of first molecules that are not bound to the nanoparticles should preferably be removed after conjugation. This can be done by one or more cycles of precipitation and resuspension of the nanoparticles, as is known by a skilled person.
After the nanoparticles are effectively coated with the first molecule, the nanoparticles are incubated with the second molecule in a subsequent step (step d) of the current methods. Normally, the second molecule is provided in a (relatively) purified form in a liquid composition, which may be an aqueous composition and / or a buffer. In certain embodiments, the second molecule can be present in a biological sample.
Incubation of the nanoparticles with the second molecule allows interaction of the second molecule with the first molecule conjugated to the nanoparticles. The proximity of the second molecule to the nanoparticles changes the dielectric medium that surrounds the nanoparticles, which normally leads to changes in certain optical properties of the nanoparticle, such as the absorbance. For this reason, measuring one or more of these properties can provide information about the interactions between the first and second molecules.
Accordingly, a further step (step (e) referred to above) of the methods set forth herein includes monitoring the interaction between the first and second molecules when contacted (in step d) by exposing the nanoparticles with at least one excitation light source and by monitoring one or more optical properties of the above-mentioned nanoparticles. The light source normally emits light or radiation at one or more wavelengths between 220 and 1000 nm. In certain embodiments, an excitation light source is used that emits light or radiation with a strength between about 1 nanowatt and 100 watts. In more particular embodiments, the excitation light source is a (xenon) flash lamp or a laser.
The monitoring of the optical parameters is used to detect a change therein, e.g. a change in absorption at a certain wavelength. Any suitable optical parameter can be evaluated or monitored in this step, the representative parameters being comprised of, but not limited to: absorbance, refractive index, absorption, scattering, fluorescence, luminescence and the like. The optical parameter can be monitored with any suitable device and protocol, with suitable protocols well known to those skilled in the art. The presence or absence of a change in the optical parameter is then used to determine whether or not the first molecule interacts with a second molecule, which in certain embodiments is used to give an indication of the presence of a relevant analyte (second molecule) in a sample. Accordingly, in a further step (step (f) referred to above) of the methods of the present invention, a change of one or more optical properties of the above-mentioned nanoparticles is detected. This change is a result of the presence of an interaction between the first molecule and the second molecule. Where there is interaction between the first and the second molecule, this will indeed be detected, as the optical properties of the nanoparticle will change. Where there is no interaction between the first and the second molecule, no change in optical properties will be detected.
The methods described herein include the step of detecting a signal, in particular a change in signal at one or more wavelengths. The terms "monitor", "determine", "measure", "assess", "detect" and "evaluate" are used interchangeably and refer to any form of measurement, and do not include the detection of any change. Such measurements can include both quantitative and quantitative determinations, both relative and absolute, and also include determining the amount of a substance present, as well as determining whether or not it is present.
For this reason, as heirin uses, the term "detecting" means determining a signal (or a change therein), both qualitatively and quantitatively.
In certain embodiments, the one or more optical properties of the nanoparticles are measured at two or more wavelengths between 220 and 1000 nm, preferably between 350 and 1000 nm. Measurement at two or more wavelengths allows for more accurate data. In certain embodiments, these wavelengths are discrete wavelengths within that range.
The methods of the present invention normally further comprise the step of analyzing the detected signal or change in signal and determining a particular property of the first or second molecule or of their interaction based on it.
In certain embodiments, the step of determining a change in one or more optical properties of the above nanoparticles includes determining the dissociation constant (Kd) or the binding or association constant (Ka) for the association-dissociation equilibrium between the first and second molecule. The association * dissociation equilibrium can be represented by equation 1: M1 + M2 M1M2 (equation 1) wherein M1M2 represents an association or complex of M1 and M2.
The reaction is characterized by the association rate constant kon and the dissociation rate constant coff. In equilibrium, the forward-binding transition M1 + M2 -> M1M2 is balanced by the backward-decomposing transition M1M2 -> M1 + M2. Starting from a first-order reaction, this is MM1] [M2] = coffe [M1 M2] (equation 2) where [M1], [M2] and [RL] respectively have the concentration of unbound free M1, the concentration of unbound free M2 and the concentration of M1M2 complexes. The association constant Ka and the dissociation constant Kd are defined by:
Ka = 11 Kd = konlkoff = [M1 M2] / ([M1] [M2]) (equation 3)
Accordingly, if the concentrations of M1, M2 and M1 M2 are known to be in equilibrium, Ka and Kd can be calculated. Alternatively, Ka and Kd can be calculated from a binding isotherm (see below).
More information, such as the association energy, can be obtained on the basis of the association or dissociation constant.
In a further aspect, the present invention provides for the use of the present method of determining an interaction between a first and a second molecule for the preparation of binding isotherms for the binding of said second molecule to said first molecule, wherein the binding affinity, binding constant Ka or the dissociation constant Kd of said second molecule for said first molecule is evaluated based on said binding isotherm.
A binding isotherm can be constructed based on the detected changes in optical properties, such as the refractive index or absorbance i and the amount (or concentration) of second molecule added to the nanoparticles.
The methods of the present invention are also important in the context of screening methods. Therefore, in certain embodiments, the present invention provides screening methods in which the detection is performed according to the present invention. In further embodiments, the methods are "high throughput" screening methods, more particularly methods that are performed at least in part in a device for "high throughput" screening.
The term "screening" refers to determining the presence of something relevant, e.g., an analyte, an event, etc. As such, the methods of the present invention can be used to screen a sample for the presence or absence of one or more target analytes in the sample. As such, the invention provides methods for detecting the presence of one or more target analytes in a sample. In addition, the present methods can also be used to screen for compounds that modulate the interaction of a particular specific binding element pair. The term modulate includes both reducing (e.g., suppressing) and increasing the interaction between the two molecules. For example, where the colloid exhibits a first element of a binding pair and the colloid is contacted with the second element in the presence of a candidate agent, the effect of the candidate agent on the interaction of the binding element pairs can be evaluated or evaluated to become.
In yet a further aspect, the present invention relates to the instrumentation for implementing the methods of the present invention. More specifically, the set of instruments comprises nanoparticles to which a first molecule was conjugated. More specifically, the nanoparticles are conjugated in such a way that there is no complete coverage of the nanoparticles with the first molecule. In certain embodiments, the coverage is less than 70%, less than 60%, more particularly between 30 and 50%.
The nanoparticles of the present invention can be obtained in the manner described above, by conjugating them with less than 70% of the amount required for complete coverage of the nanoparticles with the first molecule.
The invention further provides kits for carrying out the methods of the present invention. More specifically, kits for carrying out the methods of the present invention contain a medium comprising a plurality of metal nanoparticles; instructions for the use of the above-mentioned nanoparticles in the methods according to the present invention; - optionally, the above-mentioned first molecule; and - optionally, the second molecule mentioned above.
In certain embodiments, the above-mentioned nanoparticles are provided with functional groups, preferably selected from carboxyl, amine, azide, carbonyl and hydroxyl.
The kits of the present invention optionally also contain solvents, buffers and / or stabilizers. The kits optionally also contain one or more linker and / or non-linker molecules as described herein.
In certain embodiments, the nanoparticles are golden nano-rods. Many methods for the production of golden nanoparticles are known by skilled persons. Normally, these methods require the use of golden nanoparticles and cetyl trimethyl ammonium bromide (CTAB) as a coordinating molecule, and result in nanoparticles completely coated with CTAB. Although, in the methods of the present invention, particles completely coated with CTAB may not be suitable for conjugation to the first molecule. For this reason, an exchange of the CTAB coating with a coating of various molecules, such as mercaptocarboxylic acids, may be required. However, such an exchange normally requires long reaction times and / or various phase conversions between the polar and non-polar phase. The inventors have found optimized coating methods that are particularly suitable for obtaining nanoparticles conjugated to a first molecule. These methods are of general interest for the coating of nanoparticles, but can also be used in the methods of the present invention. Thus, in a further aspect, the present invention provides methods for coating a metal nanoparticle, including the use of metal I nanoparticles with a CTAB coating to obtain nanoparticles with a mercaptocarboxylic acid coating. In certain embodiments, the methods of the invention include the following steps: i) providing a liquid composition comprising metal nanoparticles that are at least partially coated with CTAB. Normally, the metal nanoparticles provided in this step are completely coated with one or more molecules in which the coating contains CTAB. The coating may further contain other molecules, such as benzyl dimethylammonium chloride.
ii) adding a thiol-polyethylene glycol to the above-mentioned composition, whereby a liquid composition containing metal nanoparticles coated with a thiol-polyethylene glycol is obtained.
iii) the purification of the above-mentioned composition obtained in step ii) by separating the above-mentioned nanoparticles from free thiol-polyethylene glycol and free CTAB.
iv) adding a mercaptocarboxylic acid to the above-mentioned composition obtained in step iii, whereby a liquid composition containing metal nanoparticles coated with a mercaptocarboxylic acid.
In contrast to the known methods, which require hydrophobic solvents in some steps, all steps of the method for coating a metal nanoparticle according to the present invention can be carried out in an aqueous phase. This significantly reduces potential health and safety risks and facilitates the scaling up of nanoparticle production. Furthermore, it also leads to reduced costs with regard to chemical products and waste management. Accordingly, in certain embodiments, the liquid composition in steps i), ii), iii) and iv) is an aqueous composition.
In certain embodiments, mercaptocarboxylic acid added in step iv) is a composition of the formula (I) described above.
In certain embodiments, the thiol-polyethyleneglycol added in step ii) is a compound of formula (IV) as described above, wherein nc is such a number that the molecular weight of composition (IV) is between 100 Da and 10 kDa, preferably between 1kDa and 10kDa, for example 5kDa; and R 1 is C 1-6 alkoxy, preferably methoxy or ethoxy, wherein said C 1-6 alkyl can optionally be replaced by one or more groups such as hydroxyl.
The nanoparticles coated with a mercaptocarboxylic acid obtained in step iv) as described above can be used to conjugate a molecule thereto. Accordingly, the present invention further provides a method for conjugating a molecule to a metal nanoparticle, comprising, inter alia: i) providing a liquid composition comprising metal nanoparticles that are at least partially coated with CTAB.
ii) adding a thiol-polyethylene glycol to the above-mentioned composition, whereby a liquid composition containing metal nanoparticles coated with a thiol-polyethylene glycol is obtained.
iii) the purification of the above-mentioned composition obtained in step ii) by separating the above-mentioned nanoparticles from free thiol-polyethylene glycol 5 and free CTAB.
iv) adding a mercaptocarboxylic acid to the above composition obtained in step iii), whereby a liquid composition containing metal nanoparticles coated with a mercaptocarboxylic acid.
v) contacting the above-mentioned metal particles obtained in step iv) with the above-mentioned molecule.
In a further aspect, the present invention provides a method for conjugating a molecule to a nanoparticle, comprising, inter alia: a) providing nanoparticles containing one or more metals; b) providing the above-mentioned nanoparticles with one or more functional groups, or linking the first molecule to a molecule that contains a metal-binding functionality; c) conjugating said first molecule to said nanoparticles, wherein the amount of said first molecule attached to said nanoparticles is less than 70% of the amount required for complete coverage of said nanoparticles with said first molecule;
The following examples are provided to illustrate the present invention and are by no means intended, and in no way should be interpreted, as limiting the scope of the present invention.
EXAMPLES
A) Preparation of nanoparticles coated by 11-mercaptoundecanoic acid a1) Preparation of nanoparticles coated with CTAB
The method used for the preparation of the golden nano-rods is similar to the method described by Yu and Irudayaraj (Anal. Chem. 2007, 79, 572-579). In summary, a seed solution was prepared by adding 0.5 mM hydrogen I tetrachloroaurate (HAuCU) to 0.1 M CTAB, after which a 0.01 M NaBH 4 was added. A growth solution was then prepared by adding 42mM AgNO3 and then 1mM HAuCU to the 0.1M CTAB solution. Immediately after the preparation of the growth solution, an ascorbic acid solution is added to the growth solution. After a change in color, the seed solution is added, whereby golden nanoparticles coated with CTAB are generated.
a2) Removal of oversized CTAB
After one week, excess CTAB was removed from the nano-rod suspension by centrifuging the suspension at 10,000 g for 30 minutes. Subsequently, the supernatant (containing the majority of the oversized CTAB) was removed and the precipitated nanoparticles in the sediment were resuspended in (double distilled) water.
a3) Exchange of CTAB with mPEG-SH
After removal of excess CTAB, the CTAB coating of the nanopots was exchanged with a sulfhydryl-functionalized methoxy polyethylene glycol (mPEG-SH, available from Nanocs as "PEG3-0021") coating. First a 10 g / L solution of mPEG-SH in (double distilled) water was prepared. To this solution was added a same volume of purified nanoparticle suspension with agitation (stirring). The mixture was then subjected to ultrasonic vibrations for 5 minutes, followed by agitation for 12 hours at room temperature. a41 Removal of oversized MPEG-SH
The suspension obtained in step a3) was centrifuged at 8000g for 30 minutes at 25 ° C. After centrifugation, the supernatant was removed and the precipitated nanoplasts were resuspended in a (1x concentrate) tris / borate / EDTA (TBE) buffer. The centrifugation and resuspension cycle was repeated three times. After the final centrifugation step, the nanostars were resuspended in a (1x concentrate) TBE buffer. a51 Exchange of the mPEG-SH coatinq with MUDA
For the exchange of the mPEG-SH coating of the nanostars with a mercaptoundecanoic acid (MUDA) coating, a 60 g / L solution of MUDA in ethanol was prepared. This solution was added to the nanoparticle suspension obtained in step a4) resulting in a concentration of approximately 1.2 g / l MUDA concentration. The suspension was then subjected to ultrasonic vibrations for 5 minutes, followed by agitation for two hours. The suspension was then centrifuged at 5000g for 30 minutes at 25 ° C, followed by the
Removal of the supernatant and resuspension of the precipitated nanostars in 25mL TBE buffer. A 60 g / L MUDA solution in ethanol was then added to the nanoparticle suspension (resulting in a final MUDA concentration of 1.2 g / l), followed by agitation for 12 hours. a61 Removal of oversized MUDA
3 The nanoparticle suspension obtained in step a5) was centrifuged at 5000g for 30 minutes at 25 ° C, followed by the removal of the supernatant and resuspension of the precipitated nanoparticles in the TBE buffer. The cycle of centrifugation and resuspension was repeated twice. After the final centrifugation step, the sediment was resuspended in a 10 mM MES buffer comprising 0.002% (per volume) of Tween (polysorbate). The suspension was then dialyzed against 10mM MES buffer comprising 0.002% (per volume) Tween for one hour (3L buffer per 25mL nanoparticle suspension).
In order to obtain nano rods coated with a mixture of MUDA and another molecule, the method described above can be used, with the difference that in step a5) use is made of a solution containing MUDA and that other molecule.
B) Conjugation of nanoparticles with HSA
The amount of HSA required to achieve complete coverage of nanoparticles was determined by means of a concentration titration. A slurry was provided containing carboxylated (i.e., MUDA-coated) gold nanoparticles, as described in Example A). The carboxyl groups were activated with EDC and sulfo-NHS, and the nano-bar suspension was mixed with increasing amounts of HSA in a range of 0 to 200 pg of protein per mL of nano-bar suspension.
Absorbance spectra of these samples were recorded and ARU, this is the change in the ratio (OD (+max + 80)) / (ODX ***). i.e. the ratio of the optical density at Xmax + 80 nm (OD (Xmax + 80)) and the optical density at the peak value (Οϋλ ^ χ) was displayed graphically with respect to the amount of HSA per mL nanoparticle suspension. The ratio of these optical densities provides information on the amount of conjugated HSA. Figure 1A shows the ARU vs. graphs. the HSA amount and shows that the increased amounts of added HSA result in an increased change of ARU, which can be understood as an increased amount of conjugated HSA that surrounds the particles. The attachment of protein to the nanomaterial indeed leads to a difference in the refractive index around the nanomaterial and thereby to a redshift of the Ämax which can be detected by reading an absorbance spectrum. The effect of protein conjugation on the optical density (OD) of a suspension of golden nano-bars is shown in Figure 1B. Higher amounts of conjugated protein lead to increasing red shifts of the spectrum, i.e. shifts to higher wavelengths. Figure 1A shows that after reaching the maximum amount (100%) of protein that can be attached to the nanomaterial, the spectrum no longer changes, resulting in a plateau in the display of ARU versus the amount of protein.
The lowest amount of protein on the plateau is the minimum amount of protein required for complete coverage of the nanomaterial with the relevant protein.
In a particular embodiment of the present invention, the optimum amount of the protein to be used is the amount corresponding to 30-60% of the plateau. In the present example, this corresponds to 9-25 pg / mL of proteins, as shown in Figure 1.
In this range, a sufficiently low amount of protein is adhered to the bars to avoid depletion of the ligand, and a sufficiently high amount of protein is adhered to the bars to provide a significant redshift of Amax after binding a binding partner.
C) Effect of binding site density on the determination of Kd
In order to study the effect of the number of binding sites provided on the nanoparticles on the measured binding properties, such as the dissociation constant (K <j), a carboxylated nanoparticle suspension as used in Example B was divided into two groups. The two groups were conjugated with HSA by activating the carboxyl groups with EDC / sulfo-NHS and mixing the suspension with 600 pg / mL HSA (sample 1) and 18.75 pg / mL HSA (sample 2), respectively. The unreacted carboxyl groups present on the surface of the nano-bars were blocked by the addition of BSA. In accordance with the results of Example A), a complete coverage of the nano-rods with HSA was obtained in sample 1, while only a partial coverage was obtained in sample 2.
The two solutions were contacted with Anti-Human Serum Albumin monoclonal antibody (ab18081, available from abcam®) in identical conditions. After an incubation time of 30 minutes, the absorption spectra of the suspensions were recorded.
Figure 2 shows the effect of adding different amounts of the antibody on the optical properties of the two equilibrium suspensions. In all cases, the increased addition of antibody resulted in an increased change in the ratio of OD (Amax + 80)) / ODAmax, indicative of increased amounts of HSA molecules that bind to the antibody.
Dose-response curves can be constructed on the basis of the absorbance data, from which the dissociation constant Kd for the HSA-antibody association-dissociation equilibrium was calculated. The results are shown in Table 1. With 5 complete coverage (sample 1), the Kd calculated from the experiments deviate considerably from the correct value as determined for sample 2. This deviation can be attributed to the depletion of the ligand.
Table 1 - Results of K <j determination with different binding site density
Similar results are obtained with the use of other blocking agents. Table 2 shows the Kd values obtained with the use of methoxy-PEG-amine (mPEG-NH 2), BSA and ovalbumin. The incubation time was 60 minutes for each blocking agent. The results, shown in Table 2, indicate that the obtained values for are equal to the margin of error.
Table 2 - Results of K <j determination with different blocking agents
A similar group of golden nano bars was divided into three groups. The groups were conjugated with HSA by mixing the suspension with 600 pg / mL HSA (sample 1), 37.5 pg / mL HSA (sample 2) and 18.75 pg / mL HSA (sample 3), respectively. The unreacted carboxyl groups present on the surface of the nano-bars were blocked by the addition of BSA. In accordance with the results of Example B), a complete coverage of the nano-rods with HSA was obtained in sample 1, while only a partial coverage was obtained in samples 2 and 3.
The three solutions were contacted with Anti-Human Serum Albumin monoclonal antibody (ab18081, available from abcam®) in identical conditions. After an incubation time of 60 minutes, the absorption spectra of the suspensions were recorded, from which the dissociation constant K <i for the HSA-antibody association-dissociation equilibrium was calculated. The results are shown in Table 3. With complete coverage (sample 1), the K <j calculated from the experiments again deviate considerably from the correct value as determined for samples 2 and 3. This deviation can also be attributed here to the depletion from the ligand.
D) MUDA functionalization of a protein and mPEG-SH exchange
The relevant first molecule in this example is BSA. BSA was covalently modified on the primary amine with a thiol linker, in particular MUDA. For this reaction, the carboxyl group of MUDA was activated with EDC and sulfo-NHS.
Subsequently, an excess of the modified BSA (BSA-MUDA) was added to a suspension of purified mPEG-SH-coated nano-rods, as obtained in step a4) of the above-described example A). This results in a (partial) exchange of mPEG-SH by BSA_MUDA on the surface of the nanoparticles. The exchange is slow and takes a few hours to several days.
Figure 3 shows a concentration titration of mPEG-SH-coated nanostars with BSA_MUDA. Addition of larger amounts of BSA_MUDA normally leads to an increased exchange of mPEG-SH with BSA_MUDA, which is indicated by an increased ARU. The measurement of the same samples after 120 minutes and after 3 days shows that longer exchange times lead to an increased exchange.
E) Conjugation via Click chemistry or Staudinger ligation i e1 1 Click chemistry
Figure 4 shows a conjugation between a first molecule (1) and a nanoparticle (2) according to a particular embodiment of the present invention. The first molecule (1) is a protein and the nanoparticle (2) is a golden nano-rod, provided with azide functional groups. In a first step (A), the protein (1) is provided with an alkyl group by reacting an amine group of the protein with the succinimidyl group of composition (3). In a second step (B), the fictionalized protein (4) is conjugated to the nanoparticle (2) via an azide alkyne Huisgen cycloaddition reaction, resulting in a nanoparticle conjugate (5).
e2ï Staudinaer lation
Figure 5 shows a conjugation between a first molecule (1) and a nanoparticle (2) according to a particular embodiment of the present invention. The first molecule (1) is a protein and the nanoparticle (2) is a golden nano-rod, provided with azide functional groups. In a first step (A), the protein (1) is provided with a triarylphosphine group (containing an ester group situated on the phosphorus) by reacting an amine gorep of the protein with the succinimidyl group of composition (6). In a second step (B), the functionalized protein (7) is conjugated to the nanoparticle (2) via a Staudinger reaction, whereby a nanoparticle conjugate (8) is obtained.

权利要求:
Claims (15)
[1]
A method for determining an interaction between a first and a second molecule, comprising: a) providing nano rods containing one or more metals, and having an aspect ratio ranging between 1.5 and 5; b) providing said nano-rods with one or more functional groups, or linking said first molecule to a molecule that contains a metal-binding functionality; c) conjugating the above-mentioned first molecule with the above-mentioned nano-rods, wherein the amount of the above-mentioned first molecule attached to the above-mentioned nano-rods is between 30% and 70% of the amount required for full coverage of the above-mentioned nano-rods with the above-mentioned first molecule; d) incubating the above-mentioned nanoparticles with the second molecule mentioned above; e) the monitoring of step d) by illuminating the above-mentioned nano-bars with at least one excitation light source and the monitoring of one or more optical properties of the above-mentioned nano-bars; and f) the detection of a change of one or more optical properties of said nano-rods wherein said change is a result of the presence of an interaction between said first molecule and said second molecule.
[2]
The method according to claim 1, wherein said nanoparticles contain gold, silver or copper.
[3]
The method of claim 1 or 2, wherein step f) comprises determining an association-dissociation equilibrium between said first and second molecule.
[4]
The method of any one of claims 1 to 3 above, wherein step f) comprises measuring said one or more optical properties at two or more wavelengths ranging between 350 and 1000 nm.
[5]
The method of claim 4, wherein step f) comprises measuring the absorbance of the nanostats at two or more wavelengths ranging between 350 and 1000 nm.
[6]
The method according to any of the preceding claims from 1 to 5, wherein step b) comprises the provision of nano-rods, with on their surface: one or more molecules containing a metal-binding functionality and a functional group selected from carboxyl, amino, azide , alkynyl, carbonyl and hydroxyl; and one or more molecules containing the above-mentioned metal-binding functionality and not containing the above-mentioned functional group selected from carboxyl, amine, azide, alkynyl, carbonyl and hydroxyl.
[7]
The method of any one of claims 1 to 6 above, wherein step c) includes, inter alia: c1) optionally, selecting a suitable pH and ionic strength for conjugation of said first molecule with said nano-rods via a buffer test; c2) the determination of the amount of said first molecule required for the conjugation of said first molecule with said nano-rods; c3) conjugation of the above-mentioned first molecule with the above-mentioned nanostats, based on the information obtained in step c2) and optionally c1).
[8]
The method according to claim 7, wherein step c2) comprises a concentration titration of said nano-rods with said first molecule, optionally at the pH and ionic strength selected in step c1).
[9]
The method of any one of claims 1 to 8 above, wherein said step b) comprises coupling of said first molecule to a linker molecule with a metal-binding functionality, and step c) comprises conjugation of said first molecule to said nano-rods, via the above-mentioned left molecule.
[10]
The method of claim 9, wherein said linker molecule is a mercaptocarboxylic acid.
[11]
The method of any one of claims 1 to 10 above, wherein said first molecule is a protein and the surface of said nano-bars is provided with carboxyl groups.
[12]
The method of claim 11, further comprising reacting carboxyl groups on the surface of said nano-rods with a carboxyl blocking reagent.
[13]
The method of any one of claims 1 to 12, wherein the amount of said first molecule adhered to said nano-rods is between 30% and 50% of the amount required for complete coverage of said nano-rods with said first molecule.
[14]
A method for coating a metal nanoparticle with a compound, comprising: i) providing a liquid composition containing metal nanoparticles that are at least partially coated with cetyl trimethyl ammonium bromide (CTAB); ii) adding a thiol-polyethylene glycol to the above-mentioned composition, whereby a liquid composition is obtained which contains metal nanoparticles coated with thiol-polyethylene glycol; iii) purification of the above composition obtained in step ii), by separating the above nanoparticles from free thiol-polyethylene glycol and free CTAB; iv) adding a mercaptocarboxylic acid to the above-mentioned composition obtained in step iii), whereby a liquid composition containing metal nanoparticles coated with mercaptocarboxylic acid is obtained, and v) bringing the above-mentioned particles obtained in step iv) into contact with the above connection.
[15]
The method of claim 14, wherein said liquid composition in steps i), ii), iii) and iv) is an aqueous composition.

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同族专利:

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公开号 | 公开日

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US20150024402A1|2015-01-22|

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EP2814774A1|2014-12-24|

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IN2014MN01800A|2015-07-03|

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AU2013220314A1|2014-08-14|

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AU2013220314B2|2016-03-10|

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CA2864614A1|2013-08-22|

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US20170248589A1|2017-08-31|

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WO2013121011A1|2013-08-22|

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GB201202631D0|2012-04-04|

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HK1203919A1|2015-11-06|

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US9678066B2|2017-06-13|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO2010029175A1|2008-09-12|2010-03-18|Modpro Ab|Detection method and device based on nanoparticle aggregation|

---

US20060275310A1|2004-09-30|2006-12-07|Nano Science Diagnostics, Inc.|Method and detection and decontamination of antigens by nanoparticle-raman spectroscopy|

---

US8426152B2|2007-01-03|2013-04-23|Lamdagen Corporation|Enzymatic assay for LSPR|

---

DE602008005013D1|2007-05-29|2011-03-31|Pharma Diagnostics Nv|REAGENTS AND PROCEDURES FOR DETERMINING PK / ADME TOX PROPERTIES OF NEW CHEMICAL UNITS AND ACTIVE CANDIDATES|

---

US20120164073A1|2007-11-30|2012-06-28|Old Dominion University|Stable nanoparticles, nanoparticle-based imaging systems, nanoparticle-based assays, and in vivo assays for screening biocompatibility and toxicity of nanoparticles|

---

GB201003088D0|2010-02-24|2010-04-14|Univ Exeter|Method for the preparation of a novel nanoparticle conjugate|WO2016057474A1|2014-10-06|2016-04-14|Indiana University Research & Technology Corporation|Systems and methods for localized surface plasmon resonance biosensing|

---

CL2017001945A1|2017-07-30|2018-10-19|Univ Tecnica Federico Santa Maria Utfsm|Method, process, composition and kit to measure concentration of dissolved molecules in the continuous phase of a colloid.|

---

US10758983B1|2018-04-17|2020-09-01|Government Of The United States, As Represented By The Secretary Of The Air Force|Concentrated synthesis of monodispersed gold nanorods|

---

CL2018001473A1|2018-06-01|2019-10-25|Univ Tecnica Federico Santa Maria Utfsm|Kit and methods to evaluate the adsorbent properties of the surface of a material|

法律状态:
2019-11-20| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190228 |

优先权:

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申请号 | 申请日 | 专利标题

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US201261599455P| true| 2012-02-16|2012-02-16|

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US201261599455|2012-02-16|

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GBGB1202631.6A|GB201202631D0|2012-02-16|2012-02-16|Method of measuring interaction between molecules|

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GB201202631|2012-02-16|

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